Magnetising Pollen to break the Plant Transformation Bottleneck?

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Published on: December 19, 2017

The potential for crop improvement by ‘traditional’ genetic modification and by ‘game-changing’ gene-editing technologies is easy to appreciate. The introduction a foreign gene or the alteration of endogenous gene function in order to modify the way in which a plant responds to a particular environmental stimuli is the underlying goal of most applied plant scientists. Our improved knowledge of how these techniques work, advances in the speed and cost DNA synthesis alongside the adoption of the principles of synthetic biology in the engineering of molecular constructs means that generation of DNA parts for genetic modification is, in theory at least, now facile endeavor.


However there is an ‘eleplant in the room’ of every grant proposal that promises to generate an altered variety of any desired orphan crop. Our ‘eleplant’ is the efficiency, or lack thereof, in plant transformation. This issue was the topic of a 2016 Perspective piece in The Plant Cell in which the example of Sorghum was cited, an important food crop that is unfortunately recalcitrant to transformation, taking up to 12months to generate T1 transformants. This bottleneck will continue to be an issue when discussing new targets for genetic modification as callus-based mechanisms of transformation are famously extremely challenging, with one method good for the goose might be not so good for the gander.

From http://www.plantcell.org/content/28/7/1510

These challenges have been solved for many major crops but even with this knowledge, regeneration of transgenic crops only usually takes place in labs with specific knowledge and experimental pipelines (in the UK at facilities at NIAB or the JIC)*.


It is in this climate that a recent paper by Zhao et al in Nature Plants might be another true game changer. They have modified the magnetofection procedure that has been used very successfully to introduce DNA into animal cells in order to modify existing pollen transformation techniques. This protocol involves mixing DNA with magnetic nanoparticles that can be introduced using a magnetic field into pollen grains through small apertures in the pollen wall. These transformed pollen can then be used to fertilise emasculated plants as normal, from which transgenic seeds can then be selected in the usual manner.

From https://www.nature.com/articles/s41477-017-0072-y

This technique relies upon the pollen aperature being greater than 5um and Zhao et al demonstrate that this was possible in a range of flowering plants including pepper, pumpkin, zucchini and lily. The majority of their experimental work highlighted the introduction of a gene expressing BT toxin into cotton and the subsequent identification of insect resistant plants. The viability of magnetotransformed pollen was unaffected and after the initial fertilization the transgene segregated with normal mendelian ratios.


Importantly for future uptake of this technology, the authors were able to successfully transform Elite varieties that are recalcitrant to callus-transformation, thus greatly reducing the time usually needed for crossing between easily transformable and elite lines. Success rates even for floral dip transformation are lower than 1% so the reported 2-10% in this study, over three years of experiments, strongly suggests that this technique has enormous potential for crop genetic modification.

The only minor drawback is that due to the high success rate, extra generations of selfing transgenic plants might be necessary to obtain pure breeding lines due to the integration of multiple independent insertions.

These experiments have been conducted with a single research lab so it remains to be seen whether these success rates are recapitulated in other locations that have similar but potential significant alterations in their experimental setup.


Importantly the authors do not attempt to use this technique to transform any grass species, a taxonomic group that supplies the vast majority of global animal calories. This will be important to ascertain yet might prove challenging or impossible due to the size of grass pollen grains. Only time will tell whether this is possible.

There is little doubt that this work will raise significant interest in academic and industrial labs across the globe.

Watch this space whether this will prove the breaking of the transformation bottle(neck).

*- Of course Arabidopsis is immune from such concerns as it can be transformed by floral dip, due to an unusually open gynoecium during development.

A commentary article on the Zhao et al paper is also available in Nature Plants.

Arabidopsis Research Roundup: December 18th

This festive Arabidopsis Research Roundup begins with a commentary article from a global consortium of plant scientists who propose a framework of future training for researchers who will take advantage of the experimental tools available in Arabidopsis. Secondly is study from Caroline Dean (JIC) that defines the role of the LHP1 protein in epigenetic control of gene expression. Thirdly John Doonan (Aberystwyth) is a co-author of work that defines an important component of mitotic spindle formation. Next is a study led by Zinnia Gonzalez-Carranza in Nottingham that offers further insights into the function of the HWS gene. The fifth study comes from the lab of Alexander Ruban (QMUL), further investigating the importance of NPQ in photosynthetic control. The sixth paper from the Van Ooijen lab (Edinburgh) characterises the role of sumoylation in the control of CCA1 activity. The penultimate paper from the Harberd lab in Oxford defines the importance of DNA mismatch repair on genome sequence integrity whilst the final paper characterises the next phase in the long story of Arabidopsis ALF4 function and includes Charles Melynk (SLCU) as a co-author.


