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Dec 27
0

Genome editing: an introduction to CRISPR/Cas9

By | Blog, Future Directions
Damiano Martignago

Dr Damiano Martignago, Rothamsted Research

This week’s blog post was written by Dr Damiano Martignago, a genome editing specialist at Rothamsted Research.

 

Genome editing technologies comprise a diverse set of molecular tools that allow the targeted modification of a DNA sequence within a genome. Unlike “traditional” breeding, genome editing does not rely on random DNA recombination; instead it allows the precise targeting of specific DNA sequences of interest. Genome editing approaches induce a double strand break (DSB) of the DNA molecule at specific sites, activating the cell’s DNA repair system. This process could be either error-prone, thus used by scientists to deactivate “undesired” genes, or error-free, enabling target DNA sequences to be “re-written” or the insertion of DNA fragments in a specific genomic position.

The most promising among the genome editing technologies, CRISPR/Cas9, was chosen as Science’s 2015 Breakthrough of the Year. Cas9 is an enzyme able to target a specific position of a genome thanks to a small RNA molecule called guide RNA (gRNA). gRNAs are easy to design and can be delivered to cells along with the gene encoding Cas9, or as a pre-assembled Cas9-gRNA protein-RNA complex. Once inside the cell, Cas9 cuts the target DNA sequence homologous to the gRNAs, producing DSBs.

CRISPR/Cas9

The guide RNA (sgRNA) directs Cas9 to a specific region of the genome, where it induces a double-strand break in the DNA. On the left, the break is repaired by non-homologous-end joining, which can result in insertion/deletion (indel) mutations. On the right, the homologous-directed recombination pathway creates precise changes using a supplied template DNA. Credit: Ran et al. (2013). Nature Protocols.

 

Genome editing in crops

Together with the increased data availability on crop genomes, genome editing techniques such as CRISPR are allowing scientists to carry out ambitious research on crop plants directly, building on the knowledge obtained during decades of investigation in model plants.

The concept of CRISPR was first tested in crops by generating cultivars that are resistant to herbicides, as this is an easy trait to screen for and identify. One of the first genome-edited crops, a herbicide-resistant oilseed rape produced by Cibus, has already been grown and harvested in the USA in 2015.

Wheat powdery mildew

Researchers used CRISPR to engineer a wheat variety resistant to powdery mildew (shown here), a major disease of this crop. Image credit: NY State IPM Program. Used under license: CC BY 2.0.

 

Using CRISPR, scientists from the Chinese Academy of Sciences produced a wheat variety resistant to powdery mildew, one of the major diseases in wheat. Similarly, another Chinese research group exploited CRISPR to produce a rice line with enhanced rice blast resistance that will help to reduce the amount of fungicides used in rice farming. CRISPR/Cas9 has also been already applied to maize, tomato, potato, orange, lettuce, soybean and other legumes.

Genome editing could also revolutionize the management of viral plant disease. The CRISPR/Cas9 system was originally discovered in bacteria, where it provided them with molecular immunity against viruses, but it can also be moved into plants. Scientists can transform plants to produce the Cas9 and gRNAs that target viral DNA, reducing virus accumulation; alternatively, they can suppress those plant genes that are hijacked by the virus to mediate its own diffusion in the plants. Since most plants are defenseless against viruses and there are no chemical controls available for plant viruses, the main method to stop the spread of these diseases is still the destruction of the infected plant. For the first time in history, scientists have an effective weapon to fight back against plant viruses.

Cassava brown streak disease

The cassava brown streak disease virus can destroy cassava crops, threatening the food security of the 300 million people who rely on this crop in Africa. Image credit: Katie Tomlinson (for more on this topic, read her blog here).

 

Genome editing will be particularly useful in the genetic improvement of many crops that are propagated mainly by vegetative reproduction, and so very difficult to improve by traditional breeding methods involving crossing (e.g. cassava, banana, grape, potato). For example, using TALENs, scientists from Cellectis edited a potato line to minimize the accumulation of reducing sugars that may be converted into acrylamide (a possible carcinogen) during cooking.

 

Concerns about off-targets

One of the hypothesized risks of using CRISPR/Cas9 is the potential targeting of undesired DNA regions, called off-targets. It is possible to limit the potential for off-targets by designing very specific gRNAs, and all of the work published so far either did not detect any off-targets or, if detected, they occurred at a very low frequency. The number of off-target mutations produced by CRISPR/Cas9 is therefore minimal, especially if compared with the widely accepted random mutagenesis of crops used in plant breeding since the 1950s.

 

GM or not-GM

Genome editing is interesting from a regulatory point of view too. After obtaining the desired heritable mutation using CRISPR/Cas9, it is possible to remove the CRISPR/Cas9 integrated vectors from the genome using simple genetic segregation, leaving no trace of the genome modification other than the mutation itself. This means that some countries (including the USA, Canada, and Argentina) consider the products of genome editing on a case-by-case basis, ruling that a crop is non-GM when it contains gene combinations that could have been obtained through crossing or random mutation. Many other countries are yet to issue an official statement on CRISPR, however.

Recently, scientists showed that is possible to edit the genome of plants without adding any foreign DNA and without the need for bacteria- or virus-mediated plant transformation. Instead, a pre-assembled Cas9-gRNA ribonucleoprotein (RNP) is delivered to plant cells in vitro, which can edit the desired region of the genome before being rapidly degraded by the plant endogenous proteases and nucleases. This non-GM approach can also reduce the potential of off-target editing, because of the minimal time that the RNP is present inside the cell before being degraded. RNP-based genome editing has been already applied to tobacco plants, rice, and lettuce, as well as very recently to maize.

