Clocked: local SNPs in global pops

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R4RNA arc diagrams (top) for predicted secondary structure comparison of the (upper) G/G/U/G/C and (lower) A/U/G/C/A haplotypes, with (bottom) SNPs aligned along the LHY 5’UTR region (exons; boxes and introns; horizontal lines). [from Fig 1 (b and c) of James, Sullivan and Nimmo, PCE, 2018]
Our study on the correlation between ‘natural variation’ in a clock gene sequence with bioclimatic parameters is out now as OpenAccess in the journal Plant, Cell & Environment.

The paper is called ‘Global spatial analysis of Arabidopsis natural variants implicates 5′UTR splicing of LATE ELONGATED HYPOCOTYL in responses to temperature

The starting point for this work was the idea that the 5’UTR of the core clock gene LATE ELONGATED HYPOCOTYL, also known as LHY, could function as a thermosensor given that we previously saw temperature sensitive alternative splicing of LHY.

We tested our theory using the 1001 genomes resource, a whole-genome sequence database for at least 1001 strains of the reference plant, Arabidopsis thaliana. Arabidopsis is native to Europe, but can now be found in the United States, North Africa and temperate Asia. We examined subtle differences, or polymorphisms, in the DNA sequences of >1001 accessions. These are often referred to as single nucleotide polymorphisms (SNPs). We found that different strains tended to ‘shake out’ as particular ordered assemblies of the SNPs, called haplotypes [for example, in the picture above the G/G/U/G/C haplotype is compared to the A/U/G/C/A haplotype] .

We were interested to see if the distinct haplotypes aligned with particular features of where these plants were growing – maybe the haplotypes grouped according to latitude, longitude, or altitude? Or would they group according to climate, such as temperature? seasonality? or even rainfall? For this we made use of the WorlClim database – a free public resource offering global climate data for ecological modelling.

The key findings were that:

  1. One of the haplotypes has hallmarks of being a signature of ‘relict’ accessions (survivors of the last ice-age and the subsequent expansion of new populations). This version is the most distinct in the respect that, worldwide, the accessions bearing this haplotype are found in regions of low rainfall. They are also associated with the highest elevations with low mean annual temperatures and a wider range of maximum–minimum temperatures
  2. Two of the remaining three haplotypes seem to associate with milder annual mean temperatures and lower altitude and wetter habitats
  3. The fourth haplotype, seems to be a low temperature specialist. This haplotype is commonly found in the mountainous Pyrenees region of northern Spain and is prominent at the limit of Arabidopsis growth in northern Sweden
  4. By measuring the extent of LHY spliced upon cooling in representative strains from two haplotypes we established that haplotype does indeed affect the splicing of LHY transcripts in response to cooling
  5. We propose that the LHY haplotypes possess distinct 5′UTR pre‐mRNA folding thermodynamics and/or specific biological stabilities based around the binding of trans‐acting RNA splicing factors

There is much interest in identifying plant thermometers and how they have evolved to cope with new temperature environments. Our new work shows that subtle differences in the DNA sequence of global populations of Arabidopsis plants influences the scalable splicing sensitivity of the mRNA for this central clock component, thereby finely tuning the clock to specific temperature environments.

We anticipate that these findings will be of interest and relevant to crop breeding programs that aim to produce stable food crops in the face of changing climate. 

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Free the roots

 

Been growin’ the Arabidopsis seedlings in Eppendorf tubes filled with agar media. The young roots do what they do best and grow downwards through the agar media (this is called ‘gravitropism‘). The race is on though….if we don’t help them now they will be trapped in a plastic hell of diminishing nutrients.

That’s when we come to the rescue and clip the bottom of the agar tubes so that the growing roots can escape to freedom. This part of the process of growing Arabidopsis hydroponically is between the two earlier posts ‘Seed spotting‘ + ‘Green shoots emerging‘ and this later one: ‘Looking for roots‘. We use a tube cutter from VWR.

 

Once all the tubes are clipped, a puddle of hydroponic ‘root juice’ is added to the tray. This is double the strength of the hydroponic media used to make the agar in the tubes. We do this to help tease the roots down and out of the tube…it’s their reward for their escape to freedom.

Got a good crop this time…hopefully that’ll mean we can do lots of interesting experiments.

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Looking for roots

 

Lets have a look at how our little Arabidopsis plants are getting on. These were the plants that were growing in the yellow ‘nursery’ boxes. The young seedlings are about two weeks old, now growing hydroponically in blackened boxes to keep the roots dark.

Here, were having a wee look to see if the roots have emerged from the bottom of the cut Eppendorf tubes. Yes ! there are roots coming out into the hydroponic media (a minimal medium without any sucrose).

The boxes are in environmentally controlled growth cabinets (Sniggers cabinets) where we can control light intensity and temperature and humidity. I have the plants growing in 12 hours of light and 12 hours of dark (12h LD) at 20oC. These are quite standard “lab” conditions. I suppose 12h LD would be equivalent to the equinoxes in Nature…..would we ever get 20oC in Scotland in March or September ? (I very much doubt!)

