Plant science, by JoVE!

JoVE 2

Researchers from the University of Warwick published a methodology paper with a twist this week. The paper, published online by the Journal of Visualized Experiments (JoVE), gives step-by-step instructions and video demonstration of a method for purifying a protein and identifying proteins that perform its post-translational modifications.

Authors Sophie Piquerez, Alexi Balmuth, Jan Skenář, Alex JonesJohn Rathjen and Vardis Ntoukakis developed the method in order to characterize the interactions between nucleotide-binding leucine-rich-repeat proteins and the Prf/Pto complex in effector-triggered immunity. In principle the method could be applied to any protein – the protein of interest is epitope-tagged, immunoprecipitated and analysed by MS.

A video journal lends itself to new or improved methodology rather than high impact conclusions. As with a lot of JoVE articles, the scientifically significant results obtained using the protocol have already been published; in this case in Ntoukakis et al. 2013 (PLOS Pathogens, 10.1371/journal.ppat.1003123).

The authors conclude the abstract by saying the paper demonstrates:

  1. Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry;
  2. It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate;
  3. The immunoprecipitation step is essential to get enough protein to do the MS. (more…)

A tale of two models

James Lloyd (University of Leeds) was the lead author on last month’s Plant Journal paper on nonsense-mediated mRNA decay, which demonstrated that working on a model plant can sometimes be a hinderance rather than a help. In this guest post he explains how he and his supervisor Brendan Davies overturned widely held assumptions about NMD in plants – by working on moss.

Physcomitrella patens, the model moss, growing on agar.

Plant biology has been greatly advanced by the use of Arabidopsis thaliana as a model. Fantastic community resources, such as mutant collections and numerous genome sequences from natural accessions have aided researchers in all areas of plant sciences. However, A. thaliana is far from perfect. Nonsense-mediated mRNA decay (NMD) is a little heard of pathway to regulate transcript stability and it has recently been shown to be involved in pathogen response in A. thaliana (Rayson et al., 2012).

The mechanism of mRNA decay has largely been worked out in animals and revolves around the phosphorylation of an RNA helicase called UPF1 by a kinase SMG1. However, fungi and A. thaliana both lack the SMG1 kinase, so it has been a mystery how the fungal and plant proteins are phosphorylated. We have recently shown that SMG1 is not animal specific but is an ancient component of the NMD machinery, appearing in the genomes of all plants examined, with the exception of A. thalianaSMG1 is even found in the genome of A. lyrata, a close relative of A. thaliana.

Arabidopsis thaliana, the model plant.

The lack of SMG1 in the genome of A. thaliana meant that we had to use another plant as our model to understand the role of SMG1 in the plant kingdom. Enter moss! Physcomitrella patens is the model moss, with a sequenced genome and a high rate of homologous recombination it is a relatively simple task to identify and delete any gene in the genome. The rate of homologous recombination is much lower in flowering plants than it is in moss, meaning that short (around 1 Kb) of moss genomic DNA from upstream and downstream of a gene of interest can be cloned around an antibiotic selection gene and then transformed into moss cells and a large proportion of antibiotic resistant plants have the gene of interest deleted.

We deleted SMG1 from moss and found that NMD was compromised thus placing plant SMG1 in the NMD pathway (Lloyd and Davies, 2013). Therefore, many plants including crops like rice and maize are likely to rely on SMG1 to control gene expression through NMD. Future research will hopefully reveal why A. thaliana has lost SMG1 and if another kinase has replaced SMG1 in this plant.

A. thaliana has been useful in characterising other components of the NMD pathway in plants, such as UPF1 and understanding the biological role in plants (such as controlling the pathogen response). However, it was limited in helping us understand the NMD pathway of commercially important crops and it was moss to the rescue!

Animal researchers have long used multiple, evolutionarily diverse models, including but not limited to fruit flies, C. elegans, zebrafish, frogs and mice. Despite big differences between invertebrates like fruit flies and vertebrates like humans, a great deal about human biology has been learnt by using these organisms in the lab.