In conclusion, genome editing techniques, and CRISPR/Cas9 in particular, offers scientists and plant breeders a flexible and relatively easy approach to accelerate breeding practices in a wide variety of crop species, providing another tool that we can use to improve food security in the future.
For more on CRISPR, check out this recent TED Talk from Ellen Jorgensen:

About the author

Dr Damiano Martignago is a plant molecular biologist who graduated from Padua University, Italy, with a degree in Food Biotechnology in 2009. He obtained his PhD in Biology at Roma Tre University in 2014. His experience with CRISPR/Cas9 began in the lab of Prof. Fabio Fornara (University of Milan), where he used CRISPR/Cas9 to target photoperiod genes of interest in rice and generate mutants that were not previously available. He recently moved to Rothamsted Research, UK, where he works as Genome Editing Specialist, transferring CRISPR/Cas9 technology to hexaploid bread wheat with the aim of improving the efficiency of genome editing in this crop. He is actively involved with AIRIcerca (International Association of Italian Scientists), disseminating and promoting scientific news.

Dec 19
0

Aquaporins capable of functioning as all-in-one osmotic systems

By | Blog, GPC Community
Caitlin Byrt

Dr Caitlin Byrt, University of Adelaide

This week’s post was written by Dr Caitlin Byrt, University of Adelaide, whose research focuses the roles of water-channeling proteins – aquaporins – and ion transport in plants.

 

Aquaporins are water-channel proteins that move water molecules through cell membranes. They are found in every kingdom of life. Cell membranes are semi-permeable to water, but often require more rapid movements of water across membranes; cells achieve this using aquaporins.

Aquaporins play key roles in your kidneys, which typically filter each of the three liters of plasma in your body 60 times per day – that’s 180 liters of plasma each day! Around three times your body weight in water passes through your own aquaporins each day.

Water on leaf

Around 50% of global rainfall passes through plants, and half of this moves through the aquaporins. Image credit: Dennis Seiffert. Used under license: CC BY-ND 2.0.

Aquaporin function

Have you got on the scales recently? Nearly 70% of your body weight is water. Water is the major component of cells in all of your tissues and this is the same for plants. Around 50% of global precipitation passes through plants, and half of this moves through aquaporins, so aquaporins account for the largest movement of mass for any protein on earth.

Often, in cell membranes, four aquaporin proteins will come together to form a tetramer to assist with the transportation of water across the cell membrane. There are types of aquaporins that only transport water, and others that transport glycerol, neutral acids or gasses. Historically, plant science literature has reported that the molecular structure of aquaporins prevents any charged particles, such as ions, from permeating. This is different in the animal world where there are reports of aquaporins that are permeable to ions. For example, in humans one of the most highly expressed aquaporins, AQP1, can function as a dual water and ion channel.

 

Testing plant aquaporins in frog cells

Recently, we observed that one of the most highly expressed plant aquaporins is permeable to ions when expressed in heterologous systems such as Xenopus laevis (frog) oocyte (egg) cells or yeast cells. This indicates that plants may also have types of aquaporins that can function as a dual water:ion channels.

 

Xenopus oocytes

The function of plant aquaporins can be studied by expressing them in different systems such as the Xenopus laevis oocyte cells pictured here. Photo credit: Dr Caitlin Byrt.

 

If you want to know if a particular plant aquaporin can function as a water channel you can test it by expressing the aquaporin in a laboratory oocyte expression system. We use a tiny needle to inject RNA coding for plant aquaporins of interest into the oocyte, and for control oocytes we inject the same amount of water. The oocytes are kept in a saline solution and we usually study them one or two days after injecting the RNA to allow time for them to synthesize the protein.

If you place oocytes expressing an aquaporin into water alongside control oocytes, then the aquaporin-expressing oocytes will burst much quicker than the controls because water rushes in through the aquaporin and causes the cell to swell rapidly. To explore whether a protein conducts ions, we use electrodes to measure the currents generated when charged ions pass across the oocyte membrane. We can also use ion-specific electrodes to explore which ions are transported.

 

AtPIP2;1 can transport water and ions

The plant aquaporin we studied is coded in the genome of the model plant Arabidopsis; it is a plasma membrane-located protein called AtPIP2;1. The AtPIP2;1 protein is known to be highly prevalent in root epidermal cell membranes, and it also functions in the guard cells of leaves, which act like tiny valves to regulate the uptake of carbon dioxide for photosynthesis and the release of water vapor.

 

AtPIP2 Arabidopsis

The model plant Arabidopsis has an aquaporin, AtPIP2;1, that can function as a dual water:ion channel. Photo credit: Dr. Jiaen Qiu.

 

We observed that AtPIP2;1 expression induces both water and ion (salt) movement across the cell membrane of oocytes. We know that the ionic conductance can be carried in part by sodium ions and that it is inhibited by calcium, cadmium and protons. This means AtPIP2;1 is a candidate for a previously reported calcium-sensitive non-selective cation channel responsible for sodium ion entry into Arabidopsis roots in saline conditions.

We are investigating the physiological role of ion permeable aquaporins in plants, and exploring how plants regulate the coupling of ion and water flow across key membranes. The regulation of ion permeability through plant aquaporins could be important in the control of water flow and regulation of cell volume. There is increasing discussion around the hypothesis that plants could drive water transport in the absence of water potential differences using salt and water co-transport, and this makes us wonder whether ion-permeable aquaporins may be involved. Testing whether ion-permeable aquaporins can function as an ‘all-in-one’ osmotic system in plants is an exciting new direction for research in this field.