Friesner J et al (2017) The Next Generation of Training for Arabidopsis Researchers: Bioinformatics and Quantitative Biology. Plant Physiol. doi: 10.1104/pp.17.01490. Open Access

The current GARNet PI Jim Murray and past GARNet coordinator Ruth Bastow are authors in this international consortium that suggests future directions for the global Arabidopsis community. This consortium is led by Joanna Friesner and concludes that it is critical that the next generation of plant scientists receive appropriate training in bioinformatics and quantitative biology so as to take advantage of the remarkable array of datasets that are now available to Arabidopsis researchers.


Berry S, Rosa S, Howard M, Bühler M, Dean C (2017) Disruption of an RNA-binding hinge region abolishes LHP1-mediated epigenetic repression Genes Dev. doi: 10.1101/gad.305227.117 Open Access

Caroline Dean (John Innes Centre) leads this study that investigates the role of the polycomb associated protein LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) in the regulation of the repressive histone mark H3K27me3. They demonstrate that the intrinsically disordered hinge region of LHP1 is responsible for RNA-binding and that disruption of this region prevents the formation of sub-nuclei foci, provides a potential link to wider epigenetic regulation.


Lee YJ, Hiwatashi Y, Hotta T, Xie T, Doonan JH, Liu B (2017) The Mitotic Function of Augmin Is Dependent on Its Microtubule-Associated Protein Subunit EDE1 in Arabidopsis thaliana. Current Biol. doi: 10.1016/j.cub.2017.11.030

Open Access

John Doonan and colleagues at Aberystwyth University are co-authors on this study regarding the role of the Microtubule-Associated Protein Subunit EDE1, which is a member of the Augmin complex, during mitosis. EDE1 specifically localised with the augmin complex during spindle formation, a role that cannot be replaced by the homologous protein AUG8. This work reveals that specificity of the augmin complex can be determined by interaction with subunits that only contribute to complex function during particular phases of the cell cycle.


Zhang X, Jayaweera D, Peters JL, Szecsi J, Bendahmane M, Roberts JA, González-Carranza ZH (2017) The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway. PLoS One. doi: 10.1371/journal.pone.0189788 Open Access

Zinnia Gonzalez-Carranza (Nottingham) is the corresponding author on this study that follows on from work published earlier in 2017 regarding the role of the HAWAIIAN SKIRT gene is plant development. In this latest work they identify mutations in the previously characterized Exportin-5 HASTY gene as suppressors of the hws mutant phenotype. Further investigation shows that HWS genetically interacts with other genes involved in miRNA pathway indicates that HWS somehow interacts with biogenesis, accumulation or function of these small RNAs.


Townsend AJ1, Ware MA1, Ruban AV (2017) Dynamic interplay between photodamage and photoprotection in photosystem II. Plant Cell Environ doi: 10.1111/pce.13107

In this paper Alexander Ruban (QMUL) is the corresponding author on work that expands his groups contribution to the understanding of the role non-photochemical quenching (NPQ) plays during photoinhibition. In this work they compare the activity of NPQ versus endogenous photosystemI repair mechanisms in the maintenance of photosynthetic activity during photoinhibitory conditions. Overall they conclude that NPQ is a more important mechanism for photoprotection under short periods of illumination.


Hansen LL, Imrie L, Le Bihan T, van den Burg HA, van Ooijen G (2017) Sumoylation of the Plant Clock Transcription Factor CCA1 Suppresses DNA Binding. J Biol Rhythms doi: 10.1177/0748730417737695 Open Access

This paper from the Van Ooijen lab accompanies one that was featured in last weeks ARR and extends their finding that sumoylation plays an important role in control of the circadian clock. In this paper they show that the CCA1 clock protein is sumoylated and that perturbing this modification alters the binding of CCA1 to a target promotor, even though it’s localization or stability were unaffected. Using an in vitro system they show that sumoylation is a direct determinant of CCA1 binding to its target promotor suggesting that this PTM fine tunes the activity of this key circadian control element.


