**3. Genetic strategies to modify plant biomass properties**

The potential for improvement of plant biomass production has not yet been extensively explored because traditional breeding of crop plants (e.g., maize, rice, and soybean) has been focused on selection of the high grain-yield traits. Thus, to convert biomass effectively into fuels, fine chemicals, and commodity materials, a range of approaches using genetic strategies have been explored. This can be addressed from at least two points of view: modifying biomass properties to reduce processing costs or increasing biomass yield and reducing agricultural inputs. An example of these possibilities is given in Figure 2.

**Figure 2.** Possible approaches to enhance productivity of a crop biomass. Diagram indicating the two main routes for enhancing plant biomass yield and quality-related traits via genetic strategies. The first objectives to increase the bio‐ mass yield per land area (*i.e.* the biomass yield and its stability). The second objectives to modify biomass characteris‐ tics and composition to generate conversion process-friendly products for fuels and biochemicals.

### **3.1. Genetic strategies to modify biomass quality-related traits**

The quality-related traits herein considered refer to sugar- and starch-based and lignocellulosic feed-stocks. In addition, we will also consider in this section plant oils. Bio-based polymers can be generated, besides from polysaccharides and lignin of biomass crops, also from lipids, oils, and fatty acids (FAs) synthesized in oil crops (e.g., oil palm, canola, soybean).

#### *3.1.1. Sugar- and starch-based feedstocks*

The prevalence of metabolic fluxes inside plant cells is focalized on the production and usage of sugars, the primary products of the photosynthetic process, and their conversion into storage and structural carbohydrates, namely starch and cellulose.

Starch is the dominant constituent of many harvestable organs (e.g., tubers or grain) and is the second-largest form of biomass produced by vascular plants. It is a versatile and useful biopolymer not only because it is a cheap, natural material, but because of the ease with which its physicochemical properties can be altered through chemical or enzyme modifications and/ or physical treatments. Starch can be efficiently hydrolyzed by α-amylases that break starch mainly into oligosugars and glucoamylases that yield glucose monomers in biotechnological production processes [7].

#### *3.1.1.1. Biosynthesis and genetics of starch*

In recent years, our understanding on the nature and starch accumulation has largely in‐ creased, resulting in a vast body of published literature. The reader is referred to several reviews on these topics for detailed information [e.g., 8, 9]. The main findings emerging from these studies indicate that in chloroplasts, starch is accumulated for only short periods of time and thus is named 'transitory starch'; whereas in amyloplasts, starch is accumulated for longterm storage and therefore termed 'reserve starch'. In storage tissues starch is deposited as insoluble, semi-crystalline granules but also occurs to a lesser extent in most vegetative tissues of plants. It is composed of two distinct fractions: amylopectin (highly branched, 75–80% of starch dried mass) and amylose (mostly linear, 20–25% of dried mass). Both are made of α-1,4 glucosidic bond glucose residues branched via α-1,6 glucosidic linkages. The clustered nature of the α-1,6 branch points allows glucan side-chains to form double-helical structures, compacting large amounts of glucose. Consequently, extremely large structures can be synthesized and packaged in an insoluble state.

Advances in genetics and biochemistry have led to significant discoveries in how starch is synthesized in plants [10]. Three enzymes in this pathway, localized in the plastids, are playing a cardinal role in the synthesis of amylose and amylopectin: ADP-glucose pyrophosphorylase (AGPase, involved in the initiation of starch biosynthesis), starch synthase (SS, involved in elongation and granule formation), and branching enzyme (BE, involved in branching and granule formation). Further studies across plant species have indicated that these enzymes carrying out starch synthesis are encoded by well-conserved families of genes. Moreover, a number of mutations that cause defects in various steps in the pathway of starch biosynthesis were described and used to clone genes involved in this biosynthesis. Table 1 gives a list of the enzymes and genetic loci of cloned genes involved in the pathway of starch synthesis in maize endosperm. Furthermore, these mutants have provided information to achieve modi‐ fied natural starches by reshaping endogenous processes by using, for instance, antisense RNA technology, ectopic expression or mutant enzymes, or by introducing or modifying enzymes or molecules that are implicated indirectly in starch synthesis [12].

**3.1. Genetic strategies to modify biomass quality-related traits**

storage and structural carbohydrates, namely starch and cellulose.

*3.1.1. Sugar- and starch-based feedstocks*

102 Plants for the Future

production processes [7].

*3.1.1.1. Biosynthesis and genetics of starch*

synthesized and packaged in an insoluble state.

The quality-related traits herein considered refer to sugar- and starch-based and lignocellulosic feed-stocks. In addition, we will also consider in this section plant oils. Bio-based polymers can be generated, besides from polysaccharides and lignin of biomass crops, also from lipids,

The prevalence of metabolic fluxes inside plant cells is focalized on the production and usage of sugars, the primary products of the photosynthetic process, and their conversion into

Starch is the dominant constituent of many harvestable organs (e.g., tubers or grain) and is the second-largest form of biomass produced by vascular plants. It is a versatile and useful biopolymer not only because it is a cheap, natural material, but because of the ease with which its physicochemical properties can be altered through chemical or enzyme modifications and/ or physical treatments. Starch can be efficiently hydrolyzed by α-amylases that break starch mainly into oligosugars and glucoamylases that yield glucose monomers in biotechnological