 

Caitlin Byrt and Steve Tyerman

Dr. Caitlin Byrt, Professor Steve Tyerman and colleagues are investigating whether aquaporins permeable to ions are present in a range of different plant species. Photo credit: Wendy Sullivan

 

More information:

Byrt, C.S., Zhao, M., Kourghi, M., Bose, J., Henderson, S.W., Qiu, J., Gilliham, M., Schultz, C., Schwarz, M., Ramesh, S.A., Yool, A., and Tyerman, S.D., 2016. Non‐selective cation channel activity of aquaporin AtPIP2; 1 regulated by Ca2+ and pH. Plant, Cell & Environment.

Yool, Andrea J., and Alan M. Weinstein. New roles for old holes: ion channel function in aquaporin-1. Physiology 17.2 (2002): 68-72.

Nov 05
1

Using plants to convert explosives to fertilizers: an interview with Neil Bruce

By | Blog, Interviews
Neil Bruce

Professor Neil Bruce

This week we spoke to Professor Neil Bruce, whose research at the University of York (UK) focuses on metabolic pathways. His insights into the detoxification of pollutants by plants and microorganisms has led to promising new solutions to help clean up polluting explosives from military testing.

 

Could you begin by telling us a little about your research interests?

I have very broad research interests that often revolve around finding enzymes for biotechnological applications. A particular focus of my lab is the biochemistry and molecular genetics of plant and microbial metabolism of xenobiotic (foreign) compounds, such as environmental pollutants. Elucidating these metabolic pathways often results in the discovery of new enzymes that catalyze interesting chemistries. Being a biologist at heart, I’m interested in the evolutionary origin of these enzymes, but also by studying their structure and function I’m exploring how these enzymes can be engineered to further improve their properties for a particular application, such as environmental remediation or biocatalysis.

 

 

You spoke at the GARNet 2016 meeting about engineering plants to remediate explosives pollution. Could you explain what this problem is and how it affects both people and the environment?

Explosive compounds used in munitions are highly toxic and the potential for progressive accumulation of such compounds in soil, plants, and groundwater is a significant concern at military sites. It is estimated that in the US alone, 10 million hectares of military land is contaminated with components of munitions. The explosives mainly used in artillery, mortars and bombs are 2,4,6-trinitrotoluene (TNT) and Composition B (containing TNT and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)). The US Department of Defense estimated that the clean-up of unexploded ordnance, discarded military munitions and munition constituents on its active ranges would cost between $16 billion and $165 billion. Explosives pollution is, however, a global problem, with large amounts of land and groundwater contaminated by TNT and RDX, including polluted sites in the UK that date back to the First and Second World Wars. Explosives pollution will continue to be a pressing issue while there is a requirement for military to train and the existence of armed conflict requires munitions to be manufactured. There is an urgent need to develop sustainable in situ technologies to contain and treat these pollutants.

 

TNT toxicity in plants

TNT is toxic to plants because of the actions of an enzyme called monodehydroascorbate reductase, which breaks TNT down into a toxic form. Plants lacking this enzyme, such as the mdhar6 mutant plants on the right, can grow very well on TNT-polluted soil. Credit: Johnston et al. (2015).

 

How did you develop the idea of using plants to remove explosives pollution? What benefits do plants have over the microorganisms from which the enzymes are obtained?

We have worked closely with the UK Ministry of Defence and US Army to understand the fate of explosives in the environment. Knowledge of their effects on biological systems is important, as this information can be used to support the management of contaminated sites. We have, therefore, been uncovering the molecular mechanisms behind these detoxification processes in plants, and have used this knowledge, in combination with studies on the bacterial degradation of pollutants, to successfully engineer transgenic plants able to remediate toxic explosive pollutants in a process called ‘phytoremediation’.

An innovative aspect of our work has been the use of genetic engineering to combine the biodegradative capabilities of explosives-degrading bacteria with the high biomass, stability and detoxification systems inherent in plants. While it is possible to find explosives-degrading bacteria on polluted land, they do not degrade the explosives fast enough to prevent leaching into the groundwater. Our engineered transgenic plant systems, however, can efficiently remove toxic levels of TNT and RDX from contaminated soil and water.

 

You mentioned that you are currently testing transgenic switchgrass to remove RDX and TNT pollution in the US. Why did you choose this species and have you considered developing other species suited to different environments?

Plants appropriate for the phytoremediation of explosives need to be adaptable to conditions on military ranges, for example, they need good fire tolerance, and to be able to grow over a wide geographical range. Switchgrass meets these criteria, and is also deep-rooting, can be grown on marginal lands, and researchers can benefit from established methods for genetically engineering switchgrass. We have also been engineering other grass species and have considered fast-growing deep-rooting trees such as poplar.

 

Turning explosives into fertilizers

In a poetic twist, rather than turning fertilizers into explosives, Professor Bruce’s phytoremediating plants convert explosives into fertilizer. Credit: Neil Bruce.

 

How quickly can engineered plants remove this pollution?

In the lab these plants can remove levels of explosives pollution found in the environment within a matter of days. We are currently carrying out field trials with our transgenic plants on a military site in the US, to observe their phytoremediation effectiveness in the real world. If these trials are successful, a number of demonstration studies on contaminated sites will be required to convince end users of the benefits of phytoremediation for remediating and maintaining military land. These demonstration studies will also allow us to evaluate any risks, which will be important to obtain further approval from the US Department of Agriculture to be able to use these plants on a larger scale.

