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 5â€ČUTR 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 5â€ČUTR 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!)

 

 

 

Are ye’ a cold kinase? Or are ye’ no a cold kinase?

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I’ve been wrestling with a couple of ‘kinase’ papers of recent. They were both published in December 2017 in the journal Developmental Cell.

Firstly there is Zhao et al. with ‘MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability

…and the other paper is Li et al. with ‘MPK3- and MPK6-Mediated ICE1 Phosphorylation Negatively Regulates ICE1 Stability and Freezing Tolerance in Arabidopsis

Both papers examine the early response of plants to temperature and the involvement of protein kinases – principally the mitogen-activated protein kinases (MAPKs) e.g. the MAP kinase kinase kinases (MAP3Ks; also called MAPKKKs or MEKKs), MAP kinase kinases (MAP2Ks; also called MKKs or MEKs), and MAPKs…..you can already see how complicated this can get.

When plants adapt to cold there are large changes in the expression of thousands of genes, and its now well established that these changes are mediated by what are known as the CBF genes and they do this by regulating another subset of genes known as the COR genes. Its a bit like a domino effect. Once cold is triggered the dominoes start falling leading to COR gene expression and the plants physiological response to temperature.

Whereas quite a lot is now known about the later stages of the domino line at the molecular level, less is known about the ‘who’ and ‘what’ starts to triggering the domino line. Moving slightly up from the CBFs are the ICEs (ICE1 and ICE2).  ICE1 is a transcription factor that binds to CBF promoters and activates their expression. ICE is therefore seen as a very interesting ‘domino’ and how it is knocked over (..or activated) in this cascade is of great interest.  Several suspects were on the wanted list, including the MAPKs: MPK3, MPK4 and MPK6 and MEKK1 and MKK2, and these are the focus of both these papers.

I won’t go into much more detail. However, here are the highlights from both papers:

Li et al.:

1. Cold activates mitogen-activated protein kinases MPK3 and MPK6

2. MPK3/MPK6 phosphorylate and destabilises ICE1 protein, and

3. MPK3/MPK6 activation attenuates plant freezing tolerance

Zhao et al. :

1. The MKK4/5-MPK3/6 cascade negatively regulates freezing tolerance

2. The MEKK1-MKK2-MPK4 cascade positively regulates freezing tolerance

3. MPK3/6-mediated phosphorylation of ICE1 promotes ICE1 degradation

I think the other important thing is that Zhao et al. show that MPK4 positively regulates the cold response by constitutively suppressing MPK3 and MPK6 activity i.e. MPK4 blocks the ‘destruction’ of ICE1 by MPK3 and MPK6.

Anyway there is also a commentary article in the same issue in Developmental Cell on the background and features of these two papers called ‘MAP kinase Signaling Turns to ICE’

Must check it out. I think it will articulate these results much better than I can…

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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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abundance of transcript=amount of protein?

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Thought that this was an interesting paper, published recently in PNAS.

The paper shows that alternative splicing produces different transcript isoforms for the 5’UTR region of the human gene encoding α-1-antitrypsin called SERPINA1, such that splicing of 5’UTR modulates the inclusion of long upstream ORFs (uORFs). What’s new with all this I hear you say. Well, the authors go on to show that while SERPINA1 transcripts produce the same protein isoform, they do so with different translation efficiencies. Differences in uORF content and 5’UTR secondary structure combine to differentiate the translational efficiencies of SERPINA1 transcripts.

α-1-antitrypsin is of interest because deficiencies in this protein are associated with chronic obstructive pulmonary disease (COPD), liver disease, and asthma. This work points to the possibility that genetic alterations in noncoding gene regions, such as the 5’UTR region, could result in α-1-antitrypsin deficiency.

The work also reinforces the idea that the amount of protein produced from a gene is not a simple function of the abundance of the transcript.

The reference is: Proc Natl Acad Sci U S A. 2017 Nov 21;114(47):E10244-E10253. doi: 10.1073/pnas.1706539114. Epub 2017 Nov 6.

The image used is their Figure 3. SHAPE-MaP structure probing data for SERPINA1 transcripts.

We are Detectorists

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Loved the whimsical comedy of the ‘Detectorists‘ series with the excellent MacKenzie Crook (as Andy Stone) and Toby Jones (as Lance Stater). It follows Lance and Andy’s lives as their personal situations and ambitions orbit their central obsession with metal detecting.

It made me think of some analogies with Science. Obsession – yeah, tick. Do we ever stop thinking about our research topic? From hours of scanning the landscape only to uncover ‘ring-pull’ duds (yeah tick – see ‘failed experiments, no?). Or is it that we are just centimetres away from that Anglo-Saxon gold hoard.  Maybe we have selected the wrong ‘field’ in the first place. I think that, like Lance and Andy, most of us dream of landing ‘the big one’ –  maybe that discovery that will be transformative? We can but dream?

Especially loved the concept of the “discovery dance” – what kind of celebration would you do when the the game changing discovery comes your way….can you practice it? should you practice it?…or should it come as a natural reaction?

Here’s a link to a brief YouTube clip:

I think we are all Detectorists at heart. Think I’ll get practicing my dance….

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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.