In recent years, our understanding on the nature and starch accumulation has largely in‐ creased, resulting in a vast body of published literature. The reader is referred to several reviews on these topics for detailed information [e.g., 8, 9]. The main findings emerging from these studies indicate that in chloroplasts, starch is accumulated for only short periods of time and thus is named 'transitory starch'; whereas in amyloplasts, starch is accumulated for longterm storage and therefore termed 'reserve starch'. In storage tissues starch is deposited as insoluble, semi-crystalline granules but also occurs to a lesser extent in most vegetative tissues of plants. It is composed of two distinct fractions: amylopectin (highly branched, 75–80% of starch dried mass) and amylose (mostly linear, 20–25% of dried mass). Both are made of α-1,4 glucosidic bond glucose residues branched via α-1,6 glucosidic linkages. The clustered nature of the α-1,6 branch points allows glucan side-chains to form double-helical structures, compacting large amounts of glucose. Consequently, extremely large structures can be

Advances in genetics and biochemistry have led to significant discoveries in how starch is synthesized in plants [10]. Three enzymes in this pathway, localized in the plastids, are playing a cardinal role in the synthesis of amylose and amylopectin: ADP-glucose pyrophosphorylase (AGPase, involved in the initiation of starch biosynthesis), starch synthase (SS, involved in elongation and granule formation), and branching enzyme (BE, involved in branching and granule formation). Further studies across plant species have indicated that these enzymes carrying out starch synthesis are encoded by well-conserved families of genes. Moreover, a number of mutations that cause defects in various steps in the pathway of starch biosynthesis were described and used to clone genes involved in this biosynthesis. Table 1 gives a list of

oils, and fatty acids (FAs) synthesized in oil crops (e.g., oil palm, canola, soybean).


a) ae = Amylose extender; bt1 = brittle1; bt2 = brittle2: du1 = dull1; sbe1= starch branching enzyme1; sh1= shrunken1; sh2= shrunken2; su1= sugary1; su2= sugary; wx = waxy. pu1= pullulanase1.

b) Changes relative to normal. ↑↓= increase or decrease, respectively. Sugars = the alcohol-soluble sugars.

**Table 1.** Summary of mutant effects in specific maize endosperm mutants where an associated enzyme lesion has been reported. [Modified from Motto et al., 2011, [11].

Notably, starch biosynthesis is a remarkable regulated process, both at transcriptional and post-transcriptional level; it is also interconnected with an ample variety of cellular processes and metabolic pathways [10]. Its regulation involves a complex and an as yet not well clear assemblage of factors that are adjusted to the physiological status of the cell. For example, marked regulatory properties were found for enzymes involved in starch biosynthesis, especially for AGPases, which is subject to multilevel regulation. AGPases are heterotetramers that contains two large (51 kD) and two slightly smaller (50 kD) subunits that are both required for optimal enzyme activity but have nonequivalent roles in enzyme function; the large subunit plays more of a regulatory role, while the small subunit has both catalytic and regulatory properties.

Evidence from comprehensive expression profiling in various plant species has revealed a pathway-wide regulation of the expression of genes affecting sucrose-starch interactions [13]. Furthermore, the coordinated regulation of gene expression in source and sink tissues appears, to a large extent, orchestrated by the sugar status of the cell. Although the sensing and signaling mechanisms mediating these processes are largely unknown, studies have suggested the presence of members of the WRKY(term derived from the most prominent feature of these proteins the WRKY domain), or WRKY domain) and AP2/EREBP (APETALA 2/ethylene response element binding protein) families of transcription factors (TFs) [14,15] and of an ethylene receptor participating in source-sink communication and sucrose-mediated regula‐ tion of starch synthesis [16]. Additional research has shown that starch biosynthesis may also be regulated by post-translational protein modification, including allosteric regulation by metabolites, redox regulation, protein–protein interactions and reversible protein phosphor‐ ylation [17]. It is suggested that some of the newly discovered aspects of fine control of the starch biosynthetic pathway may apply to many other proteins that are directly and indirectly involved in polymer synthesis and degradation. Thus, to achieve a significant progress in the rate of starch synthesis, it would be important to increase the expression of a set of enzymes and transporters in the starch pathway.

#### *3.1.1.2. Modification of starch-specific properties*

Altering the quality of the starch by plant breeding and molecular biology has already been achieved via the commercial exploitation of some starch mutants that include types that cook to form clear colloidal solutions rather than opaque gels (e.g., waxy maize or wheat) or others that are useful industrially (e.g.*,* amylose extender maize), and finally others that accumulate less starch and more sugar (e.g., sweet maize, sweet potato). However, the industrial applica‐ tions of starch to formulate commercial products are yet limited due to poor reactivity of glucose, which is the elementary unit of starch. The addition, during starch biosynthesis, of glucose residues possessing reactive side-chains or charged groups would expand the number of commercially usable chemical alterations and, consequently, enlarge the future uses of starch industrial applications [12]. Thereby, the need for starch possessing specific properties by diverse industries is fueling starch biotechnology research [18]. Figure 3 gives examples of possible genetic and biochemical modifications directed for improving starch characteristics more adapted to industrial end uses.