 

What other projects are you working on? Could you elaborate on any recent discoveries?

As well as explosives, we are also working on the use of plants to extract platinum group metals (PGMs) from mining waste. PGMs are used in an ever-expanding array of technologies and demand is spiralling upwards; however, these are rare and expensive to mine. It is essential that these metal reserves are utilized and recycled responsibly, not dispersed and lost into the environment. Plants can take up metals from their environment and, in the case of PGMs, can deposit them as nanoparticles within their tissues. Importantly, we have recently shown that plants containing palladium nanoparticles can also be used to make efficient biocatalysts, and we are currently using synthetic biology in plants to improve palladium uptake and nanoparticle formation.

 

More information:

Johnston, E.J., Rylott, E.L., Beynon, E., Lorenz, A, Chechik, V. and Bruce, N.C. (2015) Monodehydroascorbate reductase mediates TNT toxicity in plants. Science. 349: 1072-1075.

Gunning, V., Tzafestas, K., Sparrow, H., et al. (2014) Arabidopsis glutathione transferases U24 and U25 exhibit a range of detoxification activities with the environmental pollutant and explosive, 2,4,6-trinitrotoluene. Plant Physiol. 165: 854-865.

Rylott, E.L.. Budarina, M.V., Barker, A., Lorenz, A., Strand, S.E. and Bruce, N.C. (2011) Engineering plants for the phytoremediation of RDX in the presence of the co-contaminating explosive TNT. New Phytologist, 192: 405-413.

Sep 30
2

1000 Plants

By | Blog, Interviews

The 1000 plants initiative (1KP) is a multidisciplinary consortium aiming to generate large-scale gene sequencing data for over 1000 species of plants. Included in these species are those of interest to agriculture and medicines, as well as green algae, extremophytes and non-flowering plants. The project is funded by several supporters, and has already generated many published papers.

Gane Wong is a Professor in the Faculty of Science at the University of Alberta in Canada. Having previously worked on the Human Genome Project, he now leads the 1KP initiative. Dennis Stevenson, Vice President for Botanical Research, New York Botanical Garden, and Adjunct Professor, Cornell University (USA), studies the evolution and classification of the Cycadales. He became involved in the 1KP initiative as an opportunity to sample the breadth of green plant diversity.

We spoke to both Professor Stevenson (DS) and Professor Wong (GW) about the initiative. Professor Douglas Soltis from Florida Museum of Natural History also contributed to this blog post with input in editing the answers.

What do you think has been the biggest benefit of 1KP?

DS: This has been an unparalleled opportunity to reveal and understand the genes that have led to the plant diversity we see around us. We were able to study plants that were pivotal in terms of plant evolution but which have not previously been included in sequencing projects as they are not considered important economically

The 1KP project presented a fantastic opportunity to explore plant biodiversity. Photo by Bob Leckridge. Used under Creative Commons 2.0.

The 1KP project presented a fantastic opportunity to explore plant biodiversity. Photo by Bob Leckridge. Used under Creative Commons 2.0.

GW: The project was funded by the Government of Alberta and the investment firm Musea Ventures to raise the profile of the University of Alberta. Notably there was no requirement by the funders to sequence any particular species. I was able to ask the plant science community what the best possible use of these resources would be. The community was in full agreement that the money should be used to sample plant diversity.

Hopefully our work will change the thinking at the funding agencies regarding the value of sequencing biodiversity.

What techniques were utilized in this project to carry out the research?

GW: Complete genomes were too expensive to sequence. Many plants have unusually large genomes and de novo assembly of a polyploid genome remains difficult. To overcome this problem, we sequenced transcriptomes. However, this made our sample collection more difficult as the tissue had to be fresh. In addition, when we started the project, the software to assemble de novo transcriptomes did not work particularly well. I simply made a bet that these problems would be solved by the time we collected the samples and extracted the RNA. For the most part that’s what happened, although we did end up developing our own assembly software as well!

The 1KP initiative is an international consortium. How has the group evolved over time and what benefits have you seen from having this diverse set of skills?

GW: 1KP would not be where it is today without the participation of scientists around the world from many different backgrounds. For example, plant systematists who defined species of interest and provided the tissue samples worked alongside bioinformaticians who analyzed the data, and gene family experts who are now publishing fascinating stories about particular genes.

 DS: One of the great things about this project is how it has evolved over time as new researchers became involved. There is no restriction on who can take part, which species can be studied or which questions can be asked of the data. This makes the 1KP initiative unique compared to more traditionally funded projects.

GW: We continually encouraged others to get involved and mine our data for interesting information. We did a lot of this through word of mouth and ended up with some highly interesting, unexpected discoveries. For example, an optogenetics group at MIT and Harvard used our data to develop new tools for mammalian neurosciences. This really highlights the importance of not restricting the species we study to those of known economic importance.

According to ISI outputs from this research, two of the most highly cited papers from 1KP are here and here.

You aimed to investigate a highly diverse array of plants. How many plants of the major phylogenetic groups have now been sequenced, and are you still working on expanding the data set?

DS: A lot of thought went into the species selection. We aimed for proportional representation (by number of species) of the major plant groups. We also aimed to represent the morphological diversity of those groups.

GW: Altogether, we generated 1345 transcriptomes from 1174 plant species.

Has this project lead to any breakthroughs in our understanding of the phylogeny of plants?

DS: This will be the first broad look at what the nuclear genome has to tell us, and the first meaningful comparison of large nuclear and plastid data sets. However, due to rapid evolution plus extinction, many parts of the plant evolutionary tree remain extremely difficult to solve.

