Weeding the Gems

In 2013 the BBSRC provided funding for 13 Networks in Industrial Biotechnology and Bioenergy (NIBB) each of which had the principle aims of supporting ‘Proof of Concept’ style projects and providing a forum for like-minded researchers to interact with each other and with industry. Each of these NIBBs are set up in much the same way although the manner in which they have developed has slightly varied.

GARNet has recently promoted the activities of the High Value Chemicals from Plants network and in order to learn about another group of researchers I recently attended the 1st International Conference of the Lignocellulosic Biorefinery Network (LBNet). This three-day residential meeting brought together both academics and representatives from industry who use a variety of experimental approaches to tackle the challenges associated with utilising the biomass held within plant cell walls. The conference schedule was split into broad sections that each focused on structural aspects of the cell wall, namely ‘Lignin’, ‘Matrix Polysaccharides (MS)’ and ‘Cellulose’. Within each of these was a ‘Construction’ and ‘Deconstruction’ sections which broadly, although not exclusively could be summarised as ‘Plant’ or ‘Bacterial’ respectively!

GARNets favourite model plant is not usually thought of as a potential source of usable biomass so it was encouraging to observe that much of the plant science discovery research was indeed occurring in Arabidopsis. This again provides evidence for the utility of Arabidopsis as a model plant despite recent moves to encourage researchers to also embrace other experimental systems.

Following an introduction from the LBNet chairman Simon McQueen-Mason, the ‘Lignin Construction’ session kicked off with both Claire Halpin (Dundee) and Wout Boerjan (VIB, Ghent) describing the use of Arabdopsis mutants to identity components of lignin signaling pathways. Lignin provides a major difficulty for the saccharification of sugars from plant biomass so a prime objective in this field is to identify plants that have reduced lignin composition yet do not show any yield penalty.

Taken from http://science.sciencemag.org/content/341/6150/1103.full

Professor Boerjan introduced a very successful collaboration between his and the Halpin group wherein they identified a new branch to the lignin biosynthesis pathway and the associated cse1 Arabidopsis mutant that appears to grow normally.

The Boerjan group has also been involved in an associated piece of research that is an outstanding example of the potential to translate Arabidopsis research into other plants. Following identification of the ccr Arabidopsis mutant, which has less lignin and provides more sugar through saccharification, they generated transgenic poplar trees that had reduced expression of CCR. Subsequent field trials excitingly showed that the amount of sugar released from these plants per volume biomass was improved but unfortunately their overall yield was reduced. However Professor Boerjan also described more recent work in which the Arabidopsis ccr mutant could be rescued by expression of CCR in only xylem elements, allowing the plants to be of normal size but release their sugars more easily. Work is now ongoing to translate this approach into poplar.

The Halpin group are also working on both enhancer and suppressor mutant screens to identify novel regulators of lignin biosynthesis and have discovered some intrigued genetic lesions that are involved in an unexpected biosynthetic pathway. In addition Professor Halpin described a GWAS experiment that her lab has undertaken using 640 barley cultivars. This has identified some novel transcription factors that might be involved in lignin biosynthesis.

Arguably the most exciting result on this topic was provided by Jan Lycakowski from the lab of Paul Dupree who gave a flash talk about his work to characterise a set of Arabidopsis gut triple mutants that are unable to correctly add glucuronic acid (GlcA) to cellulose. These plants have no apparent yield penalty, have normal looking cell walls (thought to be due to the addition of different post-translational modifications instead of GlcA) but yet will release their sugars at a greater rate than wildtype Arabidopsis. It remains to be seen whether this promising result is recapitulated in more industrially relevant plants.
Other talks in the ‘Lignin Construction’ session exemplified that researchers are using many different plant species in order to identify factors that control generation of cell wall biomass. This included examples from hemp (Alexandra Lanot, York), maize (Luisa Trindade, Wageningen), wheat (Kamaljit Moirangthem, Nottingham) and Miscanthus (Ricardo Da Costa, Aberystwyth).
The ‘MS construction’ session largely focused on the role of the sugar xylan in the construction of the cell wall and included a description from Tom Simmons of his attempts to use solid state NMR to resolve the complex structure of the cell wall. In addition Paul Knox (Leeds) gave an update from his labs successful attempts to generate a set of ‘Plant Probes’, antibodies that are specific for motifs within the cell wall. This certainly represents a very useful research tool for interested potential users.