Another strategy to improve the efficiency of starch as a feed-stock is to reduce the energy requirements for the starch in the biorefining conversion process of plant biomass to chemicals Genetic Strategies to Enhance Plant Biomass Yield and Quality-Related Traits for Bio-Renewable Fuel and... http://dx.doi.org/10.5772/61005 105

↑: an enhancement in the level of an enzyme;

↓: a decrease in the level of an enzyme;

+: addition of reactive groups.

and metabolic pathways [10]. Its regulation involves a complex and an as yet not well clear assemblage of factors that are adjusted to the physiological status of the cell. For example, marked regulatory properties were found for enzymes involved in starch biosynthesis, especially for AGPases, which is subject to multilevel regulation. AGPases are heterotetramers that contains two large (51 kD) and two slightly smaller (50 kD) subunits that are both required for optimal enzyme activity but have nonequivalent roles in enzyme function; the large subunit plays more of a regulatory role, while the small subunit has both catalytic and regulatory

Evidence from comprehensive expression profiling in various plant species has revealed a pathway-wide regulation of the expression of genes affecting sucrose-starch interactions [13]. Furthermore, the coordinated regulation of gene expression in source and sink tissues appears, to a large extent, orchestrated by the sugar status of the cell. Although the sensing and signaling mechanisms mediating these processes are largely unknown, studies have suggested the presence of members of the WRKY(term derived from the most prominent feature of these proteins the WRKY domain), or WRKY domain) and AP2/EREBP (APETALA 2/ethylene response element binding protein) families of transcription factors (TFs) [14,15] and of an ethylene receptor participating in source-sink communication and sucrose-mediated regula‐ tion of starch synthesis [16]. Additional research has shown that starch biosynthesis may also be regulated by post-translational protein modification, including allosteric regulation by metabolites, redox regulation, protein–protein interactions and reversible protein phosphor‐ ylation [17]. It is suggested that some of the newly discovered aspects of fine control of the starch biosynthetic pathway may apply to many other proteins that are directly and indirectly involved in polymer synthesis and degradation. Thus, to achieve a significant progress in the rate of starch synthesis, it would be important to increase the expression of a set of enzymes

Altering the quality of the starch by plant breeding and molecular biology has already been achieved via the commercial exploitation of some starch mutants that include types that cook to form clear colloidal solutions rather than opaque gels (e.g., waxy maize or wheat) or others that are useful industrially (e.g.*,* amylose extender maize), and finally others that accumulate less starch and more sugar (e.g., sweet maize, sweet potato). However, the industrial applica‐ tions of starch to formulate commercial products are yet limited due to poor reactivity of glucose, which is the elementary unit of starch. The addition, during starch biosynthesis, of glucose residues possessing reactive side-chains or charged groups would expand the number of commercially usable chemical alterations and, consequently, enlarge the future uses of starch industrial applications [12]. Thereby, the need for starch possessing specific properties by diverse industries is fueling starch biotechnology research [18]. Figure 3 gives examples of possible genetic and biochemical modifications directed for improving starch characteristics

Another strategy to improve the efficiency of starch as a feed-stock is to reduce the energy requirements for the starch in the biorefining conversion process of plant biomass to chemicals

properties.

104 Plants for the Future

and transporters in the starch pathway.

more adapted to industrial end uses.

*3.1.1.2. Modification of starch-specific properties*

DBE: debranching enzyme; GBSSI: granular-bound starch synthase I; SBE: starch branching enzyme; SBE A: class A SBE; SS: starch synthase.

**Figure 3.** Alteration of starch and their enduses. The makeup, modification and levels of starch can be modified through genetic and biochemical strategies. The resulting variations may modify the characteristics and applications of starch (Reproduced with permission from Slattery et al., 2000, [12]).

including *in planta* production of enzymes useful for starch degradation. For instance, gelatinization is the first passage for bioethanol production from starch. It is reliable that a modified starch possessing a lower gelatinization temperature might need a minor supply of energy in the biorefining conversion process. Rice research has highlighted that the expression of a recombinant amylo-pullulanase-formed starch that when heated to 85°C was perfectly transformed into soluble sugars [19].

In addition to altering starch quality, it is also possible to modify starch quantity via biotech‐ nological approaches by increasing starch content, and thus yield, in storage organs of crops [10]. These strategies include enhancement of AGPase activity, extending the supply of starch precursors to the amyloplast, increasing the supply of sucrose to heterotrophic cells, expanding sucrose breakdown within the heterotrophic cell, enhancing the expression of starch synthase class IV, blocking starch breakdown, altering the expression of global regulators, enhancing trehalose-6-phosphate content, blocking the activity of ADPG breakdown enzymes, and increasing starch content in heterotrophic organs.

Altering contents of molecules or enzymes that are not directly affecting starch biosynthetic processes may also be a useful strategy to change positively starch quality and quantity. Production of starches possessing novel properties might permit to maintain natural starch properties that could be damaged by post-harvest operations. Surely, the generation of these starches might abolish the necessity for post-harvest changes [9].

Additional investigations are required to unveil how the different levels of regulation (e.g., transcriptional, allosteric, and post-translational) interact to control the subtle structure of starch and starch granules. Only when this level of knowledge will be achieved, the complete capacity for the comprehensive arrangement of starch molecules with specific functionality will be practicable.

#### *3.1.2. Lignocellulose feed-stocks*

Lignocellulosic biomass, derived from crops and agricultural residues, is a promising renew‐ able source for the production of fuels and bio-based materials. It is estimated that there is an annual worldwide production of 10–50 billion tons of dry lignocellulose, accounting for about half of the global biomass yield [20]. Thus global availability and unsuitability for human nutrition have promoted lignocellulosic feed-stocks into the focus for biorefinery applications, as a fundamental source of fermentable carbohydrates for biofuel productions and for a broad array of chemicals and biodegradable compounds. Nevertheless, the generation of fermentable sugars from lignocellulose is one of the major constraints for the industrialization of lignocel‐ lulose biorefining. This is attributable to the compact and rigid structure of lignocellulose commonly referred as biomass recalcitrance [21], a distinguishing feature closely related to the chemical and physical characteristics of the cell walls that is crucial for plant survival [22].

