agronomy Review Low Lignin Mutants and Reduction of Lignin Content in Grasses for Increased Utilisation of Lignocellulose Cecilie S. L. Christensen and Søren K. Rasmussen * Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark; [email protected] * Correspondence: [email protected]; Tel.: +45-353-334-36 Received: 20 March 2019; Accepted: 17 May 2019; Published: 21 May 2019 Abstract: Biomass rich in lignocellulose from grasses is a major source for biofuel production and animal feed. However, the presence of lignin in cell walls limits its efficient utilisation such as in its bioconversion to biofuel. Reduction of the lignin content or alteration of its structure in crop plants have been pursued, either by regulating genes encoding enzymes in the lignin biosynthetic pathway using biotechnological techniques or by breeding naturally-occurring low lignin mutant lines. The aim of this review is to provide a summary of these studies, focusing on lignin (monolignol) biosynthesis and composition in grasses and, where possible, the impact on recalcitrance to bioconversion. An overview of transgenic crops of the grass family with regulated gene expression in lignin biosynthesis is presented, including the effect on lignin content and changes in the ratio of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units. Furthermore, a survey is provided of low-lignin mutants in grasses, including cereals in particular, summarising their origin and phenotypic traits together with genetics and the molecular function of the various genes identified. Keywords: brown midrib; cell wall; gold hull and internode; grass family; lignin; monolignol pathway; mutational breeding; orange lemma; transgenic cereals 1. Introduction Cereals are a basic food supply for humans and animals worldwide and include rice, maize, wheat, barley and sorghum. They are mainly grown for their nutritional grains that provide dietary calories for human consumption, animal feed and alcoholic beverages. However, whole-crop silage is also a major product in agriculture and is used for animal fodder. Straw from grain production is often considered a by-product, but it is still essential for animal bedding and feed or can be returned to the soil to maintain soil fertility. Additionally, cereals are used in bioindustries for the production of biofuel, textiles, paper, and biochemicals (for a detailed list see [1,2]). The worldwide demand for cereals is growing, but a decrease in their production is starting to be seen [3]. It is therefore crucial to understand the barriers to efficient utility and breeding for new varieties with improved (utility) benefit as feedstuff for animals and bioproducts. In particular, the concept of the multi-purpose crop, in which the grains are used for food and feed and the straw for bioenergy seeks to overcome the food–feed–fuel dilemma by improving the ligno-cellulosic material from straw in second-generation bioethanol production [4]. Lignocellulose is the main component of plant cell walls and the most abundant organic material on earth. It is primarily composed of energy-rich polysaccharides in the form of cellulose, hemicellulose and pectin, rigid phenolic polymers forming lignin and structural (glyco) proteins. The structure is vital for plant growth and serves as a scaffold providing structural and mechanical strength to the plant and protection against external stresses; it encloses each cell individually and facilitates water and solute Agronomy 2019, 9, 256; doi:10.3390/agronomy9050256 www.mdpi.com/journal/agronomy Agronomy 2019, 9, 256 2 of 21 flux in theAgronomy vascular 2018 systems, 8, x FOR PEER [5, REVIEW6]. Besides these properties, lignocellulose is also an essential2 of 22 source of animal feedThe and structure used is in vital various for plant bioindustries growth and serves [2]. as a scaffold providing structural and mechanical The compositionstrength to the ofplant the and lignocellulosic protection against material external distressesffers; dependingit encloses each on cell the individually biomass source,and but it usually consistsfacilitate ofs water 20–50% and cellulose,solute flux in 20–30% the vascular hemicellulose, systems [5,6].7–30% Besides ligninthese properties and 5–35%, lignocellulose pectin, with lower is also an essential source of animal feed and used in various bioindustries [2]. amounts of structuralThe composition proteins of the that lignocellulosic all depend material on the differs plant depending species, on as the reviewed biomass bysource, [5,7 but,8]. itPlant cells are made upusually by twoconsist typess of 20 of–50% cell cellulose, walls, i.e., 20– primary30% hemicellulose, cell walls 7–30 (PCW)% lignin and and secondary 5–35% pectin cell, with walls (SCW) placed betweenlower