Belfield EJ, Ding ZJ, Jamieson FJC, Visscher AM, Zheng SJ, Mithani A, Harberd NP (2017) DNA mismatch repair preferentially protects genes from mutation. Genome Res. doi: 10.1101/gr.219303.116

Past GARNet Advisory board member Nick Harberd (Oxford) leads this multi-generational study on the effect of DNA mismatch repair (MMR) on maintenance of an entire genome. They perform whole genome sequencing across five generations of Arabidopsis plants with a mutation in the MMR pathway and show that particular types of nucleotide error are more prevelant amongst the total 9000 mutations that accumulate. Interestingly they show that single nucleotide variants are more likely to accumulate in genic regions, indicating that protein coding areas of the genome are preferentially protected from damage.


Bagchi R, Melnyk CW, Christ G, Winkler M,, Kirchsteiner K, Salehin M, Mergner J, Niemeyer M, Schwechheimer C, Calderón Villalobos LIA, Estelle M (2017) The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases. EMBO J. doi: 10.15252/embj.201797159

During his time as a research fellow at the Sainsbury lab in Cambridge. Charles Melynk contributed to this research that is a throwback to the early day of Arabidopsis mutant analysis. The alf4 was first described as a possible auxin mutant in 1995 and this work brings this study full circle by characterising the ALF4 protein as a novel regulator of SCF complexes, which are known to be involved in auxin and GA signaling. ALF4 specifically functions by interacting with the SCF-core component RBX1. Future work will determine whether this effect is specific to SCFs involved in hormone signaling or whether it is a more general effect.

Arabidopsis Research Roundup: December 8th.

This weeks Research Roundup begins with two papers from the University of Edinburgh on very different topics of Arabidopsis research. Firstly Alistair McCormick and Sofirtios Tsaftaris introduce a new low-cost phenotyping platform whilst Gerben Ooijen’s group has analysed the role of SUMOylation in the control of the circadian clock. The next three papers each involve wide UK collaborations and either look at plant nutrient composition (Nottingham, Dundee, York), the role of N-end rule pathway in the control of seed storage mobilisation (Rothamsted, Nottingham, Oxford, Birmingham, Cambridge) or the development of a new tool for the study of phloem sieve elements (Leeds, Rothamsted, Cambridge, Newcastle). The penultimate paper from Daniel Zilbermann (JIC) highlights the global mechanisms of methyltransferase function in Arabidopsis and mice whilst the final paper from Alexandre Ruban (QMUL) and co-authors continues his groups work to unpick the specifics of NPQ.


Dobrescu A, Scorza LCT, Tsaftaris SA, McCormick AJ (2017) A “Do-It-Yourself” phenotyping system: measuring growth and morphology throughout the diel cycle in rosette shaped plants. Plant Methods. doi: 10.1186/s13007-017-0247-6

Open Access

University of Edinburgh colleagues Alistair McCormick and Sofirtios Tsaftaris lead this work that presents a low cost phenotyping system for the analysis of the growth rate and phenotypic characteristics of Arabidopsis thaliana rosettes. The software that they have developed allows the accurate segmentation of multiple rosettes within a single image and overall offers a straightforward solution for automated phenotyping across a range of growth environments.


Hansen LL, van den Burg HA, van Ooijen G (2017) Sumoylation Contributes to Timekeeping and Temperature Compensation of the Plant Circadian Clock. J Biol Rhythms. doi: 10.1177/0748730417737633

Gerben van Ooijen (University of Edinburgh) is the corresponding author of this work that has identified SUMOylation as a novel mechanism of regulating circadian clock genes in Arabidopsis. Plants with defects in sumoylation have altered circadian periods that exhibit incorrect temperature compensation. Overall these results indicate that sumoylation importantly buffers clock function in response to changing temperatures.


