Understanding and manipulating lignin biosynthesis in barley

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences


The pathway whereby plants make lignin, a strengthening and waterproofing substance in cell walls, has been extensively studied in dicot plants (tobacco, Arabidopsis and poplar) but little is known about the pathway in grasses. Cell walls and lignin in grasses have several unique features that cannot be understood by studying dicots. We want to study the genes that are involved in making lignin directly in an economically-important grass, barley. We will be able to identify most of the genes quite easily as they are likely to be very similar to the genes already identified in Arabidopsis. However several novel genes may be important in grasses, particularly those involved in generating the features that make grass cell walls unique. We expect that these genes will be switched on at the same time and in the same place as the known lignin genes giving us a handle with which to identify them. For each known or novel gene, we want to determine the function it has in making lignin and whether plants without that gene have any advantages for agriculture or industry. For example, the amount of lignin in a grass influences how difficult it is for animals who are fed that grass to digest it, as lignin itself is fairly indigestible and it surrounds the other, more digestible, components of plant cell walls. Maize plants where specific genes involved in making lignin have been 'knocked-out' or made less efficient through mutation, have reduced amounts of lignin and are more digestible than normal counterparts. We want to see if this is true also in barley by identifying and studying barley mutants in the genes that make lignin. As well as teaching us more about how plant cell walls are made in grasses, this work may lead to new ideas about how to improve the barley crop. In fact, useful mutants identified in the course of this work could be directly incorporated into barley breeding programmes.

Technical Summary

The pathway whereby plants make lignin has been extensively studied in dicots but relatively little work has focussed on the pathway in grasses. Cell walls and lignin in grasses have several unique features - such as the incorporation of H-lignin units and ferulates into the wall - that cannot be understood by studying dicots. We want to study the genes that are involved in making lignin directly in barley, a monocot cereal grass with significant economic importance to the UK. We will use a bioinformatics approach to identify barley sequences in EST databases that have homology to Arabidopsis lignin genes. We will identify and sequence cDNA clones for these genes and perform phylogenetic analysis on each gene family in order to better identify the family members most likely to be involved in developmental lignification and to be able to compare the gene family structure with that of Arabidopsis. In addition, we aim to identify novel genes potentially involved in lignification via their co-expression with known lignin genes in microarray analyis of gene expression profiles from a wide range of barley tissues. For each known or novel gene, we want to determine its function on the lignin pathway and whether mutants in that gene have any advantages for agriculture (primarily in digestibility) or industry (for biomass or bioethanol production). The rational behind the applied aspects of this work are that maize mutants in specific lignin genes (brown midrib mutants) have reduced lignin content and improved digestibility. We will identify barley mutants for lignin genes from within a mutant population available at SCRI by TILLING. We will fully characterise the mutants to determine the nature of the lesion and to analyse lignin and potential digestibility changes. Mutants with good agronomic performance but improved digestibility could be rapidly incorporated into barley improvement programmes.


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Description This project carried out the first study of the set of barley genes involved in making lignin, a component of plant cell walls. In addition, we isolated and characterised mutants in selected genes and used them to study gene function. Some of these had improved digestibility and potential value for making better barley for animal feed or for biofuel production.

Early in the project, we compiled a complete inventory of the lignin biosynthetic genes in barley by BACS cloning/sequencing and searches of publicly available databases. This inventory was updated following the release of barley draft genome assemblies by IPK and the James Hutton Institute (JHI). For each of the 10 enzymes constituting the lignin biosynthetic pathway in dicots we identified the homologous gene family members in barley. We performed phylogenetic analysis comparing these genes with those from representative dicots and monocots for which a complete genome sequence is available. As part of this analysis we obtained genomic and cDNA sequences for the barley genes most likely to be involved in lignification. In collaboration with the JHI we identified the map positions of many of these genes based on their iSELECT 9000 SNP marker map.

TILLING and transgenics: The barley Optic TILLING population was screened for mutants in our priority candidate, COMT, using two separate amplicons spanning the full coding sequence. Five COMT mutants, 2 silent and 3 mis-sense mutations, were identified. Seed for one mutant did not germinate and plants harbouring the other two mutations did not show significant reductions in COMT activity. Two knockout lines were also identified for CAD2. Problems with the viability of the stored TILLING population seed precluded the evaluation of further lignin gene mutants. To rectify this situation, we produced a new barley TILLING population and developed a novel detection strategy using next generation sequencing. As an alternative to TILLING while the population was being re-made, we used RNAi in barley transgenics to characterise the function of candidate genes involved in lignification. RNAi lines were generated for PAL, COMT and CCR. The efficiency of the RNAi suppression was characterised using either enzyme assays or antibody detection of the target protein. In the case of the PAL RNAi knockdowns, activity was reduced by up to 90%. Lines with the most suppressed PAL activity were slightly shorter than control lines and had lower grain yield, forming fewer, smaller seeds. The stems of some lines also had a colour phenotype. We are currently analysing the effects of reduced PAL activity on lignin/digestibility. For CCR knockdowns, CCR protein was reduced to almost undetectable levels and significant reductions in lignin content were measured. For the COMT knockdowns, COMT suppression to low levels was accompanied by little change to lignin content but dramatic alterations to lignin composition and structure as evidenced by a halving of S/G ratio.

