A nano-sized bio-degradable clay-comprising double stranded ribonucleic acid (dsRNA) could offer a cost-effective, clean and green alternative to chemical-based plant pesticides.
Australian researchers from the University of Queensland have successfully used a gene-silencing spray, named BioClay, a combination of biomolecules and clay, to protect tobacco plants from a virus for 20 days with a single application. Their study has been published in Nature Plants.
“When BioClay is sprayed onto a plant, the virus-specific dsRNA is slowly released from the clay nanosheets into the plant. This activates a pathway in the plant that is a natural defence mechanism. The dsRNA is chopped up into small bits of RNA by enzymes of this pathway. These small bits attack the virus when it infects the plant without altering the plant genome,” explains lead researcher, Neena Mitter.
“Even with current pesticides, we lose up to 40 per cent of our crop productivity because of pests and pathogens. We are hoping that having BioClay in the mix as an environmentally friendly, sustainable crop protection measure will reduce crop losses,” Mitter adds.
“The clay-based delivery technology could represent a positive inflection point in the progress towards commercialisation of topical RNAi. This is a non-GM, environmentally benign and very specific technology.”
John Killmer, APSE
While chemical-based pesticides kill the targeted insect, they can also affect a range of other insects that are beneficial. Mitter says, “BioClay is specific and it only kills the pathogen being targeted. Currently farmers use insecticides to kill the vector that comes with the viruses, but with BioClay we can target the virus itself.”
BioClay field trials may begin in Australia by year-end. “The first test will be on a virus that infects vegetable crops — capsicum, tomato, chilli,” Mitter tells SciDev.Net.
Farmers can use the existing equipment to deliver BioClay and the researchers are hopeful that it will be a commercially viable product for farmers everywhere. The clay component is cheap to make, but not the RNA.
Several companies like APSE, a US based startup, are working on the mass production of RNAs. APSE is developing RNA manufacturing technology for RNA interference (RNAi) or gene silencing applications.
“Our technology for RNA production should be ready in 2-3 years. We are targeting US$2 per gram,” APSE’s John Killmer tells SciDev.Net.
Killmer says, “The clay-based delivery technology could represent a positive inflection point in the progress towards commercialisation of topical RNAi. This is a non-GM, environmentally benign and very specific technology.”
RNAi technology is being used by many in the agriculture industry including the biotech firm Monsanto. The company’s BioDirect technology is focused on applications of RNAi directly onto the leaves of a plant.
Monsanto’s spokesperson John Combest tells SciDev.Net, “As insects develop resistance to certain classes of pesticides, giving farmers another option to control these pests is critical. The idea is not to replace any given system of farming, whether modern GM systems or others — it’s to provide farmers with products that can complement or replace agricultural chemical products.”
Sales of quinoa (Chenopodium quinoa) have exploded in the last decade, with prices more than tripling between 2008 and 2014. The popularity of this pseudocereal comes from its highly nutritious seeds, which resemble grains and contain a good balance of protein, vitamins, and minerals. The nourishing nature of quinoa meant it was prized by the Incas, who called it the “Mother grain”.
Quinoa is a popular ‘grain’, but it is more closely related to spinach and beetroot than cereals like wheat or barley. Image credit: Flickr user. Used under license: CC BY 2.0.
Quinoa is native to the Andes of South America, where it thrives in a range of conditions from coastal regions to alpine regions of up to 4000 m above sea level. Its resilience and nutritious seeds means that quinoa has been identified as a key crop for enhancing food security, but there are currently very few breeding programs targeting this species.
The challenge of improving the efficiency and sustainability of quinoa production has so far been restricted by the lack of a reference genome. This week, a team of researchers led by Professor Mark Tester (King Abdullah University of Science & Technology; KAUST) addressed this issue, publishing a high-quality genome sequence for quinoa in Nature. They compared the genome with that of related species to characterize the evolution and domestication of the crop, and investigated the genetic diversity of economically important traits.
The evolution of quinoa
Tester and colleagues used an array of genomics techniques to assemble 1.39 Gb of the estimated 1.45-1.50 Gb full length of quinoa’s genome. Quinoa is a tetraploid, meaning it has four copies of each chromosome. The researchers shed light on the evolutionary history of this crop by sequencing descendants of the two diploid species (each containing two sets of chromosomes) that hybridized to generate quinoa; kañiwa (Chenopodium pallidicaule) and Swedish goosefoot (Chenopodium suecicum). Comparing these sequences to quinoa and other relatives, the team showed that the hybridization event likely occurred between 3.3 and 6.3 million years ago. A comparison with other closely related Chenopodium species also suggested that, contrary to previous predictions, quinoa may have been domesticated twice, both in highland and coastal environments.