Many important questions cannot be answered by simply studying A. thaliana, big differences in fundamental processes exist between accessions. Therefore, multiple models are needed in plant sciences. Moss is just one plant that can help compliment research in other plant species, using the powerful genetic tool of homologous recombination.

References:

Lloyd JPB and Davies B (2013) SMG1 is an ancient nonsense-mediated mRNA decay effector. The Plant Journal 1365-313X http://dx.doi.org/10.1111/tpj.12329

Rayson S, Arciga-Reyes L, Wootton L, De Torres Zabala M, Truman W, et al. (2012) A Role for Nonsense-Mediated mRNA Decay in Plants: Pathogen Responses Are Induced in Arabidopsis thaliana NMD Mutants. PLoS ONE 7(2): e31917. doi:10.1371/journal.pone.0031917

Images courtesy of James Lloyd. 

How do jasmonates control plant development?

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Published on: March 5, 2013

Jasmonates mediate responses to plant biotic- and abiotic-stress and influence plant development as well as secondary metabolism (for a recent review, see Avanci et al. 2010, GMR 9:484; Acosta and Farmer 2010, Arabidopsis Book PMC3244945. Today’s highlighted paper, at the moment in advanced view from Plant Physiology, sheds new light on the elusive mechanism behind the jasmonate inhibitory effect on plant organ development and growth, which has traditionally taken a back seat to its involvement in the stress response.

The authors used a range of traditional and systems-based methods to uncover the mechanism of growth inhibition by jasmonates (JA). First of all, they looked at the cell size and cell number in the first true leaves of Arabidopsis mutants altered in JA synthesis and perception, aos and coi1 respectively, in the presence and absence of methyl jasmonate (MeJA) treatment. Both cell size and cell number were reduced after treatment with MeJA in aos1 and the Col gl1 wildtype. This effect was minimal in coi-1, demonstrating the importance of COI1 in JA-mediated cell growth inhibition. Importantly, using flow cytometry they also showed that MeJA delays the switch from the mitotic cell cycle to the endoreduplication cycle, again in a COI1-dependent manner, as well as inhibiting mitotic cycle itself.

MeJA treated Arabidopsis seedlings. From left to right: col gl1, aos1, coi1

To work out the mechanism for JA’s effect on cell size, number, and ploidy, Noir, Bömer et al. performed novel global transcriptional profiling to identify the molecular players whose expression is regulated during leaf development by jasmonate via COI1. Senior author on the paper Alessandra Devoto from Royal Holloway, University of London explained, (more…)

Valentine’s Volatiles

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Published on: February 14, 2013

Plenty of flowers are beautiful and expensive, but the lovely rose scent makes roses the perfect traditional gift to your Valentine. And since it is Valentine’s Day today, and this is a plant science blog, here’s a brief review of the science of floral scents and a recently published paper on the topic (roses not included).

Despite rose breeders managing to come up with flowers with stronger, subtle, or new scents from new rose varieties, the science of floral scents is not well understood. Floral scent can be under stronger natural phenotypic selection than flowers (Parachnowitsch et al. 2012; New Phyt. 195:667), but the agents of selection may be any number of organisms including pollinators and herbivores and the main influencing factor on scent evolution is not known (Theis and Adler 2012; Ecology 93:430). 

The molecular mechanism and regulation of biosynthesis of the volatile, low-molecular weight compounds that cause floral scent is also fairly uncharacterised. They are mainly products of the terpenoid, fatty acid, and phenylpropanoid pathways. Recently a group from the Hebrew University of Jerusalem characterised a regulation mechanism of the phenylpropanoid volatile biosynthesis pathway (Spitzer-Rimon et al. 2012; Plant Cell 24:5089).