The ‘MS deconstruction’ session included the description of an exciting piece of work by Lenka Frankova who works with Stephen Fry at the University of Edinburgh. From published work, Lenka described the discovery of a unique enzyme, Hetero-trans-B-glucanase (HTG) from Equisetum plants (the ‘living fossil’ species of horsetails). HTG appears to be uniquely able to form crosslinks between cellulose and the MS xyloglucan and therefore offers a potential biochemical mechanism to strengthen cell walls. The Fry group have recently obtained a BBSRC responsive mode grant to investigate the potential role of HTG in a range of grass species. It is intriguing to speculate that this enzyme might provide a novel mechanism to strengthen cell walls, potentially providing an opportunity to add structurally rigidity to existing grass species.

In the ‘Cellulose Construction’ session both Daniel Cosgrove (Penn State) and Simon Turner (Manchester) provided an update on each of their attempts to define the nature of the plant cell wall. The Cosgrove group uses AFM and SEM to determine the arrangement of cellulose microfibrils both in the native state and under strain, the latter experiments using a novel experimental procedure where they physically stress onion epidermal cell walls.

The Turner lab works on the Cellulose Synthase Complex (CSC) and Professor Turner described a set of extremely detailed domain-swap experiments between protein members of the CSC complex. This has allowed them to propose a model for the arrangement of protein subunits within the CSC and offers insight into the mechanism by which this complex is aligned with cellulose and underlying microtubules.

The final ‘plant’ focused talk was provided by Bjorn Sundberg who, following an academic career that helped establish the reputation of the UMEA Plant Science Centre, now also works for the international forestry company StoraEnso. It was fascinating to learn about the companies transition away from paper production into research that focuses on exploiting the energy available in lignocellulose. Professor Sundberg’s role involves the global development of genetic strategies to improve Eucalyptus as a biomass stock. His description of the 20-year timescale from identification of interesting traits to first harvest is something unusual within the timeline of the academic grant cycle! The recently development of a Eucalyptus SNP-chip will hopefully improve this timescale and the company are also looking to improve transformation efficiencies, a bottleneck in the genetic engineering of many crop species.

Elsewhere in the ‘Deconstruction Session’ were talks from invited experts who discussed their work aimed at understanding and improving the process of cell wall breakdown from a microbial perspective. These included studies described by Joe Bennett and Nicola Oates from the University of York where they screened environmental microbial communities that grow on waste grain stalks with the aim of identifying novel degrading enzymes. Nicola in particular described some novel putative carbohydrate-active enzymes isolated from the Graphium species of fungus.

Overall this LBNet meeting provided an outstanding selection of talks and plenty of opportunity for discussion at the remote conference venue of Shrigley Hall, located on the edge of the Peak District. Observationally it seemed that a number of collaborations might arise from discussions. As with each of the BBSRC NIBBs, the LBNet provides ‘Proof of Concept’ and ‘Business Innovation’ funding of up to £50K. Please give some thought how your research might interact with LBNet and contact the network coordinator Vernoica Ongaro.

From a GARNet perspective it was clear from the presented Arabidopsis research that some of the genes involved in cell wall construction would not have been intuitively predicted to act in this process. Therefore it might be worth investigating whether your gene of interest somehow impacts formation of the cell wall as it could open up extra funding opportunities!

Please see this list of live tweets from the conference.

GARNet and the Gatsby Charitable Foundation are delighted to be able to offer four £500 travel bursaries for attendance of the International Conference on Arabidopsis Research which will take place in South Korea in June 2016.

To be eligible, the student must still be studying on a UK-based PhD Program at the time of the conference and must also submit a poster abstract to present at the meeting. At the time of application, students must also supply a letter from their supervisor confirming that they have financial support to attend the conference.

