Weeding the Gems

The High Value Chemicals in Plants (HVCfP) Network in a BBSRC-funded NIBB (Network in Industrial Biotechnology and Bioenergy) that is centered at the University of York. At the end of October, Cambridge Cottage at Kew Gardens hosted the HVCfP Annual Meeting that brought together over 80 interested members of the network to discuss recent research developments and their resulting commercial potential.

The schedule was comprised of invited keynote speakers as well as talks from contributors who have taken advantage of the funding opportunities provided by the network. These include the Proof of Concept (PoC) fund that are worth up to £50K as well as smaller Business Innovative Vouchers (BIV) (previously £5K, now increased to £10K). Funding opportunities are ongoing through the life of the NIBB so please check out funding (https://hvcfp.net/funding/) page for information about previous awards and current open calls.

The meeting kicked off with two keynote talks, firstly from Monique Simmons, deputy director of Kew Science who introduced the wide variety of resources associated with the Botanic Gardens. Over the past year, Kew has undergone a significant reorganization and has a renewed remit for interaction with the scientific community. Perhaps of greatest interest to GARNet readers will be the Kew DNA bank (http://apps.kew.org/dnabank/homepage.html) that contains over 40K samples from around the globe, the majority of which are available to order, either in aliquots suitable for PCR or in larger quantities if necessary.

David Hughes from Syngenta followed up with an excellent talk about the current situation surrounding the debate around Genetically Modified Crops. He gave his thoughts on why this ‘debate’ has gone on for almost 20 years even without any reliable evidence about the negative effects of developed crops. He thought the negative public response could be summed up by three emotions.

Essentialism: is the ‘essence’ of the original organism transferred to the crop

Naturalism: ‘Synthetic’ is evil, ‘natural’ is good.

Disgust: If something is not ‘100% safe’, then it must be bad.

David also advised that the power of narrative is a much more effective technique than the use of potentially bland scientific evidence, which usually falls on deaf ears amongst the public.

The day included two talks from commercial members of the HVCfP network that provided a contrast to the majority academic talks. They included a presentation from James King of Oxford Biotrans (http://oxfordbiotrans.com/), a company that is attempting to exploit biocatalytic routes to develop novel compounds. Their focus is on the use of cytochrome P450s and one of their initial challenges is to enzymatically produce nootkatone, which provides the flavor and scent from grapefruit. They aim to produce this compound via the intermediate Valecene, which can be collected from oranges. The unpredictable grapefruit harvest and the difficulties in isolation of endogeous nookatone mean that Oxford BioTrans suggest that using enzyme catalysis might be a more cost effective method for isolation of the high value product. The other SME that presented was Neem Biotech, which is located in the South Wales Valleys, a place not usually associated with biotechnology. Their major product is Vetrinol, which includes garlic extract and that is used for boosting the bovine immune system.

Cathie Martin discussing Cavity Spot

The remainder of talks featured researchers involved in PoC or BIV projects. These were all rather fascinating (especially for a researcher usually preoccupied by Arabidopsis) as they introduced novel compounds and non-model organisms that were ‘unusual’ to many in the audience. Cathie Martin (JIC) is well known for her work with purple tomatoes but here she presented her attempts to isolate the compound from Mugwort leaf extract that is responsible for reducing cavity root spot disease, which is caused by Pithium species of Oomycete. This work was performed in collaboration with ‘Root Crop Consultancy’ and resulted from an observation that carrots grown in the presence of mugwort were less susceptible to cavity spot disease.

Three talks that described research funded by PoC grants came from Gary Loake (Edinburgh), Luis Mur (Aberystwyth) and Mike Beale (Rothamstead) who each highlighted work aimed at to improve production from organisms already used to produce medicinally useful compounds. Gary Loake is hoping to develop the use of cambial meristematic cells (CMC) to improve paclitaxel biosynthesis from Taxus (Yew) (for review of CMCs http://www.sciencedirect.com/science/article/pii/S1871678415000205). The Taxus CMCs are able to produce plenty of paclitaxel so to improve this aspect they have analysed expression profiles in an attempt to identify the transcription factors that are important in this process. The ultimate aim would be to use synthetic biology approaches to potentially modify the expression of these TFs in order to maximise paclitaxel production.