Biomass crops, either woody species (e.g., pine, poplar, eucalyptus) or grasses (e.g., sugarcane, sorghum, miscanthus, maize stover) consist primarily of cell walls. These are formed by intricate assemblages of celluloses, hemicelluloses, pectins, lignins, and proteoglycans [23]. A diagrammatic illustration of the framework of lignocellulose structure forming cell walls can be seen in Figure 4.

#### *3.1.2.1. Cell wall composition and architecture*

The main component of cell walls is cellulose, which makes up 15–30% of the dry biomass of primary cell walls and up to 40% of the secondary cell walls. Cellulose is a β(1- 4)-linked chain of glucose molecules, with a degree of polymerization varying in length between 8000–15,000 residues [24]. Its building blocks, qualified as elementary fibrils, which are supposed to accommodate approximately 36 β-D-glucan chains, are covered with other non-cellulosic polysaccharides to form microfibrils; these are subsequently cross-linked with hemicellulose/ pectin matrixes to generate macrofibrils that confer structural stability at the cell wall. Hemicelluloses (20–30%), the second most abundant constituent of lignocellulosic biomass, are heterogeneous polymers derived from varying combinations of both hexoses (D-glucose, D-mannose, D-galactose) and pentoses (D-xylose and L-arabinose), including heteromannans, xyloglucan, heterxylans, and mixed-linkage glucan [25]. In contrast to cellulose, the polymer Genetic Strategies to Enhance Plant Biomass Yield and Quality-Related Traits for Bio-Renewable Fuel and... http://dx.doi.org/10.5772/61005 107

Altering contents of molecules or enzymes that are not directly affecting starch biosynthetic processes may also be a useful strategy to change positively starch quality and quantity. Production of starches possessing novel properties might permit to maintain natural starch properties that could be damaged by post-harvest operations. Surely, the generation of these

Additional investigations are required to unveil how the different levels of regulation (e.g., transcriptional, allosteric, and post-translational) interact to control the subtle structure of starch and starch granules. Only when this level of knowledge will be achieved, the complete capacity for the comprehensive arrangement of starch molecules with specific functionality

Lignocellulosic biomass, derived from crops and agricultural residues, is a promising renew‐ able source for the production of fuels and bio-based materials. It is estimated that there is an annual worldwide production of 10–50 billion tons of dry lignocellulose, accounting for about half of the global biomass yield [20]. Thus global availability and unsuitability for human nutrition have promoted lignocellulosic feed-stocks into the focus for biorefinery applications, as a fundamental source of fermentable carbohydrates for biofuel productions and for a broad array of chemicals and biodegradable compounds. Nevertheless, the generation of fermentable sugars from lignocellulose is one of the major constraints for the industrialization of lignocel‐ lulose biorefining. This is attributable to the compact and rigid structure of lignocellulose commonly referred as biomass recalcitrance [21], a distinguishing feature closely related to the chemical and physical characteristics of the cell walls that is crucial for plant survival [22].

Biomass crops, either woody species (e.g., pine, poplar, eucalyptus) or grasses (e.g., sugarcane, sorghum, miscanthus, maize stover) consist primarily of cell walls. These are formed by intricate assemblages of celluloses, hemicelluloses, pectins, lignins, and proteoglycans [23]. A diagrammatic illustration of the framework of lignocellulose structure forming cell walls can

The main component of cell walls is cellulose, which makes up 15–30% of the dry biomass of primary cell walls and up to 40% of the secondary cell walls. Cellulose is a β(1- 4)-linked chain of glucose molecules, with a degree of polymerization varying in length between 8000–15,000 residues [24]. Its building blocks, qualified as elementary fibrils, which are supposed to accommodate approximately 36 β-D-glucan chains, are covered with other non-cellulosic polysaccharides to form microfibrils; these are subsequently cross-linked with hemicellulose/ pectin matrixes to generate macrofibrils that confer structural stability at the cell wall. Hemicelluloses (20–30%), the second most abundant constituent of lignocellulosic biomass, are heterogeneous polymers derived from varying combinations of both hexoses (D-glucose, D-mannose, D-galactose) and pentoses (D-xylose and L-arabinose), including heteromannans, xyloglucan, heterxylans, and mixed-linkage glucan [25]. In contrast to cellulose, the polymer

starches might abolish the necessity for post-harvest changes [9].

will be practicable.

106 Plants for the Future

be seen in Figure 4.

*3.1.2.1. Cell wall composition and architecture*

*3.1.2. Lignocellulose feed-stocks*

**Figure 4.** Structure of lignocellulose. Cellulose, hemicellulose, and lignin form structures called microfibrils, which are organized into macrofibrils that mediate structural stability in the plant cell wall. (Reproduced with permission from Rubin 2008, [23].

chains are branched and achieve comparatively short molecular lengths (500–3000 residues). The utilization of hemicellulose, in general, and its main component xylose, in particular, are at the center of research efforts in metabolic engineering to optimize lignocellulosic feed-stocks for biorefining technologies. Lignin (15–25%) is the third most-abundant biopolymer in cell walls and the largest available resource of natural aromatic polymers [26]. It is a heteropolymer mainly composed of three major phenolic monomers, namely p-coumaryl alcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S). Combinations of these structural units or monolignols are incorporated into lignins with species, tissue, and developmental specificity. Lignin performs an important role in strengthening cell walls by cross-linking polysaccharides, thus providing support to structural elements in the overall plant body and conferring an exceptionally resistant to biological attacks, properties that interfere, however, with enzymatic conversion of polysaccharide components.