Hornworts are non-vascular plants that grow in damp, humid places. Photo by Jason Hollinger. Used under Creative Commons License 2.0.

Hornworts are non-vascular plants that grow in damp, humid places. Photo by Jason Hollinger. Used under Creative Commons License 2.0.

One significant breakthrough was the discovery of horizontal gene transfer from a hornwort to a group of ferns. This was unexpected and very interesting in terms of the ability of those ferns to be able to accommodate understory habitats.

GW: With regard to horizontal gene transfer, there are papers in the pipeline that will illustrate the discovery of even more of these events in other species. We have also studied gene duplications at the whole genome and gene family level. This is the most comprehensive survey ever undertaken, and people will be surprised at the scale of the discoveries. However, we will be releasing our findings shortly as part of a series and it would be unwise for us to give the story away here! Keep a look out for these!

Sep 18
0

Cassava brown streak: lessons from the field

By | Blog, GPC Community

This week’s post was written by Katie Tomlinson, a PhD student at the University of Bristol, UK, who spent three months as an intern at the National Crops Resource Research Institute in Uganda. She fills us in on the important research underway at the Institute, and how they communicate their important results to local farmers and benefit rural communities.  

Over the summer, I had a great time at the National Crops Resources Research Institute (NaCRRI) in Uganda. I’m currently in the second year of my PhD at the University of Bristol, UK, where I’m researching how the cassava brown streak disease (CBSD) viruses are able to cause symptoms, replicate and move inside plants. I was lucky enough to be given a placement at NaCRRI as part of the South West Doctoral Training Partnership Professional Internship for PhD Students (PIPS) scheme, to experience the problem for myself, see the disease in the field, meet the farmers affected and investigate the possible solutions.

 

Cassava brown streak disease

Cassava brown streak disease symptoms on tubers. Image credit: Katie Tomlinson.

 

Cassava is a staple food crop for approximately 300 million people in Africa. It is resilient to seasonal drought, can be grown on poor soils and harvested when needed. However, cassava production is seriously threatened by CBSD, which causes yellow patches (chlorosis) to form on leaves and areas of tubers to die (necrosis), rot and become inedible.

Despite being identified in coastal Tanzania 80 years ago, CBSD has only been a serious problem for Uganda in the last 10 years, where it was the most important crop disease in 2014–2015. The disease has since spread across East Africa and threatens the food security of millions of people.

NaCRRI is a government institute, which carries out research to protect and improve the production of key crops, including cassava. The focus is on involving farmers in this process so that the best possible crop varieties and practices are available to them. Communication between researchers and farmers is therefore vital, and it was this that I wanted to assist with.

 

Scoring cassava brown streak disease

Scoring cassava plants for Cassava brown streak symptoms. Image credit: Katie Tomlinson.

 

When I arrived I was welcomed warmly into the root crop team by the team leader Dr Titus Alicai, who came up with a whole series of activities to give me a real insight into CBSD. I was invited to field sites across Uganda, where I got to see CBSD symptoms in the flesh! I helped to collect data for the 5CP project, which is screening different cassava varieties from five East and Southern African countries for CBSD and cassava mosaic disease (CMD) resistance. I helped to score plants for symptoms and was fascinated by the variability of disease severity in different varieties. The main insight I gained is that the situation is both complex and dynamic, with some plants appearing to be disease-free while others were heavily infected. There are also different viral strains found across different areas, and viral populations are also continually adapting. The symptoms also depend on environmental conditions, which are unpredictable.

I also got to see super-abundant whiteflies, which transmit viruses, and understand how their populations are affected by environmental conditions. These vectors are also complex; they are expanding into new areas and responding to changing environmental conditions.

It has been fascinating to learn how NaCRRI is tackling the CBSD problem through screening different varieties in the 5CP project, breeding new varieties in the NEXTGEN cassava project, providing clean planting material and developing GM cassava.

 

Tagging cassava plants

Tagging cassava plants free from Cassava brown streak disease for breeding. Image credit: Katie Tomlinson.

 

And there’s the human element…

In each of these projects, communication with local farmers is crucial. I’ve had the opportunity to meet farmers directly affected, some of whom have all but given up on growing cassava.

 

Challenging communications

Communicating has not been easy, as there are over 40 local languages. I had to adapt and learn from those around me. For example, in the UK we have a habit of emailing everything, whereas in Uganda I had to talk to people to hear about what was going on. This is all part of the experience and something I’ll definitely be brining back to the UK! I’ve had some funny moments too… during harvesting the Ugandans couldn’t believe how weak I was; I couldn’t even cut one cassava open!

 

Real world reflections

I’m going to treasure my experiences at NaCRRI. The insights into CBSD are already helping me to plan experiments, with more real-world applications. I can now see how all the different elements (plant–virus–vector–environment–human) interact, which is something you can’t learn from reading papers alone!

Working with the NaCRRI team has given me the desire and confidence to collaborate with an international team. I’ve formed some very strong connections and hope to have discussions about CBSD with them throughout my PhD and beyond. It’s really helped to strengthen collaborations between our lab work in Bristol and researchers working in the field on the disease frontline. This will help our research to be relevant to the current situation and what is happening in the field.

 

Some of the NaCRRI team

Saying goodbye to new friends: Dr. Titus Alicai (NaCRRI root crops team leader), Phillip Abidrabo (CBSD MSc student) and Dr. Esuma Williams (cassava breeder). Image credit: Katie Tomlinson.