amounts the middle of structural lamella proteins and thethat plasmaall depend membrane on the plant (Figure species,1 as). reviewed PCWs surround by [5,7,8]. all plant cells and arePlant continuously cells are made up formed by two duringtypes of cell walls growth., i.e., prim Theary structure cell walls is(PCW) thin and and secondary flexible, cell suitable for walls (SCW) placed between the middle lamella and the plasma membrane (Figure 1). PCWs elongatingsurround cells, but all plant still cells suffi andciently are continuously strong to formed withstand during arising cell growth. turgor The pressurestructure is [thin9,10 and]. It consists primarily offlexible, cellulose suitable and for hemicellulose, elongating cells, withbut still higher sufficiently amounts strong of to pectinwithstand and arising proteins turgor in p dicotsressure compared to monocots[9,10] [5. ,11It consist]. SCWss primarily are formed of cellulose between and hemicellul the PCWose and, with the higher plasma amounts membrane of pectin inand specialised cells suchproteins as sclerenchyma in dicots compared and xylemto monocots vessels [5,11]. after SCWs cell are formed elongation between has the beenPCW and completed. the plasma They are membrane in specialised cells such as sclerenchyma and xylem vessels after cell elongation has been composedcompleted of a greater. They amountare composed of cellulose of a greater and amount hemicellulose of cellulose and than hemicellulos PCW, ande than pectin PCW, isand also partly replaced bypectin lignin. is also These partly components replaced by lignin form. These a thicker components cross-linked form a thicker matrix cross than-linked in matrix PCWs. than As in mentioned above, thePCWs. function As mentioned of lignocellulose above, the function is to provide of lignocellulose mechanical is to provide strength mechanical to the strength cells and to the to facilitate fluid transport.cells and Lignin to facilitate is the fluid fundamental transport. L componentignin is the fundamental for forming component that scaff forolding forming structure that and its scaffolding structure and its occurrence has also been documented in PCW and the middle lamella occurrence[5,6] has. also been documented in PCW and the middle lamella [5,6]. Figure 1. SchematicFigure 1. Schematic illustration illustration of theof the lignocellulosic lignocellulosic matrix matrix in the in thesecondary secondary cell wall cell of the wall grass of the grass family. The main polymers shown are cellulose, hemicellulose, and lignin (shown simplified and not family. Theto mainscale: polymersfor microscopic shown pictures are see cellulose, [12]). They hemicellulose, are organised in and structures lignin called (shown microfibrils simplified that and not to scale: for microscopicgive structural picturesstability to see the [ 12plant]). cell They wall. are Lignin organised is the component in structures providing called the microfibrils recalcitrant that give structuralstructure stability embedding to the plant cellulose cell wall. together Lignin with is thehemicellulose. component Lignin providing is mainly the composed recalcitrant of structure embeddingp- cellulosehydroxyphenyl together (H), withguaiacyl hemicellulose. (G) and syringyl Lignin is(S) mainly units, composedwhich are ofderived p-hydroxyphenyl from (H), 4-hydroxycinnamyl alcohols also known as monolignol, p-coumaryl alcohol, coniferyl alcohol and guaiacyl (G) and syringyl (S) units, which are derived from 4-hydroxycinnamyl alcohols also known as sinapyl alcohol. The monolignols are synthesised in the cytosol from phenylalanine and tyrosine monolignol,(grasses p-coumaryl only) through alcohol, the coniferyl phenylpropanoid alcohol andpathway sinapyl and alcohol.monolignol The-specific monolignols pathway,are then synthesised in the cytosolexported from across phenylalanine the plasma membrane and tyrosines into the (grasses secondary only) cell wall through and oxidized the phenylpropanoid by cell wall-bound pathway and monolignol-specificperoxidase (PRX) pathway,and laccase then (LAC exported), before polymerization across the plasma into themembranes lignin polymer into. (Illust therat secondaryion: cell Martin Mook). wall and oxidized by cell wall-bound peroxidase (PRX) and laccase (LAC), before polymerization into the ligninThe polymer. recalcitrant (Illustration: structure Martin of lignin Mook). is the major limitation of utilising SCWs´ nutritional polysaccharides for animal feedstock and producing bioproducts. Lignin also serves as a mechanical The recalcitrantdefence barrier structureand is known of to lignin accumulate is the under major pathogenic limitation attacks of [13 utilising–15]. It has SCWs also been´ nutritional polysaccharidesdemonstrated