### *3.1.2.2. Biogenesis and genes involved in cell wall assembly*

Genetic progress to improve cell wall composition and structure is a crucial objective for two motives: i) cell walls constrict cell size and shape and thus have a remarkable role in plant growth, affecting biomass production and ii) cell walls are recalcitrant to degrada‐ tion by microorganisms to liberate sugars for fermentation, consequently affecting bio‐ mass quality [27].

According to Carpita and McCann [28], plants devote approximately 10% of their genome (i.e., ≈2,500 genes) to construction and dynamic rearrangement of their cell walls during growth. Specifically, the previous authors have grouped ≈1200 cell wall-related genes that are impli‐ cated in the synthesis, assembly, and disassembly of the plant cell walls into six categories/ stages of cell wall biogenesis consisting of substrate generation, polysaccharide synthesis, membrane trafficking, assembling and turnover, secondary cell wall formation, and signaling. What emerges from this study is that the differences among angiosperms in cell wall compo‐ sitions are reflected in the structure of these gene families.

In higher plants, cellulose is synthesized by large multimeric plasma membrane-associated cellulose synthase rosettes, termed CESA [29]. The subunits are encoded by the *CESA* genes represented by multiple, usually 10 or more, members, the majority of which appear to be involved in primary wall formation as judged from mutational genetic studies and gene expression profiling. In addition to *CESA* genes, chemical and genetic screens have also identified various genes that indirectly contribute to cellulose biosynthesis, such as *COBRA* (encoding a protein anchored to the membrane through glycophosphatidylinositol, GPI), *KOBITO (*encoding a membrane associated protein of unknown function), and *KORRIGAN* (encoding a membrane-anchored β-glucanase) [29]. A member of the *COBRA* gene family, *CobL4* from Arabidopsis, and its orthologs *Brittle culm-1* (*Bc1*) from rice, and *Brittle stalk-2 (Bk2*) from maize, have been shown to specifically affect cellulose formation in secondary cell walls.

The complexity of events contributing to the activation of *CESA* at the plasma membrane and its motility suggest that the list of players in this biosynthesis is far from complete and might include accessory proteins and cell wall-sensing mechanisms that appear to affect cell wall biogenesis [29]. Furthermore, gene expression studies have revealed that CESA proteins are expressed spatially and temporally throughout plant development, indicating that specific transcription factors belonging to NAC (no apical meristem), MYB (myeloblastosis), WRKY, and leucine zipper families, play a role in cell wall biogenesis. Therefore, a better understand‐ ing of the regulation, activation, and assembly of the CESA complex as well as discovery and characterization of CESA accessory proteins and plant-specific TFs will further clarify targets for genetic manipulation.

The biosynthesis of hemicelluloses requires the coordinated expression of several glycan synthases and glycosyltransferases (GTs) for polymer backbone and side-chain formation, respectively [25]. In this context, it was found that several *CELLULOSE SYNTHASE-LIKE F* (*CSLF*) genes encoding Golgi-localized GLs, are involved in hemicellulose biosynthesis. More specifically, evidence indicates that 25 *xylem-specific GT* genes from 7 GT families support this biosynthesis [30]. Furthermore, it was clarified that several *GT* gene families (i.e., *GT43, GT47,* and *GT61*) cooperate in xylan biosynthesis. Additionally, the identification of Arabi‐ dopsis *IRX* mutants has implicated that *GT8, GT43*, and *GT47* families as potential *glucuro‐ noxylan* (*GX*) biosynthetic genes. Further research has documented that the biosynthesis of GAXs would require at least three GTs: xylosyl-transferase (XylT), arabinosyl-transferase (AraT), and glucuronosyl-transferase (GlcAT) [31]. Similarly, it was reported that in xylan synthesis glycosyl hydrolases may also play a role, as well as a number of transcription factors [32]. These include, in particular, master switches such as Secondary Cell Wall Associated NAC Domain 1 (SND1) and other TFs directly downstream, such as multiple MYB factors and a KNOTTED1-like homeodomain protein. Furthermore, in maize, *UDP-glucose 6-dehydrogenase* genes were found to encode central enzymes of hemicellulose biosynthesis and appear essential for cell wall formation in young organs. Although, the information reported above has pointed out the importance of the hemicelluloses in plant growth and development, future research is required to combine these single components and their assemble. This will permit to improve our understanding on the biosynthesis of this important class of plant cell wall components. It is expected that altering the expression of those genes will very likely change the amounts and properties of hemicellulose, which in turn, may lead to decrease recalcitrance.

*3.1.2.2. Biogenesis and genes involved in cell wall assembly*

sitions are reflected in the structure of these gene families.

mass quality [27].

108 Plants for the Future

walls.

for genetic manipulation.