Aberystwyth University has a strong tradition of research in Avena species (Oat) so Dr Mur is using these resources in order to understand and maximise the production of Avenanthramide, which has well-established medicinal benefits. The HVCfP money has funded the use of HPLC to analyse their germplasm for altered production of Avenanthramide. Broadly they have found that domesticated hexaploid Avena species have higher levels of the compound compared to wild diploid varieties. They are now conducting expression analysis in order to understand the molecular reason for these alterations.

Mike Beale is taking advantage of the National Willow Collection (NWC) to undertake a phytochemical and pharmacological screen in order to identify novel compounds. Willow (Salix) is famously the original source of aspirin so Dr Beale and colleagues are using NMR and MS in an attempt to analyse unpurified extracts from 200 species of Willow. By screening against a battery of cancer cell-lines, fungi and bacteria, they hope to identify extracts that can be further interrogated to find compounds of interest.

The other two talks resulting from BIV-funding were both focused on algae, although with organisms of different sizes! David Bailey from IOTA Pharmaceuticals outlined their plans to exploit the potential of using the macroalgae Sargassum muticum (seaweed) as a production chassis while Paul Knox (Leeds) and John Dodd (AlgaeCytes) described their early work to identify novel polysaccharides found in microalgae.

Matt Davey discussing Living Stones

The meeting ended with a series of flash presentations given by representatives of GARNet, the Liverpool GeneMill, the University of Essex Cell screening facility and finally from Matt Davey (Cambridge). His talk highlighted the incredible potential that is provided by plant species as he is developing commercial relationships to study the UV absorption properties of Living Stone species (Lithops). Initial experiments have shown that direct UV light is very successfully absorbed by Lithops species and this is of interest to the cosmetic industry.

Although much of the research presented in this meeting is in its infancy, the HVCfP Network can be pleased with the process it has made. With another set of PoC and BIV funding recently announced the network is supporting research in a wide range of organisms aimed at producing a varying array of compounds. The network has been providing funding since 2014 so the first set of PoC funding is now coming to an end. The true success of the network will be realised over the next two years, determined by the amount of follow-on funding that can be obtaining to support the research conducted in these seed-projects. We will watch this space with interest….

Paper Review: Quantitative characterization of genetic parts and circuits for plant synthetic biology. Nature Methods (2015) doi:10.1038/nmeth.3659

Professor June Medford andd Dr Ashok Prasad (Colorado State) have been in the vanguard of the development of tools for plant synthetic biology over the past few years. Here we highlight a recently published article in Nature Methods that presents their quantification of genetic parts and circuits designed for plant synthetic biology.

To date, quantitatively defined gene circuits have been almost exclusively characterised in unicellular organisms. Some of the initial challenges in transferring this characterisation to multi-cellular organisms includes use of orthogonally useful parts, that are active irrespective of developmental or organismal context, as well as developing methods for the quantification of input and output characteristics. This latter aim is hindered by the difficulties of plant transformation that adds significant levels of variation to this process. In this study the authors aimed to overcome some of these challenges by characterising parts using a medium-throughout method that analyses luciferase expression in protoplasts all within in a 96-well plant format.

This set of synthetic parts focused on the analysis of different repressive elements fused to a constitutive promotor region. These repressors were designed with orthogonal activity in mind and included both previously characterised and novel elements (Figure 1a). The output utilised a dual luciferase assay where one species of the Luc enzyme was constitutively expressed and compared to the expression level that was influenced by the designed repressor region (Figure 1b). This allowed tuneable expression that is dependent on the type of repressor elements that were present, all within the context of high levels of initial constitutive expression. In total, 128 different promotor combinations were tested in an Arabidopsis protoplast expression system. These expression levels were further varied by altering the levels of induction provided by two different compounds (DEX and OHT).

Figure 1 from Nature Methods

Initial experimentation generated significant ‘noise’, which is not ideal for generating predictable tools for use in synthetic biology. By analyzing a number of possible sources of experimental variation they found that the ‘batch effect’ was most significant, which described the differences between protoplast samples prepared on different days (attempts to resolve the precise aspect of the experimental technique that caused the variation were unsuccessful). Although it is somewhat harkening to know that the transformation efficiency or uptake of chemical inducers does not contribute as much to experimental variation, it is somewhat worrying that the fundamental preparation of protoplasts provided the highest amount of variation. However the authors were able to develop a mathematical model tat they used in their analysis that accounted for the ‘batch effect’. Ultimately the authors found that the repressor elements worked as expected but that some molecular motives acted more predictably in different pairings, the details of which can be found in the paper.