 

Sep 02
0

Interview with Dr. Winfried Peters: Bringing forgotten ideas on plant biomechanics into the 21st century

By | Blog, Interviews

This week we spoke to Dr. Winfried S. Peters from Indiana University/Purdue University Fort Wayne (IPFW). His research mainly focuses on the biomechanics of plant cells, which led him to take a second look at some of the ideas of botanists in the 19th and early 20th century and use modern techniques to make exciting new discoveries.

Winfried Peters

Dr Winfried S. Peters, Indiana University/Purdue University Fort Wayne (IPFW), next to several tons of land-plant sieve elements!

 

Could you begin by describing your research interests?
I am interested in the biophysical aspects of the physiology of plants and animals. In plants, my research focuses on the mechanics of growth and morphogenesis, and on the cell biology of long-distance transport in the phloem. For both topics, a solid background in the history of the field can be quite helpful – I love studying the old literature to reconstruct the ideas botanists had a century or two ago regarding the functioning of plants.

At the recent New Phytologist Symposium, entitled “Colonization of the terrestrial environment 2016”, you presented fascinating work on the sieve tubes of kelp, which resemble the phloem tubes of vascular plants. What is the purpose of these tubes?
In large photosynthetic organisms, not all parts of the body are truly autototrophic. Some tissues produce more material by photosynthesis than they need, while others produce less than they require or none at all– think of green leaves and growing root tips. Over-producing tissues can act as sources and export photoassimilates to needy sink tissues. Sieve tubes are arrays of tubular cells that mediate this exchange, enabling the rapid movement of photosynthate-rich cytoplasm between sources and sinks.

What techniques did you utilize to investigate the function of these tubes, and what did this reveal?
During my recent sabbatical, I became involved in this project in the lab of my friend and long-term collaborator, Professor Michael Knoblauch. Michael heads the Franceschi Microscopy and Imaging Center at Washington State University, where we studied sieve tubes of the Bull Kelp (Nereocystis luetkeana) using a variety of state-of-the-art microscopy techniques. Most importantly, we employed fluorescent dyes to visualize transport in sieve tube networks. To do this, one needs to work with intact kelp, which is demanding given a thallus size of 12 meters and more. So we moved to Bamfield Marine Sciences Centre on Vancouver Island, where Bull Kelp is a ‘common weed’.

A particularly important result was the pressure-induced reversal of the flow direction in sieve tubes and across sieve plates. This was in line with Ernst Münch’s (1876-1946) theory, who suggested that sieve tube transport was driven by osmotically generated pressure gradients.

 

Nereocystis wounding

An intact Nereocystis luetkeana is kept in a tank (right) while sieve tube transport is studied using a fluorescence microscope. Photo credit: Michael Knoblauch.

How do the biomechanics of the kelp sieve tubes differ from the phloem tubes of higher plants?
Regarding cytoplasmic translocation, there doesn’t seem to be a difference – in higher plants as in kelps, the contents of the sieve tubes move in bulk flow – but wounding responses differ drastically. After wounding, we found that kelps have a massive swelling of the walls, which reduced the sieve tube diameter by more than 70%. By injecting silicon oil into severed kelp sieve tubes we demonstrated that wall swelling was fully reversible, and that the swelling state of the walls depended on intracellular pressure.

Wounding response in kelp

Sieve wall tubes swell after wounding due to changes in intracellular pressure. (Images taken from video below).

Have reversible wall-swelling reactions been observed in other species, and what are the implications of this finding?
We have observed the wall-swelling response in all kelp species examined. Ironically, there is no shortage of drawings and photographs of kelp sieve tubes with swollen walls in the literature over the last 130 years; however, the dynamics of cell behavior remained hidden in plain sight because fixed tissue samples rather than fully functional, whole organisms were studied. Consequently, sieve tubes with swollen walls were misinterpreted as senescent cells. There also are publications on turgor-dependent cell wall swelling in red and green algae, but these ceased around 1930.

Afterwards, wall swelling was completely forgotten, judging from the textbooks. This is remarkable, as Wilhelm Hofmeister (1824-1877), often celebrated as a founding father of plant biomechanics, denied a significant role for osmotic processes in the generation of turgor, the hydrostatic pressure within plant cells. Rather, he maintained that living cells were pressurized by the swelling of their walls. The example of the kelp sieve tube shows how easy it is to remain unaware of wall swelling when it happens right before our eyes. Maybe we should take Hofmeister’s idea seriously once again?

What are the evolutionary implications of your work?
Brown algae and vascular (land) plants are only remotely related, and their sieve tube networks certainly evolved independently of each other. It seems surprising that such sophisticated structures, which serve a complex function that integrates the physiology of the entire organism, have evolved at least twice, but think again. Real cells are not embedded in a totally homogeneous environment, and neither is the cytoplasm within the cell a homogeneous solution. Thus every cell experiences gradients of solute concentrations along its inner and/or outer surface. As a consequence, differential water fluxes across the plasma membrane will occur, resulting in movements of the cell contents. In other words, Münch flow, the cytoplasmic bulk flow driven by osmotically generated pressure gradients, is not a peculiar process operating specifically in sieve tubes, but a ubiquitous phenomenon. Sieve tubes consist of cells that simply do the things cells do, just a little more efficiently as usual. In this view, the repeated convergent evolution of sieve tube networks is not really unexpected.

But kelps resemble land plants in other ways too. As in land plants, kelp cell walls are made of cellulose (at least partly), kelp cells are connected through plasmodesmata, and the kelp life-cycle is a sporophyte-dominated alternation of generations. Evidently, none of these features represents a specific adaptation to life on dry land.


