The analysis and identification of cell-wall-related genes and enzymes is a convenient approach to study the role of cell wall components in bioenergy crops. The main gene families that are explored in biomass investigations are those that are involved in the processes of cell wall biosynthesis, growth, development, and degradation.
Lignin is produced by the phenylalanine/tyrosine metabolic pathway in plant cells. In this phenylpropanoid pathway, three enzymes, namely, phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL), catalyze the initial steps to provide the precursors for all of the downstream metabolites
[16]. Other phenylpropanoid enzymes, such as quinate/shikimate p-hydroxycinnamoyltransferase (HCT), p-coumaroylshikimate 3′-hydroxylase (C3H), caffeoyl shikimate esterase (CSE), caffeic acid O-methyltransferase (COMT), and caffeoyl-CoA O-methyltransferase (CCoAOMT), working downstream of 4CL, are also essential for lignin biosynthesis
[17]. In addition to these enzymes, others that are specific for lignin biosynthesis have been identified, including cinnamoyl-CoA reductase (CCR), ferulate 5-hydroxylase (F5H), and cinnamyl alcohol dehydrogenase (CAD)
[16]. After biosynthesis and transport, lignin is generally polymerized by the enzymes peroxidase (POD) and laccase (LAC) in the secondary cell walls with three types of monolignols, namely sinapyl alcohol, coniferyl alcohol, and p-coumaryl alcohol
[16].
Cell wall growth and development incorporate large families of enzymes, including glycosyltransferases (GTs), glycosylhydrolases (GHs), methyltransferases, and acetylesterases, part of the carbohydrate active enzymes, or CAZymes, classified in the CAZy database
[18]. Despite their importance, many
CAZy genes are still uncharacterized
[18]. Furthermore, the cellulose-synthase-like (
Csl) gene superfamily appears to be crucial in regulating β-glucan synthesis during plant development
[19]. For instance, the
CslF6 gene is expressed in many plant tissues during development
[20]. However, further investigation is necessary in order to define the precise role of 1,3;1,4-β-glucan and the
CslF gene family in cell wall composition.
The endogenous degradation process of the cell wall involves several enzymes. It is a step-by-step reaction that starts with the expansion and subsequent separation of cells in which pectins are targeted, followed by hydrolysis of the cell wall components, and degradation of hemicellulose and cellulose
[22]. Among the enzymes that are involved in cell wall degradation, there are members of the glycoside hydrolase 9 (GH9) family, the endo-β-1,4-glucanases, which cleave the β-1,4-glycosidic bonds with monomers of glucose, contributing to the cellulose deconstruction. Furthermore,
GH10 and
GH11 xylanase genes are also known to control the hemicellulose degradation
[23]. Therefore, by overexpressing these key enzymes, it may be possible to modify the cell wall structure of energy crops and thus to drive improvements in the technologies for biofuel production.
3. Gene Editing Approaches to Improve Biomass Quality
The design of genetically modified plants to synthetize less recalcitrant cell walls has been applied to improve biomass saccharification. Recently, genetic engineering approaches have been applied to modify the genes that are involved in the cell wall structure
[24]. The modification of the cell wall composition by downregulating or knocking-out the lignin biosynthetic genes, or by acting on related transcription factor mechanisms, has been attempted with the aim of reducing lignin content
[25]. However, the success rate of these approaches was limited, due to undesirable traits in plants with mutations in lignin biosynthesis, such as reduced biomass yields, low germination frequency, decreased height, and increased sensitivity to pathogens
[25].
Gene overexpression is another approach that is applied to enhance a target trait. For instance, glycoside hydrolase (GH) overexpression increased the accessibility of polysaccharides
[26]. Furthermore, changes in the pectin content, and/or its modification pattern, led to an increased saccharification, and in several crops the overexpression of plant pectinases led to an increased release of simple sugars
[27].
However, overexpressing or mutating just a single gene to decrease the lignin content does not necessarily promote saccharification
[25]. Since these processes involve several cell wall modifications, a decreased recalcitrance can arguably be obtained as a result of an enhanced and optimized modification of the entire catabolic pathway. Therefore, a proper understanding of the metabolic pathways and the genetic mechanisms through a combination of different omics analyses can be essential for gene editing success.
To this respect, the modification of lignocellulosic biomass was carried out with bioengineering technologies
[28], such as gene silencing methods, for entire gene family members
[29], or the latest genome editing methods based on targeted gene manipulation, such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems
[30]. In metabolic engineering, this tool allowed an easier discovery and evaluation of the relevant genes and pathways and has become the first choice for the genetic improvement of many organisms, including industrially relevant ones
[30].
CRISPR-based methods were applied successfully in several woody plants to effectively alter the lignocellulosic composition in order to facilitate the extractability of its components, including sugar, and to improve the pulping quality
[31].
Gene Editing in Energy Crops
The CRISPR/Cas9 approach was tested in the woody perennial poplar by editing three 4-coumarate:CoA ligase targeted genes (
4CL1,
4CL2, and
4CL5), focusing on lignin and flavonoid biosynthesis
[32]. The results showed that mutations in the
4CL1 gene slow down the lignification process, and mutagenesis in the
4CL2 gene lead to an overall 20% decrease in lignin content, indicating that
4CL1 and
4CL2 can play a primary role in this biosynthesis pathway. In addition, a CRISPR-based application was carried out in poplar to decrease the lignin content by targeting the
PtoMYB156 transcription factor
[33].
MYB156 knock-out in poplar resulted in the deposition of lignin, xylan, and cellulose during SCW formation, showing how this gene may repress phenylpropanoid biosynthesis and how it negatively regulates SCW development
[33]. Despite the negative effects on plant growth, this provided useful directions for future research.