The authors then attempted to characterise a similar set of constructs in protoplasts generated from the monocot Sorghum. Although a slight alteration was made to the 5’ UTR region of the construct, they found that using the same normalization model to remove the batch effect generated a similar set of results as in the Arabidopsis protoplasts. This indicates that Arabidopsis can act as a good model for evaluating initial expression levels that might occur in other dicots or monocots.

Finally three different promotor-repressor genetic circuits were stably transformed into Arabidopsis and then multiple independent transgenic lines were isolated for each combination. Subsequently one transgenic line from each of three genetic combinations was chosen for comparison with the transient expression system. In a method outlined in Figure 2, protoplasts from the stably transformed plants were compared with those isolated from wildtype plants that were transiently transformed with the same construct. Although absolute expression was higher in the transiently transformed protoplasts, analysis of normalized data demonstrated that equivalent repressor elements performed similarly in the differently generated samples. This allowed the authors to suggest that use of the transient system was a reasonable proxy for that observed in stably transformed plants. Therefore they conclude that the transient system can be used as a more rapid screening procedure for newly developed synthetic elements.

This is an important piece of analysis that is absolutely necessary before the full potential of plant synthetic biology can be realised. This type of design, build, test procedure is common-place when using a unicellular chassis but has not been studied in this detail in a plant system.

However the amount of normalization that is required to ensure the data is directly comparable certainly highlights difficulties may lie ahead as researchers work toward the ultimate goal of developing synthetic genetic elements that are usable across many multi-cellular plant chassis. Although the GARNet blog has highlighted some excellent tools that have been recently developed for plant synthetic biology, this study acts as a cautionary tale for those entering this field and are unaware of the unpredictable nature of even the most commonly used experimental techniques.

Overall there is little doubt that this is an exciting time for plant synthetic biology even though the generation of predictable, consistent gene expression across a variety of chassis will remain rather challenging in the immediate future.

Agroinflitration of Nicotiana benthamiana is an increasingly important technique that many plant scientists may use for transient expression of their gene of interest. These experiments might include localisation or interaction studies, monitoring of pathogen effects or simply for protein production where, in extreme cases, up to 50% of the total soluble protein will be expressed by the transgene.

As a previous user of this technique, I know that many aspects of early test experiments can be undertaken haphazardly, with little understanding or expectation regarding the optimum agrobacterium concentration, best leaf segment or inoculation timings to use. There also seems to be significant variation within the same inoculated leaves, between leafs on the same plant and especially so between leaves from different plants. Therefore it is satisfying to read a recent paper published in Plant Methods from the lab of Teemu Teeri (University of Helsinki) that attempts to provide some understanding as to the sources of variation in these infiltration experiments [1].

They conduct a simple set of experiments whereby they use an intron-containing luciferase gene as a reporter to test the factors that most influenced the infiltration efficiency. They inoculated the upper leaves of 6-week old tobacco plants with either luciferase driven by the constitutive 35S promoter or by an estradiol-inducible promoter. They showed that estradiol induced an eight-fold increase in enzyme production and that these induced levels were similar to those observed with the constitutive promoter.
In order to assess variation within experiments they compared samples from the same leaf, between leaves from the same plant and between leaves from different plants. Importantly they used a nested experimental design so that they could compare the amount of variation generated between hierarchical levels of infiltration, sampling or enzyme activity.

Somewhat surprisingly they found that the largest component of variation in these experiments was between disks on the same leaf, whereas use of different leaves or different plants generated a 2.5x lower level of variation. Within disks from a single leaf, the amount of bacteria and protein showed little alteration, even though the luciferase enzyme activity was greatly different. The reason for this difference is unknown.

Therefore based on their results the authors make the following recommendations:

1. Ensure tested plants are in the same physiological state.
2. Combine the expression values from several sample disks on the same leaf, do not rely on one disk as a representative amount of expression on that leaf.
3. Inflitrate more plants but less leaves on those plants. Sample more disks on each of the leaves that are used.
4. Technical replicates (for luciferase assays) are not as important since these measurements are more reliable.

Good luck with your experiments!

1- Bashandy et al (2015) Plant Methods11:47 DOI: 10.1186/s13007-015-0091-5