Orange lemma characterisation: The rob1/orange lemma mutants were fully characterised and the mutations in the CAD gene identified. The levels of enzyme reduction were evaluated at several stages of development. Sophisticated lignin analysis showed small reductions in lignin content (16%) associated with changes to S/G ratio and the appearance of cinnamaldehyde units in lignin. There were significant increases in straw digestibility in the mutants without any alteration in malting properties. This work is at an advanced stage of preparation for publication.

Co-expression to identify novel genes: To identify new genes potentially involved in lignin or secondary cell wall biosynthesis we have compiled a database of all barley 22K microarray experiments to date (1660 microarrays) and carried out co-expression analysis on the dataset using lignin biosynthesis genes as bait. Good candidate genes for further analysis were those that were co-expressed with multiple lignin biosynthesis genes. These included orthologues for transcription factors that shown to regulate lignin biosynthesis in maize, validating the approach.
Exploitation Route Our findings could be useful for breeding cereals with improved digestibility for animal feed, second generation biofuel production, or other industrial biotechnology applications.

We collaborated with Limagrain to introduce the orange lemma mutation into other elite barley varieties and to evaluate its effect on digestibility. Small plots of field grown plants are being evaluated for digestibility and agronomic properties.
Sectors Agriculture

Food and Drink



Description One aspect of this project i.e. the characterisation of barley orange lemma mutants, was picked up by researchers at the IAEA (International Atomic Energy Agency) They asked seeds and DNA sequence for primer generation so that they could use these in breeding programmes to introduce the mutations into varieties grown in developing countries in order to improve digestibility for animal feed. The project overall generated data that was used to support subsequent successful proposals that contributed to the £28 million BBSRC Sustainable Bioenergy Centre.
First Year Of Impact 2009
Sector Agriculture, Food and Drink
Impact Types Societal


Description Cell wall lignin programme: Manipulating lignin to improve biofuel conversion of plant biomass
Amount £3,000,000 (GBP)
Funding ID BB/G016232/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2009 
End 03/2014
Title Transgenic barley with manipulated lignin 
Description Barley Transgenics: COMT RNAi lines; CCR RNAi lines; PAL RNAi lines; CAD RNAi lines; 4CL RNAi lines, C4H RNAi lines, C3H RNAi lines, HCT RNAi lines RNAi constructs were developed in pBRACT207 vectors and barley cv Golden Promise was transformed using Agrobacterium tumefaciens to produce at least 30 independent primary transformants per gene along with 15 pBRACT207 empty vector control lines. Plants were initially screened by enzyme assay or Western blot to identify those with reduced target enzyme activity/protein. Plants with reduced target enzyme were subjected to Southern blotting to identify lines with a single transgene insertion. Homozygous lines were produced from these plants for further study and full lignin analysis. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Validation of the role of specific genes in lignin biosynthesis and of candidate genes underlying GWAS peaks 
Description Evaluation of rob1 in elite barley 
Organisation Limagrain
Country France 
Sector Private 
PI Contribution Characterisation of rob1, provision of mutants and DNA sequence for primers
Collaborator Contribution Introduction of rob1 to elite varieties and field evaluation
Impact Evaluation was no clear-cut and no outputs yet
Start Year 2010
Description Introgression of rob1 into fodder barley 
Organisation FAO/IAEA Agriculture and Biotechnology Laboratories
Country Austria 
Sector Charity/Non Profit 
PI Contribution We are collaborating with the Head of the FAO/IAEA Agricultural and Biotechnology Laboratories (Seibersdorf Laboratory) to introgress the rob1 orange lemma mutation into forage barley for use in locations such as sub-saharan Africa. The overall aim of the Plant Breeding and Genetics group within the Joint FAO/IAEA Programme - Nucear Techniques in Food and Agriculture, is to enhance food security through sustainable crop production using strategic fundamental and applied crops sciences research.
Start Year 2012
Description College of Life Sciences Open Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact School of Life Sciences Open Day to bring the public in to learn about our research. Very interactive event with many questions from the public and comments about how it has changed their attitudes to key topics.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015
Description Dundee Botanic Gardens Family Fun Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Dundee Botanic Garden Family Fun Day (usually April/May): This is an annual family activity day at the gardens which has now developed into Plant Power Day and Fascination of Plants Day. We provide interactive activities related to plant science such as DNA preparation from raspberries along with displays on barley cultivation and uses, and biofuels from plants.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016