Quinoa field. Image credit: LID. Used under license: CC BY-SA 2.0.
Washing away quinoa’s bitter taste
Quinoa seeds are coated with soap-like chemicals called saponins, which have a bitter taste that deters herbivores. Saponins can disrupt the cell membranes of red blood cells, so they have to be removed before human consumption, but this process is costly, so quinoa breeders are always looking for varieties that produce lower levels of saponins.
Sweet (low-saponin) quinoa strains do occur naturally, but the genes that regulate this phenotype were previously unknown. Tester and colleagues investigated sweet and bitter quinoa strains and discovered that a single gene (TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR-LIKE 1 [TSARL1]) controls the amount of saponins produced in the seeds. The low-saponin quinoa strains contained mutations in TSARL1 that prevented it from functioning properly. This is a key target for the improvement of quinoa in the future, although farmers will have to find new ways to protect their crops from birds and other seed predators!
The high-quality reference genome for quinoa generated by Tester and colleagues is likely to be vital for allowing many exciting improvements in the future. Breeders hoping to improve the yield, ease of harvest, stress tolerance, and saponin content of quinoa can develop genetic markers to speed up breeding for these key traits, improving the productivity of quinoa varieties and enhancing future food security.
Read the paper in Nature: Jarvis et al., 2017. The genome of Chenopodium quinoa. Nature. DOI: 10.1038/nature21370
Thank you to Professor Mark Tester (KAUST) for providing information used in this post!
I am a post-doctoral research scientist within Rothamsted Research’s BBSRC-funded 20:20 Wheat® program, which aims to provide the knowledge base and tools to increase the UK wheat yield potential from 8.4 to 20 tons of wheat per hectare within the next 20 years. Field phenotyping is one component of this program and facilitates the non-destructive monitoring of field-grown crops. Traditional methods of field phenotyping require huge human effort, which consequently limits the accuracy, frequency, and number of different measurements that can be taken at one time. Fortunately, Rothamsted has an exciting solution to this problem.
The Field Scanalyzer
Dr Kasra Sabermanesh shows off the Field Scanalyzer. Image credit: Rothamsted Research
Our field phenotyping platform, the Field Scanalyzer (constructed by LemnaTec GmbH and being further developed by ourselves), supports a motorized measuring platform with multiple sensors that can be accurately positioned anywhere within a dedicated field. The sensor array comprises a high-definition RGB camera, two hyperspectral cameras, a thermal infrared camera, a system for imaging chlorophyll fluorescence and twin scanning lasers for 3D information capture. Together, these sensors generate a wealth of data about crop growth, architecture, performance, and health. The Field Scanalyzer operates autonomously and in high-throughput, meaning it can take a lot of non-destructive measurements without human supervision, throughout the crops lifecycle, with high-accuracy and reproducibility. (You can read more about the Field Scanalyzer in our recent paper: http://www.publish.csiro.au/FP/pdf/FP16163).
We are currently using the Field Scanalyzer to identify new characteristics of crops that relate to performance, as well as identifying new genetic diversity for existing traits. Outputs from either of these research components can be delivered to breeders. We are screening approximately 400 wheat varieties, but also imaging some oilseed rape and oat plants.
The scanalyzer. Image credit: Rothamsted Research
The big data problem
The Field Scanalyzer at Rothamsted is a world first, so we initially had to develop all the necessary image acquisition protocols and image processing tools, in order to exploit its full capabilities. A number of image processing tools are available; however, they are not suitable for field-grown crops, as they were not developed for complex canopies consisting of hundreds of plants in highly dynamic ambient conditions. The platform can generate up to 100 TB data with a year’s continuous operation (using all of the sensors). That’s why I work with two other post-docs to develop robust computer vision tools to automate the way we extracting quantitative image datasets. We are also validating the accuracy of the values extracted from our images by comparing them with measurements obtained manually.