Regardless of the advances in plant genomics, a crucial limitation to the genetic improvement of some bioenergy crops is still the complexity of their genomes, which slows down the use of modern breeding approaches.
O. sativa has a compact diploid genome
[34] of approximately 500 Mb and several gene editing investigations have been carried out on this crop. Here, researchers report one of the most significant studies
[35], in which the
C3H transcription factor knockdown mutant led to an altered lignin composition that resulted in enriched p-hydroxyphenyl components, with a strong reduction in cell wall cross-linking ferulates. Such structural alterations led to an important discovery: the reduction in cell wall recalcitrance and enhanced biomass saccharification
[35].
In
Panicum virgatum, its allotetraploid genome (2n = 4x = 36) represented an impediment to generate homozygous knock-out plants. However, in one study
[36], the development of genome-editing technologies made it possible to successfully apply the CRISPR/Cas9 method. This technique was used to mutate a key gene involved in the lignin biosynthesis, the
Pv4CL1 gene, which was selected as the gene target because of its preferential expression in highly lignified stem tissues. The results showed less lignin and significantly higher glucose and xylose content in the knock-out plants compared to the wild type.
Recently, pioneering efforts have been made to genetically modify
Arundo donax L.
[37], an energy crop that is able to grow under resilient conditions that is characterized by a complex genome. Since this crop is polyploid, it is very difficult to induce and select trait promising mutations. No transgenic
A. donax crops with improved biomass characteristics have been developed yet. However, by investigating the lignin biosynthetic pathway of
A. donax, a high copy number of
PAL and
C4H genes were found giving target genes for
A. donax biomass quality improvement
[38].
Increasing cellulose biosynthesis is another important aim in biomass improvement because cellulose entirely consists of C6 sugar glucose, which is useful for saccharification. Therefore, the overexpression of cellulose synthase genes (
CESAs) is often used to obtain transgenic plants that are enriched in cellulose
[31]. However, attempts to overexpress
CESAs in secondary cell walls of aspen and barley have resulted in decreased cellulose content and reduced plant growth
[39].
Recently, the cellulose biosynthesis
CESA gene family was manipulated to increase the cellulose production in poplar. Transgenic plants were obtained by overexpressing the
PmCesA2 gene from
Pinus massoniana through an
Agrobacterium-mediated transformation
[40]. The transgenic poplar showed an enhanced growth performance and an improved cellulose production, but also an increase in lignin content, due to changes in the cell wall polysaccharide composition.
Other studies have focused on the overexpression of genes belonging to the sucrose synthase (
SUS) gene family, observing a general increased plant growth and cellulose and starch content
[41]. For instance, in hybrid poplar (
Populus alba ×
grandidentata), a small increase in cellulose was found, as well as an increase in cellulose crystallinity, which contributes to increase biomass recalcitrance
[42]; however, in tobacco, such findings led to ~20% thicker cell walls, 18% more cellulose, and 9–11% less cellulose crystallinity
[43].
In 2020, the
COBRA-like gene, which is important for cellulose biosynthesis, has been proposed as a possible target for creating transgenic plants that are rich in cellulose
[31]. The
GhCOBL9A, a
COBRA-like gene from cotton (
Gossypium hirsutum) that is overexpressed in
Arabidopsis, led to a notable increase in the total biomass and cellulose content (59%). Furthermore, the
CESA gene expression of the transgenic plants measured in the SCW showed a significant increase, suggesting the involvement of a
COBRA-like gene in the
CESA pathway of the transgenic Arabidopsis
[44]. The cell walls of cotton fibers almost entirely consist of cellulose and are an interesting model for high-level cellulose production. The approach that was adopted in this study could be a great strategy to increase the cellulose content in bioenergy crops.
Furthermore, a gene that is not directly involved in cellulose biosynthesis has been demonstrated to influence its content. Particularly, the overexpression of the rice
OsMYB103L gene, encoding the
R2R3-MYB transcription factor and controlling leaf development, caused a rise in the expression of
CESA genes and an increase in cellulose content
[45]. Conversely, knocking down this gene led to a lower expression of
CESA genes.
Several investigations have focused on the reduction in C5 sugars, such as xylose, which form linkages with cell wall hemicelluloses
[46]. Mutants in xylan biosynthesis have been generated, however, the complexity of the genome of several bioenergy crops has hindered gene editing studies. In Chen et al.
[47], the inactivation of the rice
OsIRX10 led to a decrease in xylan content in the cell walls and an improved biomass saccharification. Furthermore, the simultaneous knockdown expression of two glycosyltransferase genes (
GAUT),
PtGAUT12.1 and
PtGAUT12.2, in
P. deltoides has been shown to reduce the xylan content during wood formation and reduce the recalcitrance of cell walls
[48].
Among the genome editing investigations looking at hemicellulose, researchers focus here on two particularly promising studies with regards to the improvement of biomass yield and quality. In the first study, the silencing of
GH10 genes, which are known to control the hemicellulose degradation and are highly expressed during secondary wall deposition, led to alterations in the regulation of stress-responsive genes, releasing tensional stresses
[49]. These changes could enhance primary growth and consequently result in an improved biomass yield. In the second study, endoglucanases genes from poplar (
PtGH9B and
PtGH9C) were expressed in
Arabidopsis. The transgenic lines showed changes in the sugar content and differences in cell wall crystallinity compared to the wild type, suggesting that these endoglucanases impact secondary cell wall development by contributing to the cell wall crystallization process
[50].