Genetic progress to improve cell wall composition and structure is a crucial objective for two motives: i) cell walls constrict cell size and shape and thus have a remarkable role in plant growth, affecting biomass production and ii) cell walls are recalcitrant to degrada‐ tion by microorganisms to liberate sugars for fermentation, consequently affecting bio‐

According to Carpita and McCann [28], plants devote approximately 10% of their genome (i.e., ≈2,500 genes) to construction and dynamic rearrangement of their cell walls during growth. Specifically, the previous authors have grouped ≈1200 cell wall-related genes that are impli‐ cated in the synthesis, assembly, and disassembly of the plant cell walls into six categories/ stages of cell wall biogenesis consisting of substrate generation, polysaccharide synthesis, membrane trafficking, assembling and turnover, secondary cell wall formation, and signaling. What emerges from this study is that the differences among angiosperms in cell wall compo‐

In higher plants, cellulose is synthesized by large multimeric plasma membrane-associated cellulose synthase rosettes, termed CESA [29]. The subunits are encoded by the *CESA* genes represented by multiple, usually 10 or more, members, the majority of which appear to be involved in primary wall formation as judged from mutational genetic studies and gene expression profiling. In addition to *CESA* genes, chemical and genetic screens have also identified various genes that indirectly contribute to cellulose biosynthesis, such as *COBRA* (encoding a protein anchored to the membrane through glycophosphatidylinositol, GPI), *KOBITO (*encoding a membrane associated protein of unknown function), and *KORRIGAN* (encoding a membrane-anchored β-glucanase) [29]. A member of the *COBRA* gene family, *CobL4* from Arabidopsis, and its orthologs *Brittle culm-1* (*Bc1*) from rice, and *Brittle stalk-2 (Bk2*) from maize, have been shown to specifically affect cellulose formation in secondary cell

The complexity of events contributing to the activation of *CESA* at the plasma membrane and its motility suggest that the list of players in this biosynthesis is far from complete and might include accessory proteins and cell wall-sensing mechanisms that appear to affect cell wall biogenesis [29]. Furthermore, gene expression studies have revealed that CESA proteins are expressed spatially and temporally throughout plant development, indicating that specific transcription factors belonging to NAC (no apical meristem), MYB (myeloblastosis), WRKY, and leucine zipper families, play a role in cell wall biogenesis. Therefore, a better understand‐ ing of the regulation, activation, and assembly of the CESA complex as well as discovery and characterization of CESA accessory proteins and plant-specific TFs will further clarify targets

The biosynthesis of hemicelluloses requires the coordinated expression of several glycan synthases and glycosyltransferases (GTs) for polymer backbone and side-chain formation, respectively [25]. In this context, it was found that several *CELLULOSE SYNTHASE-LIKE F* (*CSLF*) genes encoding Golgi-localized GLs, are involved in hemicellulose biosynthesis. More specifically, evidence indicates that 25 *xylem-specific GT* genes from 7 GT families support this

Lignin is one of the most relevant objectives for genetic improvement of cellulosic biomass adapted for biofuel production: modifications in its chemical composition and quantity directly influence the pretreatment costs presently used in biofuel production starting from cellulosic biomass as feed-stock [33]. Biosynthesis of lignin involves two major processes: the monolignol pathway (via the phenylpropanoid pathway) in the cytosol and polymerization of the monomers into the cell wall [34]. The enzymes needed for monolignol biosynthesis are well described and comprise ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4- (hydroxy)cinnamoyl CoA ligase (4CL), hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT), p-coumaroylshikimate 3'-hydroxylase (C3'H), caffeoyl CoA O-methyl‐ transferase (CCoAOMT), (hydroxy)cinnamoyl CoA reductase (CCR), ferulic acid 5-hydroxy‐ lase (F5H), caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), and (hydroxy) cinnamyl alcohol dehydrogenase (CAD) [35]. Similarly, the genes involved in the synthesis from phenylalanine to hydroxycinnamates and monolignol substrates of lignin biosynthesis are well established [30]. Furthermore, it was found that many of the genes encoding key lignin biosynthetic enzymes belong to multigene families [36]. Thus, specific isoforms may be expressed in different cell types at different developmental stages or in response to changing environmental conditions, complicating attempts to alter lignin accumulation.

#### *3.1.2.3. Genetic strategies to improve lignocellulosic components*

In recent years, genetic modification of the lignin biosynthesis pathway has received great attention because of the use of model plants to dissect the biosynthetic pathway and because its content in biomass is inversely correlated with its forage digestibility and quality value in the pulping industry [33]. Moreover, these findings indicate that lignin cannot be simply removed from growing plants without causing negatives developmental effects. In several plant species (e.g., maize, switchgrass, poplar, and pine), efforts using natural mutants or silencing (RNAi) strategies directed at the down-regulation of a number of genes encoding lignin biosynthesis enzymes were not successful. This is likely due to the fact that those interventions drastically reduced lignin content in a non-selective way. Nevertheless, there are cases in which mild genetic manipulations have been used to moderately reduce lignin content or modify its composition in biomass, modestly improving saccharification efficiency, forage digestibility, and pulping yield [37].

The recent development of targeted genome-editing technologies, such as zinc-finger nucle‐ ases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered, regularly interspaced, short palindromic repeats (CRISPR) or CRISPR-associated (CAS) systems, offers exciting potential to resolve the issues of highly specific genome modifications with great efficiency and specificity [38,39]. These technologies make use of sequence-specific designer nucleases that cleave targeted loci, enabling creation of small insertions and deletions, insertion of novel DNA, or even replacement of individual alleles. A simplified model summarizing the emerging techniques for plant engineering of lignin proposed by Eudes and coworkers [33] is reported in Figure 5. According to the previous authors, this strategy will eventually offer the opportunity to design crops with optimized lignin composition and distribution while retaining all other traits related to the phenylpropanoid pathway.