Approximately 1.5 years have passed since we first began operating the Field Scanalyzer, and we have now optimized all of our image acquisition protocols and have collected a full seasonal dataset. With the good quality images stored in our database, we have developed some robust tools to automatically extract the information about some key growth stages (ear emergence and flowering), as well as quantifying height and the number of some plant organs. We are still just scraping the surface though, and have a list of traits for which we want to develop computer vision tools, in order to automatically analyze them.
Take to the skies: Drones for data collection
Some of my colleagues work with drones (UAVs) to capture information about crop height, plant density (Normalized Difference Vegetation Index), and canopy temperature from large-scale field trials containing 5000 plots. They also fly the UAVs over our Field Scanalyzer site, so we can compare data collected from the higher flying UAV with those collected from the Field Scanalyzer at close proximity. The way we see it, UAVs can image large fields in a very short time (15 min), so if we notice something interesting using the UAV at the large plot-scale, we can put the material under the Field Scanalyzer for high-resolution phenotyping. On the other hand, with the Field Scanalyzer, once we gain a better understanding of which trait/s we need to focus on, when we should be looking at them, and exactly which sensor/s are required to quantify the trait, we can deploy drones with the necessary sensors (once the sensors are portable enough) to collect this information at field-scale and at the appropriate time.
Taking to the skies: Drones are used for large-scale phenotyping at Rothamsted. Credit: Rothamsted Research.
The future of phenotyping
I envision that the future of phenotyping technology will focus on reducing the cost and size of cameras/sensors, ultimately increasing their portability and accessibility. This will result in more sophisticated cameras being attached to UAVs (as many of sensors we currently use far out-weigh a UAV’s payload). Parallel to this, research efforts are focusing on developing image processing systems that efficiently extract quantitative information about the crops from acquired images. Together, phenotyping systems such as low-flying UAVs that generate easily interpreted data outputs could be developed, which may be more widely adopted by breeders and farmers to get a deeper insight into their crop’s health and performance.
Another fantastic year of discovery is over – read on for our 2016 plant science top picks!
January
A Zostera marina meadow in the Archipelago Sea, southwest Finland. Image credit: Christoffer Boström (Olsen et al., 2016. Nature).
The year began with the publication of the fascinating eelgrass (Zostera marina) genome by an international team of researchers. This marine monocot descended from land-dwelling ancestors, but went through a dramatic adaptation to life in the ocean, in what the lead author Professor Jeanine Olsen described as, “arguably the most extreme adaptation a terrestrial… species can undergo”.
One of the most interesting revelations was that eelgrass cannot make stomatal pores because it has completely lost the genes responsible for regulating their development. It also ditched genes involved in perceiving UV light, which does not penetrate well through its deep water habitat.
Plants are known to form new organs throughout their lifecycle, but it was not previously clear how they organized their cell development to form the right shapes. In February, researchers in Germany used an exciting new type of high-resolution fluorescence microscope to observe every individual cell in a developing lateral root, following the complex arrangement of their cell division over time.
Using this new four-dimensional cell lineage map of lateral root development in combination with computer modelling, the team revealed that, while the contribution of each cell is not pre-determined, the cells self-organize to regulate the overall development of the root in a predictable manner.
Watch the mesmerizing cell division in lateral root development in the video below, which accompanied the paper:
In March, a Spanish team of researchers revealed how the anti-wilting molecular machinery involved in preserving cell turgor assembles in response to drought. They found that a family of small proteins, the CARs, act in clusters to guide proteins to the cell membrane, in what author Dr. Pedro Luis Rodriguez described as “a kind of landing strip, acting as molecular antennas that call out to other proteins as and when necessary to orchestrate the required cellular response”.
This plant root is infected with arbuscular mycorrhizal fungi. Image credit: University of Zurich.
In April, we received an amazing insight into the ‘decision-making ability’ of plants when a Swiss team discovered that plants can punish mutualist fungi that try to cheat them. In a clever experiment, the researchers provided a plant with two mutualistic partners; a ‘generous’ fungus that provides the plant with a lot of phosphates in return for carbohydrates, and a ‘meaner’ fungus that attempts to reduce the amount of phosphate it ‘pays’. They revealed that the plants can starve the meaner fungus, providing fewer carbohydrates until it pays its phosphate bill.
Author Professor Andres Wiemskenexplains: “The plant exploits the competitive situation of the two fungi in a targeted manner, triggering what is essentially a market-based process determined by cost and performance”.