Wound responses including wall swelling in a sieve tube of Nereocystis luetkeana. (Watch for the rapid cell wall swelling between 11 and 14 seconds in!) This video was taken by Professor Michael Knoblauch in collaboration with Dr Winfried S. Peters.
 

If you’d like to know more about this fascinating work, it was been published in the following articles:

Knoblauch, J., Peters, W.S. and Knoblauch, M., 2016. The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates. Annals of Botany, 117(4), pp.599-606.

Knoblauch, J., Drobnitch, S.T., Peters, W.S. and Knoblauch, M., 2016. In situ microscopy reveals reversible cell wall swelling in kelp sieve tubes: one mechanism for turgor generation and flow control? Plant, Cell and Environment, 39(8), pp.1727-1736.

 

Aug 11
0

Uncovering the secrets of ancient barley

By | Blog, Interviews

This week we speak to Dr Nils Stein, Group Leader of the Genomics of Genetic Resources group at the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). We discuss his recent work on the genomes of 6000-year-old cultivated barley grains, published in Nature Genetics, which made the headlines around the world.

Nils Stein

Dr Nils Stein, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)

Could you describe your work with the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)?

The major research focuses of my group, the Genomics of Genetic Resources, are to continue sequencing the genomes of barley and wheat, perform comparative genomics on the Triticeae tribe, isolate genes of agronomic interest, and investigate the genomics of wild barley relatives.

We are currently leading the work to generate the barley reference genome, and we are also partners in several wheat genome sequencing projects. We are genotyping-by-sequencing (GBS) all 20 000 barley accessions in the IPK Genebank, as well as 10 000 pepper accessions as part of a Horizon 2020 project (G2P-SOL) investigating the Solanaceae crop species.
Your recent collaborative paper on the genomic analysis of 6,000-year-old barley grains made headlines around the world. What did this study involve?

This was an interdisciplinary study to sequence the DNA of 6000-year-old barley grains. The grains were excavated by a team of Israeli archaeologists and archaeobotanists led by Prof. Ehud Weiss, Bar-Ilan University, the DNA was extracted and sequenced by ancient DNA specialists Prof. Johannes Krause and Dr. Verena Schünemann in Germany, and the data were analyzed by Dr. Martin Mascher in the context of our comprehensive barley genome diversity information. This allowed the resulting sequence information to be put into a population genetic and ecogeographic context.

Ancient barley

Preserved remains of rope, seeds, reeds and pellets (left), and a desiccated barley grain (right) found at Yoram Cave in the Judean Desert. Credit: Uri Davidovich and Ehud Weiss.

What led you to the realization that barley domestication occurred very early in our agricultural history?

The genome of the analyzed ancient samples was highly conserved with extant barley landraces of the Levant region, which look very similar to today’s high-yielding barley varieties. Although suggestive and tendentious, this told us that the barley crop 6000 years ago looked very similar to extant material. The physical appearance and the archaeobotanical characters of the analyzed seeds also very much resembled modern barley.

 

These barley grains contain the oldest plant genomes reconstructed to date. Did you find any differences between the samples that might give us an insight into the traits that were first selected in the early domestication of the crop?

We have only scratched the surface so far. The major domestication genes controlling dehiscence, brittleness or row-type of the main inflorescence had the same alleles in the ancient samples that are found in extant barley, confirming that these traits were selected for early in domestication. Additional analyses on other genes controlling different traits in barley are still ongoing – bear in mind that many of the genes controlling major traits in barley are still unknown, which complicates the selection of targets for analysis.

Modern barley

Modern barley cultivar. Credit: Christian Scheja. Used under license: CC BY 2.0.

 Do these grains have any genetic variation that we lack at key loci in modern barley lines, for example in stress or disease resistance?

This is matter of ongoing analysis. So far it is obvious that the most genetically similar extant landraces from the Levant region have accumulated natural mutations over the last 6000 years, resulting in additional variation that we don’t find in the ancient sample.

 

What can we expect from the barley genome projects in the future?

The International Barley Genome Sequencing Consortium is preparing a manuscript on the reference sequence of barley. This will allow further analysis of the ancient DNA data with a more complete, genome-wide view, including the consideration of a more complete gene set than has been available so far. Our Israeli collaborators (Professor Ehud Weiss and Professor Tzion Fahima) have more ancient samples of similar quality. We hope we will be able to generate a more comprehensive view of the ancient population genomics of barley in the future, to better address the question of novel ancient alleles and lost genetic diversity.

The Barley Pan-Genome analysis will soon give us a better understanding of the structural variation in the barley genome. Putting the ancient DNA information into this more comprehensive genomic context will be very exciting. We also hope to be able to compare a variety of ancient samples of different ages to more precisely date the event of barley domestication.

You can read the paper here: Genomic analysis of 6000-year-old cultivated grain illuminates the domestication history of barley ($).

Jun 26
0

Let’s get Plantae!

By | Blog, GPC Community, Plantae

So you’re hearing good things about the new plant science networking platform Plantae and want to get involved? You’ve come to the right blog post! Read on to learn how to set up your profile, find friends and get involved with the community.

Who are you?

Plantae profile

Filling in your profile is easy!

Plantae is a great place to network with researchers around the world, so you’ll want your profile to be as detailed as possible.

As a minimum, add your name, a profile photo, your professional affiliations and a summary of who you are and what you do. This will help your colleagues and friends to find you, and break the networking ice with new connections!

What makes a good bio? Give the reader a little information about your fields of interest, background, plant science outreach, new papers, favorite plant, whatever you like (related to plants and plant science, of course!). Remember that Plantae is a professional networking site, so don’t put anything on there that you wouldn’t want your boss (current or future!) to see!