**Figure 5.** Cases of novel strategies for multifaceted genetic engineering of plants. (a) Genome bioediting techniques. Black box, endogenous lignin locus (target of editing); grey arrow, fiber specific promoter used to drive the expression of the bioediting gene; red box, bioediting gene: ZFNs, TALENs or CRISPR/CAS9; red star, SNP generated when the genome bioediting gene is expressed. (b) Transgene regulation techniques. Grey arrow, fiber (pFib) or vessel (pVes) specific promoter; yellow box, gene encoding the OsL5 protein with the alternative splicing cassette shown in the same color inserted in transgenes (yellow circle); blue box, gene encoding the Cys4 protein with its cognition sequence shown in the same color inserted in transgenes (blue circle); black arrow, secondary cell wall promoter (pCWII); red box, engineered gene: gene used to manipulate lignin composition which has been engineered with transgene regula‐ tion tool (yellow circle, OsL5 alternative splicing cassette; blue circle, Cys4 cognition sequence) (Reproduced with per‐ mission from Eudes et al., 2014, [23]).

Besides traditional lignin reduction methods that directly target genes from the lignin biosynthetic pathway, novel dominant approaches are currently in development. This new trend for lignin engineering focuses on the redirection of C flux to the production of related phenolic compounds and on the replacement of monolignols with novel lignin monomers to improve biophysical and chemical properties of lignins such as recalcitrance, or industrial uses [33]. Alternatively, although lignocellulosic feedstocks might be employed for conversion to biomaterials, two principal drawbacks in the producing systems are the costs of transport and processing of biomass. A solution to this problem is to produce directly in the plant cells the microbial cellulase enzymes. This will promote directly *in planta* the conversion of fermentable sugars during the biomass transportation to bioraffineries [40]. In maize, the expression of the catalytic domain of the thermostable 1,4-b-endoglucanase of *Acidothermus cellulolyticu*s [41] corroborates the idea that plant may be used as a biofactory for cellulose-degrading enzymes. Alternatively, although lignocellulosic feed-stocks might be used for conversion to biomate‐ rials, two major limitations for this process are the costs of transport and biomass processing. A solution is to produce microbial cellulase enzymes in the plant cells to facilitate the conver‐ sion of fermentable sugars *in planta* during the biomass to biorefinery conversion process [40]. Expression of the catalytic domain of the thermostable 1,4-b-endoglucanase of *Acidothermus cellulolyticu*s in maize [41] supports the opinion that maize can be used as a biofactory for cellulose-degrading enzymes.

#### *3.1.3. Lipids*

silencing (RNAi) strategies directed at the down-regulation of a number of genes encoding lignin biosynthesis enzymes were not successful. This is likely due to the fact that those interventions drastically reduced lignin content in a non-selective way. Nevertheless, there are cases in which mild genetic manipulations have been used to moderately reduce lignin content or modify its composition in biomass, modestly improving saccharification efficiency, forage

The recent development of targeted genome-editing technologies, such as zinc-finger nucle‐ ases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered, regularly interspaced, short palindromic repeats (CRISPR) or CRISPR-associated (CAS) systems, offers exciting potential to resolve the issues of highly specific genome modifications with great efficiency and specificity [38,39]. These technologies make use of sequence-specific designer nucleases that cleave targeted loci, enabling creation of small insertions and deletions, insertion of novel DNA, or even replacement of individual alleles. A simplified model summarizing the emerging techniques for plant engineering of lignin proposed by Eudes and coworkers [33] is reported in Figure 5. According to the previous authors, this strategy will eventually offer the opportunity to design crops with optimized lignin composition and

distribution while retaining all other traits related to the phenylpropanoid pathway.

**Figure 5.** Cases of novel strategies for multifaceted genetic engineering of plants. (a) Genome bioediting techniques. Black box, endogenous lignin locus (target of editing); grey arrow, fiber specific promoter used to drive the expression of the bioediting gene; red box, bioediting gene: ZFNs, TALENs or CRISPR/CAS9; red star, SNP generated when the genome bioediting gene is expressed. (b) Transgene regulation techniques. Grey arrow, fiber (pFib) or vessel (pVes) specific promoter; yellow box, gene encoding the OsL5 protein with the alternative splicing cassette shown in the same color inserted in transgenes (yellow circle); blue box, gene encoding the Cys4 protein with its cognition sequence shown in the same color inserted in transgenes (blue circle); black arrow, secondary cell wall promoter (pCWII); red box, engineered gene: gene used to manipulate lignin composition which has been engineered with transgene regula‐ tion tool (yellow circle, OsL5 alternative splicing cassette; blue circle, Cys4 cognition sequence) (Reproduced with per‐

Besides traditional lignin reduction methods that directly target genes from the lignin biosynthetic pathway, novel dominant approaches are currently in development. This new trend for lignin engineering focuses on the redirection of C flux to the production of related

digestibility, and pulping yield [37].

110 Plants for the Future

mission from Eudes et al., 2014, [23]).

Oil from crop plants represents the bioproduct that is chemically more similar to petrol and consequently has the highest aptitude to substitute it in the chemical industry. Petrol is considered to be originated from ancient, lipid-rich organic compounds, namely spores and planktonic algae (sedimented and transformed) under high pressure and temperature, throughout millions of years [42].