Alcock TD, Havlickova L, He Z, Bancroft I, White PJ, Broadley MR, Graham NS (2017) Identification of Candidate Genes for Calcium and Magnesium Accumulation in Brassica napus L. by Association Genetics. Front Plant Sci. doi: 10.3389/fpls.2017.01968

Open Access

Neil Graham and Martin Broadley (University of Nottingham) are the corresponding authors of this study that has taken advantage of the Brassica napus Associative Transcriptomes RIPR diversity panel developed by Ian Bancroft’s lab in York. Novel loci involved with an altered response to calcium and magnesium were identified in B.napus before mineral composition was analysed in Arabidopsis mutants defective in orthologous genes. The analysed plants exhibited alteration in mineral composition, meaning that the associated Brassica loci might be targets for future breeding strategies aimed at improving plant nutrient compositions.


Zhang H, Gannon L, Hassall KL, Deery MJ, Gibbs DJ, Holdsworth MJ, van der Hoorn RAL, Lilley KS, Theodoulou FL (2017) N-terminomics reveals control of Arabidopsis seed storage proteins and proteases by the Arg/N-end rule pathway. New Phytol. doi: 10.1111/nph.14909

Freddie Theodoulou (Rothamsted Research) is the corresponding author of this research that involved a collaboration with colleagues in Cambridge, Birmingham, Nottingham and Oxford. They have performed a proteomic analysis on etiolated seedlings to identify those proteins designated for degradation by the N-end rule pathway. They analysed prt6 mutant plants that lack the function of the E3 ligase PROTEOLYSIS6 (PRT6) and discovered that N-terminal peptides from 45 protein groups were upregulated in this mutant, corresponding to the equivalent downregulation of several known N-end rule proteases. Overall the authors show that PRT6 plays an important role in the regulation of seed storage mobilisation in young seedlings and is therefore a possible future target to manipulate the plant responses to adverse environmental conditions. Dr Kirsty Hassall, a statistician at Rothamsted, is an author on this paper and in the latest edition of the GARNish newsletter explains how she interacts with plant scientists during her work.


Torode TA, O’Neill RE, Marcus SE, Cornuault V, Pose-Albacete S, Lauder RP, Kracun SK, Gro Rydahl M, Andersen MCF, Willats WGT, Braybrook SA, Townsend BJ, Clausen MH, Knox JP (2017) Branched pectic galactan in phloem-sieve-element cell walls: implications for cell mechanics. Plant Physiol. doi: 10.1104/pp.17.01568 Open Access

Paul Knox (University of Leeds) is the corresponding author of this study that includes contributions from researchers at SLCU, Newcastle and Rothamsted. This work is based around the development of a monoclonal antibody, LM26 that is able to recognize a β-1,6-galactosyl substitution of β-1,4-galactan. LM26 has allowed the identification of this unusual branched galactan that is specific to phloem elements and the authors hope that it can be a useful tool in future studies on the biology of phloem elements


Lyons DB, Zilberman D (2017) DDM1 and Lsh remodelers allow methylation of DNA wrapped in nucleosomes. Elife. doi: 10.7554/eLife.30674 Open Access

Daniel Zilberman has recently moved to the John Innes Centre and is the lead author of this work that was conducted when he was working in US. This research is a cross-kingdom analysis showing that nucleosome-free DNA is the preferred target for methyltransferases in both Arabidopsis and mice, and that nucleosomes appear to be a barrier to the function of these enzymes. Furthermore they demonstrate that linker-specific methylation that is usually absent in Arabidopsis can be introduced by removal of histone H1. This shows that flowering plants still possess this ability despite its loss, during the evolution of H1, over a billion years ago.


Tutkus M, Chmeliov J, Rutkauskas D, Ruban AV, Valkunas L (2017) Influence of the Carotenoid Composition on the Conformational Dynamics of Photosynthetic Light-Harvesting Complexes. J Phys Chem Lett. doi: 10.1021/acs.jpclett.7b02634

Alexandre Ruban (QMUL) is a co-author on this study that investigates the role that carotenoid composition plays in the control of Non-photochemical quenching (NPQ), a mechanism that protects the photosynthetic apparatus from light-damage. Arabidopsis mutants with differing carotenoid compositions were analysed for the dynamics of the conformation switches that occur during NPQ. Interestingly they show that LHCII has robust function  that is resistant to different carotenoid concentrations.

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