The transition of ancient plants from water onto land was one of the most important events in our planet’s evolution, but required a massive change in plant biology. Suddenly plants risked drying out, so had to develop new ways to survive drought.
In May, an international team discovered a key gene in moss (Physcomitrella patens) that allows it to tolerate dehydration. This gene, ANR, was an ancient adaptation of an algal gene that allowed the early plants to respond to the drought-signaling hormone ABA. Its evolution is still a mystery, though, as author Dr. Sean Stevensonexplains: “What’s interesting is that aquatic algae can’t respond to ABA: the next challenge is to discover how this hormone signaling process arose.”
Sometimes revisiting old ideas can pay off, as a US team revealed in June. In 1930, Ernst Münch hypothesized that transport through the phloem sieve tubes in the plant vascular tissue is driven by pressure gradients, but no-one really knew how this would account for the massive pressure required to move nutrients through something as large as a tree.
Professor Michael Knoblauch and colleagues spent decades devising new methods to investigate pressures and flow within phloem without disrupting the system. He eventually developed a suite of techniques, including a picogauge with the help of his son, Jan, to measure tiny pressure differences in the plants. They found that plants can alter the shape of their phloem vessels to change the pressure within them, allowing them to transport sugars over varying distances, which provided strong support for Münch flow.
BLOG: We featured similar work (including an amazing video of the wound response in sieve tubes) by Knoblauch’s collaborator, Dr. Winfried Peters, on the blog – read it here!
July
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.
In July, an international and highly multidisciplinary team published the genome of 6,000-year-old barley grains excavated from a cave in Israel, the oldest plant genome reconstructed to date. The grains were visually and genetically very similar to modern barley, showing that this crop was domesticated very early on in our agricultural history. With more analysis ongoing, author Dr. Verena Schünemannpredicts that “DNA-analysis of archaeological remains of prehistoric plants will provide us with novel insights into the origin, domestication and spread of crop plants”.
BLOG: We interviewed Dr. Nils Stein about this fascinating work on the blog – click here to read more!
August
Another exciting cereal paper was published in August, when an Australian team revealed that C4 photosynthesis occurs in wheat seeds. Like many important crops, wheat leaves perform C3 photosynthesis, which is a less efficient process, so many researchers are attempting to engineer the complex C4 photosynthesis pathway into C3 crops.
This discovery was completely unexpected, as throughout its evolution wheat has been a C3 plant. Author Professor Robert Henrysuggested: “One theory is that as [atmospheric] carbon dioxide began to decline, [wheat’s] seeds evolved a C4 pathway to capture more sunlight to convert to energy.”
Professor Stefan Jansson cooks up “Tagliatelle with CRISPRy fried vegetables”. Image credit: Stefan Jansson.
September marked an historic event. Professor Stefan Jansson cooked up the world’s first CRISPR meal, tagliatelle with CRISPRy fried vegetables (genome-edited cabbage). Jansson has paved the way for CRISPR in Europe; while the EU is yet to make a decision about how CRISPR-edited plants will be regulated, Jansson successfully convinced the Swedish Board of Agriculture to rule that plants edited in a manner that could have been achieved by traditional breeding (i.e. the deletion or minor mutation of a gene, but not the insertion of a gene from another species) cannot be treated as a GMO.
Phytochromes help plants detect day length by sensing differences in red and far-red light, but a UK-Germany research collaboration revealed that these receptors switch roles at night to become thermometers, helping plants to respond to seasonal changes in temperature.
Dr Philip Wiggeexplains: “Just as mercury rises in a thermometer, the rate at which phytochromes revert to their inactive state during the night is a direct measure of temperature. The lower the temperature, the slower phytochromes revert to inactivity, so the molecules spend more time in their active, growth-suppressing state. This is why plants are slower to grow in winter”.
A fossil ginkgo (Ginkgo biloba) leaf with its modern counterpart. Image credit: Gigascience.
In November, a Chinese team published the genome of Ginkgo biloba¸ the oldest extant tree species. Its large (10.6 Gb) genome has previously impeded our understanding of this living fossil, but researchers will now be able to investigate its ~42,000 genes to understand its interesting characteristics, such as resistance to stress and dioecious reproduction, and how it remained almost unchanged in the 270 million years it has existed.