Following receipt of a GARNet/Gatsby bursary, the successful student will be required to provide a 500 word summary about their experiences at ICAR2016 for publication in the July 2016 edition of GARNish newsletter or on the GARNet blog.

We are not able to fund travel costs directly. Successful candidates will be provided with the bursary once evidence of registration to the meeting, poster abstract submission and flight bookings are provided.

Please pass on this information to anyone who might be interested in this generous bursary.

Submit the official application form to geraint@garnetcommunity.org.uk by the closing date of 5pm on March 1st 2016. We will inform successful applicants soon after the closing date so that they can book their travel to Korea

Please download the official application form here: GARNet_Gatsby_Bursary_ICAR16

Kentaro Tamura and co-workers recently published a paper in PLoS One that should be of interest to all Arabidopsis researchers, even though it might fill them with a little concern!

Kentaro works with Ikuko Hara-Nishimura at Kyoto University with a research focus on the biology of the plant nucleus, having published a number of studies regarding the function of proteins that control nuclear size, shape and movement. Therefore they were in the process of studying the ALADIN protein (which is a putative component of the plant nuclear pore complex) when they made a surprising finding. They were analyzing a T-DNA insertion mutant that removes expression of the ALADIN gene but, during the course of their investigation, realised that mutant phenotypes that they were observing (small stature, low photosynthetic activity) were in fact due to a downregulation of the adjacent PLASTID-SPECIFIC 50S RIBOSOMAL PROTEIN 5 (PSRP5) gene. They performed necessary complementation experiments to indeed show that the T-DNA was altering expression of the gene adjacent to the one in which is was inserted.

This might not be a surprise to those researchers who have been previously thwarted in their attempts to compliment a T-DNA insertion mutant. However it certainly is a timely reminder that analysis of a mutant phenotype might require a greater depth of analysis than intially though.

Can a researcher always be confident about the phenotype that they are observing, especially when it shows a novel or unexpected effect?

What is the genetic distance for which a T-DNA insertion might be able to effect the local chromatin environment?

If ever it was needed, this certainly shows the need for providing complementation data in your study, especially if the phenotype is in any way surprising.

In some manner this paper is a reminder of the recent controversy surrounding a T-DNA insertion within the AUXIN BINDING PROTEIN1 (ABP1) gene. In this case the previously considered lethal abp1-1 T-DNA mutant was in fact due to a deletion in the adjacent BELAYA SMERT (BSM) gene, which was recently confirmed by Dai et al (2015) in Nature Plants. In the Tamura case there is no evidence of an adjacent deletion but rather for a gene silencing effect, by an as yet uninvestigated mechanism.

Ultimately both cases are an excellent reminder for researchers to make sure they fully interrogate any mutant phenotype that they are working with. This is especially true if it seems too good to be true, as in fact it might very well be!!

For the past 20 years many lab researchers will tell you that day-to-day ‘cloning’ is perhaps the most frustrating part of the work they do! However more recently, help has been at hand for those researchers with the emergence of many new cloning strategies that do not rely on conventional restriction enzyme digestions! Gateway cloning is one such method and speaking personally it certainly made things much easier in the lab (although perhaps not as simple as the designers might have you believe)!

Plant Physiology recently published an OA manuscript entitled ‘MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants‘. This paper introduces a set of Gatway compatible constructs that combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities.

This ambitious goal was been acheived by mining research literature to select >20 promotors that provide tissue-type specific expression in the Arabidopsis root. These promotors have been linked with the estrogen-inducible XVE system which, again from personal experience, offers strong expression in response to an  inducible signal. Therefore this will allow induced expression of a researchers gene of interest (GOI) in a specific cell file.

These constructs can be linked to a range of fluorescent proteins to allow visualisation of different tagged proteins in the same root as well as providing the facility to make both transcriptional or translational fusions.

In the paper the authors have tested the expression of many of these reporters and the whole set of clones are available either from the lab of Ari Pekka Mähönen (free) or from Addgene (pay). They look to be a very useful resource so please give them a go!