All phenylpropanoids share the same precursor, Phe, which is biosynthesized via the shikimate pathway. A transcriptional regulator ODORANT1 (ODO1) regulates shikimate pathway enzymes and affects metabolic flow toward phenylpropanoid production. Another transcriptional regulator, EMISSION OF BENZENOIDS II (EOBII) directly regulates ODO1’s expression, indirectly affecting the shikimate pathway and the biosynthesis of phenylpropanoid volatiles. (Van Moerkercke et al. 2001, Plant J 67:917; Verdonk et al. 2005, Plant Cell 17:1612).

Working on petunia, Spitzer-Rimon et al. identified EOBI, another regulator of floral scent. (more…)

Investigating photosynthesis

In today’s highlighted article, the authors use traditional and far more modern biochemistry to uncover why photosynthesis is inhibited by Streptomyces spp., and characterise a previously unknown step in cyclic electron flow. This is also a good opportunity to point out these great photosynthesis outreach and education resources from Science and Plants for Schools. Admittedly, they don’t have anything on AA-sensitive CEF, because it’s unlikely anyone without a plant biochemistry PhD needs to know about that! But they have brilliant basic photosynthesis teaching resources, including these amazing algae-jelly-balls.

Photosynthesis background: Broadly speaking, photosynthesis is the process by which light energy from the sun is absorbed by Photosystems I and II (PSI and PSII), where it is channelled into electron transport chains and stored in ATP and NADPH. One electron carrier is ferredoxin (Fd).

There are two types of electron flow, cyclic and linear (CEF and LEF), which generate ATP. Though they are different processes, both CEF and LEF require PSI and PSII, two other thylakoid proteins, PGR5 and PGRL1, and electron carriers Fd, plastoquinone (PQ) and plastocyanin.

In CEF there are two processes by which electrons are transferred from Fd to PQ. One is a characterised NADH dehydrogenase-like compex dependent pathway, and all that is known about the other is that it is sensitive to antimycin A (AA), a product of Streptomyces spp. which inhibits CEF.

For background on AA-inhibition and the divergent electron transfer pathways in CEF, see Joët et al. (2001; Plant Phys. 24:1919). For more information on PGR5 and PGRL1, see DalCorso et al. (2008; Cell 132:273).

Gaps in knowledge of CEF:

  • What makes that electron transfer process from Fd to PQ sensitive to AA?
  • How do the electrons from photoreduced Fd get transferred to PQ and back into the electron transport chain?
  • What do functions do PGR5 and PGRL1 perform?

New from Hertle et al.: Titration and Western blotting experiments showed that PGR5 and PGRL1 dimerize to each other. Six cysteine residues were conserved in all PGRL1 proteins. Hertle et al. made mutant PGRL1 proteins in which one or more cysteine residues were substituted for serine, and tested the varients for their capacity to bind PGR5, iron, and to promote AA-sensitive CEF. They worked out that all six cysteines were essential for AA-sensitive CEF, while specific cysteines were involved in binding iron and PGR5.

In vitro assays demonstrated that PGRL1 is capable of transferring electrons between Fd and PQ analogue DMBQ in the presence of PGR5, and this reaction was inhibited in the presence of AA. Hertle et al. show clearly that PGRL1 is physically a fit for a ferredoxin-plastoquinone reductase and make a strong case for it being the mediator between Fd and PQ in CEF, and the AA-sensitive step in the cyclic electron flow.

Highlighted article: Alexander P. Hertle, Thomas Blunder, Tobias Wunder, Paolo Pesaresi, Mathias Pribil, Ute Armbruster, Dario Leister (2013) PGRL1 Is the Elusive Ferredoxin-Plastoquinone Reductase in Photosynthetic Cyclic Electron Flow. Molecular Cell – 03 January 2013, 10.1016/j.molcel.2012.11.030

Progress in pollen research

Time lapse video of Arabisopsis pollen grains germinating and growing pollen tubes. Credit: .

As an officer for GARNet, the Arabidopsis research network, I am happy to share the news that we can now add pollen germination to the long list of things for which our Arabidopsis can be called a model plant. The research is published in New Phytologist, and is currently in early view.

