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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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.

 

Improved Gateway

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Trying to re-learn the language of GATEWAY cloning. ‘BP reactions’, ‘LR cloning’, ‘attB sites’, ‘entry clones’, ‘destination vectors’, ‘binary vectors’…I could go on. Thinking of trying a series of ‘Improved Gateway Binary Vectors (ImpGWBs)‘ to make plants express our genes of interest (GOIs) fused to markers. These are described in this paper:

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As for the choice of marker, there is a dizzying choice, so as well as trying to get to grips with GATEWAY, there is the question of what best to use as marker. I’ve had disappointing results fusing our GOIs to green fluorescent protein (GFP) in the past, so maybe time to try something else. As you can see, there is no shortage of choice:

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How long is a piece of stri…promoter?

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Trying to work out how long to make the upstream promoter region for some plasmid constructs. I’ve been using the SnapGene tool to visually stitch together DNA sequences.

How long is the promoter for a particular plant gene? 500, 1000, 2000 bp? or is it defined by particular features in the promoter? I guess for some genes the upstream region barges into other gene loci.

I tried out this web-tool, plantpromoterdb.

Seems quite useful. It displays some features of the promoter that might be considered when deciding the promoter chunk length e.g. it will show the predicted transcriptional start site, and other cis– features.

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Seems, though, that most studies take a nominal 1000 to 2000bp upstream of the translational start codon (ATG). Think it’s good to know some of the promoter ‘landscape’ though. Here’s the reference for plantpromoterdb:

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