Author Professor Yunpeng Zhaosaid, “Such a genome fills a major phylogenetic gap of land plants, and provides key genetic resources to address evolutionary questions [such as the] phylogenetic relationships of gymnosperm lineages, [and the] evolution of genome and genes in land plants”.
The year ended with another fascinating discovery from a Danish team, who used fluorescent tags and microscopy to confirm the existence of metabolons, clusters of metabolic enzymes that have never been detected in cells before. These metabolons can assemble rapidly in response to a stimulus, working as a metabolic production line to efficiently produce the required compounds. Scientists have been looking for metabolons for 40 years, and this discovery could be crucial for improving our ability to harness the production power of plants.
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.
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.
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.
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.
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 Cellectisedited 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.
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.
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.
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.
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.
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
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 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.
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.
A new book, The Hidden Life of Trees, claims that trees talk to one another. But is this really the case? The simple answer is that plants certainly exchange information with one another and other organisms such as insects. Think of the scents of newly mowed grass or crushed sage. Some of the chemicals that make up these aromas will tell other plants to prepare for an attack or summon predatory insects to defend them. These evocative smells could be seen as cries of warning or screams for help.
When plants are damaged by infection or by being eaten, they release a range of volatile molecules into the air around them. After exposure to some of these chemicals, nearby plants of the same species and even other species become less vulnerable to attack, for example by producing toxins or substances that make themselves harder to digest. These changes don’t usually happen straight away but the genes needed turn on much more quickly when they are needed.
But is this really communication, as humans understand it? It really isn’t clear whether a plant releasing chemicals intends to pass on information to another plant by doing so. I respond to the chemicals released by frying onions but that doesn’t mean that the onions are talking to me. So are these really messages or just the opportunist use of chemical information in the environment?
It seems more likely that these signals started out not as a way to send information to other trees but to get messages quickly and efficiently to other parts of the same plant. Pests or infections will often jump from one branch of a tree to the ones closest to it. But a warning telling those branches to prepare for an imminent attack might have to travel most of the way through the tree and then back up it if the message had to move through the body of the plant. This could be a journey of tens of metres in a tall tree.
A signal that can travel through the air, meanwhile, can go directly to the branches closest to the attack. A consequence of these volatile signals, however, is that they can be “overheard” by any plants the chemicals reach. So when other trees respond by also beefing up their defences, is it communication or eavesdropping?
Perhaps it is a bit of both. Maybe an internal messaging system became co-opted to help plants close enough to “listen in” as they would often be related to the tree sending the message in a classic example of evolutionary “kin selection”. However, releasing chemicals into the environment is indiscriminate and other plants and organisms can take advantage. Sometimes these chemical “messages” can attract pests or parasites. The smell of crushed sage doesn’t protect it from humans, for example … rather the opposite.
Going underground
Not all transfer of information between plants is through the air. The vast majority of plants live in symbiotic relationships with soil fungi. We tend to think of forest fungi as mushrooms and toadstools above the ground but these only pop up after sexual reproduction. The real fungus is a mat of elongated cells spreading through the forest floor.
The trees provide the fungi with sugar and the fungi help the tree to gather water and soil nutrients. And many plants can be joined underground by cells of the same individual fungus. Sometimes when one plant suffers damage, other plants connected to it through their soil fungi protect themselves against future attacks while other plants equally near that aren’t “plugged in” don’t. This fungal network is another carrier for information, a true Wood Wide Web.
But who is in control? The messages are relayed by the fungus and perhaps it is the one really using the information, gathering it from one of its host plants and passing it on to the others to protect its “revenue”. The fungus helps the plants to communicate but may do it for its own purposes, and that might include preferentially helping its best producers, whether they are related to the tree sending the message or not. Information intended for family and friends may end up being passed on to unrelated third parties to profit the carrier of the message. In this way, fungi is a bit like a social media company, listening into and benefiting from its users’ posts.
So we return to the question of whether any of these examples are communication in the sense that we would mean it. Anything that makes people think more about plants is good, but perhaps making trees seem more like us can lead us to overlook their essential nature. As a slightly hippy student, what attracted me to plant science was the way that trees and other plants fluidly adjust to their environment. Perhaps using the chemicals that reach them to shape their adaptation is just another facet of this. Worrying about whether trees communicate actually says more about us than them.
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.
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.
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.
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!
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 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 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 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.
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.