Where can I find out more about this interesting person?

Plantae social media

Don’t forget to add your social media and researcher profiles

A great feature of the Core Profile is the ability to add your social media profiles, website, and enhance the visibility of your research by adding researcher profiles, for example your ORCID, Mendeley, or ResearchGate account. To ensure that the accounts connect properly, add the full URL of each profile, not just your account name.

 

Will you be my friend?

From the Community homepage you can choose to see the recent activity of your friends, but only if you’ve added them first!

Add a friend on Plantae

How to add a friend on Plantae

To find colleagues, click on ‘Members’ and you can search for a name, or filter all members by city, state or country. Click on your friend’s name to go to their profile. On the left sidebar, you’ll see a button named ‘User Actions’, which when clicked brings up the option to add them as a friend. After they accept your request, you’re officially friends. Congratulations!

Branching out

Plantae groups

Join a group to continue networking

Now you’ve added everyone you know, it’s time to connect with people that you don’t! Get over to the Discussion boards and let everyone know how you feel about the latest hot paper or public engagement scheme. Or you could join a Group of users who share your interests, location, or love of plant-themed poetry (disclaimer: the latter is currently not a Plantae group – feel free to start it!). It’s easy to join conversations or start one of your own.

Finding funding, jobs and resources

Plantae is a hub of plant science resources, including research news, funding opportunities, job advertisements, science policy news and a wealth of education and public engagement tools. Log in regularly to see up and coming events, grant calls, opinion pieces and more, or maybe upload some of your own!

Join us!

There you have it. Now you know the basics, reach out to the Plantae network, get involved in exciting plant science discussions, make the most of funding and job opportunities, and, pretty please, fill in your profile!

Jun 20
3

A typical day for PhD students in Japan

By | Blog, GPC Community
Akiko Nakazaki

Akiko Nakazaki, PhD student at Kyoto University

Kon-nichiwa! (Hello!) I am Akiko Nakazaki, a PhD student studying plant molecular cell biology at Kyoto University in Japan.

I’m interested in plant defense – specifically the glucosinolate-myrosinase defense system, which is specific to Brassicales such as Arabidopsis thaliana. Glucosinolates are a group of secondary metabolites stored in separate cells to myrosinases, the enzymes that break them down. Upon tissue damage, the glucosinolates and myrosinases are released from their cells and combine. The glucosinolates are hydrolyzed to volatile repellent compounds such as isothiocyanates and nitriles.

Glucosinolate myrosinase defense system

When damaged, cells containing glucosinolate and myrosinase are ruptured, releasing their contents. The glucosinolate is broken down by the myrosinase into volatile compounds that repel herbivores

I was impressed by this ingenious and rational survival strategy! I want to reveal this defense system at the cellular level, and am researching it in Arabidopsis thaliana by performing microscopic observations, bioassays with insects, and so on.

A day in the lab

Are you interested in how PhD students from other countries spend their day in the laboratory? I am! Let me tell you about my typical day in the lab.

I wake up at 8:30am, and have morning coffee and toast for breakfast while reading a newspaper. Then, I get dressed and ride on my bicycle to the University. During the ride (about 10 minutes), I remind myself of the day’s schedule. I get to the lab at 10am and take my seat. All the members of the lab have their own desk and workbench. I turn on my computer and check my emails.

In the daylight, I basically do experiments and read papers. I start doing microscopic observations and lose track of time until I hear my stomach growling and realize that it is almost 2pm. I have lunch at the eating space in lab. In this room, there are always some lab members who are eating, discussing their research, playing social games, etc. After lunch, I report the result of my microscopic observations to my boss and we have a brief discussion about it.

Microscopic_observations

Then, I return to my seat and realize the primers I ordered yesterday have arrived. I perform a PCR and prepare an agarose gel for electrophoresis. While I am waiting for the PCR to end, I search PubMed and Google Scholar for new papers to read. I load the PCR products to the gel and check that the PCR worked. In the evening, I allocate myself free time for doing more experiments, reading more papers, preparing research presentations, discussions, etc.

I’ve sought a more effective way to advance my research through trial and error. For example, when I started researching in the lab I was a little too ambitious, and planned my schedule too tightly. I sometimes felt tired and depressed when my research was not right on schedule, as is often the case. In these negative moods I couldn’t enjoy my work, so I adopted a schedule with more free time. Because of this change, I’ve come to be able to work flexibly and keep a positive frame of mind.

I’m home between 10pm and midnight. At home, I have a late dinner and take a good long soak in the bath (my favorite time of day!). I go to bed at 2am.

Free weekends!

On weekends I enjoy playing badminton, learning traditional Japanese dance and shopping. I try to make plans without lab work as much as I can, however I’m not able to do avoid it sometimes when I am struggling to get new data before academic conferences and progress reports. Leaving the lab allows me to get rid of stress and feel refreshed for a healthy next week. Furthermore, I devise ways to work more efficiently on weekdays, because I am required to take time off at the weekends.

Treasure every encounter

My boss always says, “It is important to value encounters with people and things.” It wasn’t until recently that I finally understood that message! I have found that experiments may not always work well, but when I look at it from a different angle, even experiments that haven’t gone the way I’d wanted could make me aware of something new and interesting. This awareness could also be brought about through discussions with others.

I am grateful for being able to receive this opportunity. Thank you.

Akiko Nakazaki is in the first year of her doctoral program in the Department of Botany, Graduate School of Science, Kyoto University, Japan.