The importance of studying pollen for plant reproduction research is obvious, but it is also an excellent and widely used system for studying cell growth and development. Some plants, such as tobacco, have pollen that can be germinated on cue, and monitored in all sorts of ways as through germination, cell development, and pollen tube growth. Unfortunately brassicas, including Arabidopsis thaliana, do not have such amenable pollen.

A team of researchers from Oxford have developed a method that yields fast, reliable germination of A.thaliana pollen. The pollen tubes that grow are long and morphologically normal.

The method uses a cellulose-based membrane covering an agarose pad, all set up on a glass microscope slide. In the authors’ view, this protocol was more successful than other attempts because the environment surrounding the pollen mimics the stigma – so not only does this paper present a method of studying Arabidopsis pollen, but it provides novel information about the environmental cues required for pollen germination. The method was optimized for temperature and pH as well as the ratios of reagents used to make the materials.

Although this paper was about Arabidopsis and marks an important development for Arabidopsis researchers working on pollen and cell growth, it is also significant for Brassica researchers. The Brassica family contains many commercially important crops, and this method can surely be adapted to serve research into cabbage, oilseed rape, or other Brassica species.

Highlighted article: M. J. Rodriguez-Enriquez, S. Mehdi, H. G. Dickinson and R. T. Grant-Downton (2012) A novel method for efficient in vitro germination and tube growth of Arabidopsis thaliana pollen. New Phytologist (Early View) doi: 10.1111/nph.12037

The genetics of broad-spectrum resistance

Downy mildew infection of Arabidopsis thaliana seedlings

Highlighted article: Dmitry Lapin, Rhonda C. Meyer, Hideki Takahashi, Ulrike Bechtold, Guido Van den Ackerveken (2012) Broad-spectrum resistance of Arabidopsis C24 to downy mildew is mediated by different combinations of isolate-specific loci. New Phytologist DOI: 10.1111/j.1469-8137.2012.04344.x

It is a mark of how effective plant immune systems are that most bacteria, fungi, and viruses do not affect plants at all either because plant tissues are not suitable for them to live in, or they are fended off. Of course there are pathogens that are compatible with plants – and within species that share compatibility, there are pockets of resistance. Some sub-groups are resistant to specific pathogen isolates, and this is caused by dominant resistant genes. A much broader, more complicated, and less common form of resistance occurs when a particular accession is resistant to a whole pathogen species, or several species. This is broad-spectrum resistance, and it can be caused by a simple dominant gene or multiple genes. Natural broad-spectrum resistance is not simple to transfer from its origin to a commercial crop because it can come from a complex set of genes which are not necessarily all dominant. (more…)

Thinking about phytoplankton

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Published on: October 16, 2012
H. akashiwo on the right, with predator Favella sp.

I’m aware phytoplankton are not usual subjects for plant science research, but these small algae are quite plant-like, in general – although they don’t have organs to complicate things. Like plants, they photosynthesize and are able to respond to their environment. Importantly, unlike plants, phytoplankton are mobile, hence the name which in Greek means ‘drifting plants’. Being extremely tiny ‘plants’, phytoplankton present an excellent opportunity for plant scientists to consider synthetic biology, which seems more feasible on a cell-scale rather than an entire plant. The super-theme of the FP7 2013 funding call was ‘The Oceans of Tomorrow,’ and while that call closes in a few short months, synthetic biology, water security and bio-sensors are important research themes which are here to stay.

Highlighted article: Elizabeth L. Harvey and Susanne Menden-Deuer (2012) Predator-Induced Fleeing Behaviors in Phytoplankton: A New Mechanism for Harmful Algal Bloom Formation? PLoS ONE 7(9): e46438. doi:10.1371/journal.pone.0046438

This research focuses on toxic phytoplankton Heterosigma akashiwo, a known cause of harmful algal blooms (HABs; for a fairly recent review of HABs and their effects on human health, see Backer and McGillicuddy Jr., 2006). (more…)

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