 

 

 

1001 Genomes

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A resource that we are increasingly using in our research is what’s known as the ‘1001 Genomes‘.

This is where the genome sequences for over 1000 different Arabidopsis plants are available for anyone to play with.

Just like you and me, we are all slightly different. Different hair colour, different height, different shoe size. The same is true for plants. The sequences for the 1001 genomes  helps us work out how Arabidopsis has evolved, and might therefore help us understand how, for example, climate change will affect plants (and ultimately our food crops). For our research, we’re interested in how plants perceive and respond to temperature. How do plants survive and adapt to very different temperature environments?  We can use the 1001 Genomes resource to help us address this question.

The flagship paper that describes this invaluable resource is a paper published by the 1001 Genomes Consortium in the journal Cell in 2016.

The paper shows that Arabidopsis ‘relict’ plants – something akin to the plant’s Founding Fathers – were prominent in the Iberian (Spain and Portugal) Peninsula – and seemed to hang about on the periphery of the last ice age (around 12,000 years ago), whereupon there was an expansion, or ‘sweep’ into more Northerly latitudes. What changes to the genome helped Arabidopsis survive in different habitats in their sweep North?

As pointed out in their Conclusion the authors state that “temperature and precipitation vary greatly across the species’ range and between groups and one would expect differences in physiological and developmental responses of Spanish and Swedish accessions”.

Why is all this important? Well, its been demonstrated that rice grain yield declines by 10% for each 1°C increase in growing-season minimum (i.e. night-time) temperature. One approach might be to therefore grow crops at higher latitudes, but by doing this our crops will need to adapt to different day-lengths. A higher latitude results in greater seasonality i.e. larger differences in day-length and temperature at higher latitudes compared to regions nearer the equator.

Why don’t we work directly with rice, wheat, barley, potato etc? Why do we work with Arabidopsis, which is a weed after all. Well the genome size of our staple crop plants are much bigger and more complex and so obtaining 1001 potato genomes would be a truly mammoth (and expensive) task. We also have a wealth of genetic resources available for Arabidopsis,  And for me – never known for my horticultural skills – Arabidopsis is easy to grow (its a weed after all), and it lives and dies quickly (around 5 weeks) meaning that there can be a quick turn around of experiments.

There are some good web-resources that allows us to play around with the 1001+ sequences from all of these different natural variants. One that I find particularly useful is the SALK 1001 genomes browser, where you can plot all the single base pair changes across a gene region for as many of the 1001 genomes you can fit on your web-browser – see an example below.

Makes you wonder, looking at all of these small changes in DNA sequence – what do they mean for the plant, and how best do we test what these changes make?

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12h L:D

tex toc coldLL gating cartoon

Today is the vernal equinox, and traditionally marks the beginning of Spring. It’s also when  daytime and night-time are of approximately equal duration.

This has resonance with our (artificial) experimental set-up of day:night (dark:light) for our plants growing in environmentally controlled cabinets. When we present our circadian data we would typically denote ‘the day’ on our slides or papers as white and black bars denoting day and night, respectively.

I suppose today is the day we should be doing our experiments in Nature instead of the growth cabinets…

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Seed spotting

After overfilling the tubes with the agar/growth media a cross is sliced into the agar with a sterile blade (see clip above).  Doing this his will hopefully help the roots find a way down through the agar/growth media.

Then the Arabidopsis seeds (sterilised in bleach and suspended in sterile water) are pipetted onto the surface of the agar one-by-one (ok, maybe sometimes two-by two) – see clip below.

 

The trays are then sealed with micropore tape and that’s it – now to wait to see if the seeds germinate – should take around a week to see the emergence of the very young shoots.

 

Eat, Sleep, Pipette, repeat

 

Setting up the next experiment. It involves pipetting agar growth media into (literally) 100’s of tubes – see movie clip above. I try to overfill the tubes with the molten agar mixture (this is important for a later step….where I place single Arabidopsis seeds on the agar surface…watch this space).

Science is 99% perspiration, and 1% inspiration….and this is the ‘perspiration’ bit.

 

 

“now acceptable for publication” :-)

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Ahhh….the best 4 words in scientific research? It’s been a long, arduous trip but finally we shall be adding a few dents to our current knowledge of alternative splicing/splicing factors/temperature and the clock….will keep you posted.

Made me think about the time it takes to publish scientific research, and I came across this commentary article in Nature from 2016.

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I think many of us working at the coal-face of research will recognise a lot of what it says, e.g.

Many….feel trapped in a cycle of submission, rejection, review, re-review and re-re-review that seems to eat up months of their lives, interfere with job, grant and tenure applications and slow down the dissemination of results.”

Also talks about “resetting the clock” – not to do with circadian clocks, but related to the time stamp of submission and resubmission(s).

Is it taking longer to publish? One contributor to the article says that the average time for their group of papers took 9 months…[9 months is good, no?]

Anyway, for the time being lets focus on…”now acceptable for publication” 🙂