#### *3.1.3.1. Biogenesis and genes involved in oil production*

Virtually, all plant seeds contain storage lipids in the form of triacylglycerol esters (TAGs) containing three FAs with chain lengths of C8–C24, with C16 and C18 (i.e., palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid) being the most commons [43]. A number of comprehensive reviews on characteristics of structure and enzymes that are involved in oil biosynthesis and deposition in seeds have been recently published. Again the reader is referred to several excellent in-depth reviews and books for detailed information [e.g., 44, 45]. Briefly, studies in this field have indicated that plant oil is synthesized from glycerol-3-phosphate and fatty acyl-CoA in the endoplasmic reticulum as TAGs, and esters of FAs acids and glycerol. Moreover, FAs are synthesized in plastid from acetyl-CoA and then transported to the cytoplasm in the form of fatty acyl-CoA. In the ER, FAs are employed for the acylation of the glycerol-3-phosphate backbone either by the Kennedy pathway or by acyl exchange between lipids. Then, the resulting TAGs are deposited in specialized structures termed oil bodies. A schematic outline of the biosynthesis of storage lipids in seeds and in vegetative tissues is depicted in Figure 6.

Additional studies have indicated that some plants can produce, besides the common five FAs above-cited, also an array of unusual or 'novel' FAs exhibiting a wide diversity of FA struc‐ tures, including various functionalities such as hydroxylation, epoxidation, acetylation, and

**Figure 6.** Schematic representation of metabolic engineering strategies for manipulation of oil content and composition in vegetative and seed tissues. Blue: target genes suitable for overexpression; Red: target genes for inactivation by mu‐ tation or RNAi constructs. Genes encoding enzymes using acyl-CoA substrates are underlined. FAS = plastid localised fatty acid synthase (Reproduced with permission from Napier et al., 2014, [46]).

conjugation that impart properties required for specific industrial uses [46, 47]. Furthermore, studies in this field have discovered a large diversity occur in the function of various FAmodifying enzymes, such as FA desaturase2 (FAD2), thioesterases, fatty-acid elongases, cytochrome P450s, acyl-CoA desaturases, acyl-ACP desaturases, sphingolipid-∆8 desaturases, and S-adenosylmethionine methyltransferases [48]. The properties of many of these enzymes showing new functionality have recently been reviewed in detail by Voelker and Kinney [49], while for gene required for lipids synthesis visit http://www.canr.msu.edu/lgc/.

#### *3.1.3.2. Genetic strategies to modify oil content and composition*

At present, the industrial value of the current seed oils is limited by their FA composition that is not well suited for the manufacture of specialty chemicals and polymers. Therefore, due to complex inheritance of oil content and composition in seeds, several metabolic engineering approaches have been employed. Although, several examples of transgenic plants with altered oil composition were obtained, none of these plants had a high level of an unusual FAs necessary for industrial application (≈ 90–95% of the total FAs) [48]. These results suggest that plants vary considerably in the ability of their background metabolic machinery to handle the newly synthesized FAs. It is probable that further research will uncover genes for specialized forms of the various acyltransferase and TAG assembly enzymes capable of efficiently handling the unusual FAs. It is suggested that co-expression of such genes along with the previously introduced FAs acid biosynthetic pathways should contribute to further increases in accumulation of novel FAs transgenic plants in the future and lead to the development of economically viable crop sources of industrial raw materials.

In addition, to search for naturally occurring enzymes that can be used for the transgenic production of industrially useful FAs, other strategies have been considered such as diverting carbon flow from starch to TAG, up-regulating FA synthesis, modifying expression of individual TAG biosynthetic enzymes, and the use of TFs [43, 46]. In this context, genetic investigations have found various seed-specific regulatory genes that may play an important role by controlling oil deposition in the seed. For instance, the *WRINKLED1 (WRI1*), an AP2/ EREBP transcription factor initially described in Arabidopsis, plays a regulatory function [50]. These studies have also identified several motifs that are important for WRI1 binding and transactivation. This is further highlighted by the study of Shen and co-workers [51] in which the maize ortholog of *LEAFY COTYLEDON1 (LEC1*) (another TF involved in the regulation of oil accumulation, upstream of *WRI1*) was expressed in transgenic maize under the control of an oleosin promoter. The resulting *ZmLEC1* overexpression lines of maize manifested out‐ standing levels of seed oil (up to 48% greater than null segregants), mainly due to extended storage lipids in the embryo. Although this high-oil phenotype was stable over various generations, it was also unfortunately associated with detrimental agronomic traits, such as reduced seed germination and plant growth, suggesting unacceptable and unpredictable pleiotropic effects caused from impaired seed-specific expression of *ZmLEC1*. To overcome these negative effects, the previous workers performed analogous experiments by overex‐ pressing ZmWRI1. This research resulted in a high oil phenotype (≈ 31% higher than nulls) without the negative side effect on germination and growth. Notably, the strength of the promoter adopted to drive their expression of *ZmWRI*1 gave a favorable impact on the enhancement in seed oil deposition. Alternatively, the use of a weaker promoter produced only an accumulation in the seed approaching 17%.

conjugation that impart properties required for specific industrial uses [46, 47]. Furthermore, studies in this field have discovered a large diversity occur in the function of various FAmodifying enzymes, such as FA desaturase2 (FAD2), thioesterases, fatty-acid elongases, cytochrome P450s, acyl-CoA desaturases, acyl-ACP desaturases, sphingolipid-∆8 desaturases, and S-adenosylmethionine methyltransferases [48]. The properties of many of these enzymes showing new functionality have recently been reviewed in detail by Voelker and Kinney [49],

**Figure 6.** Schematic representation of metabolic engineering strategies for manipulation of oil content and composition in vegetative and seed tissues. Blue: target genes suitable for overexpression; Red: target genes for inactivation by mu‐ tation or RNAi constructs. Genes encoding enzymes using acyl-CoA substrates are underlined. FAS = plastid localised

At present, the industrial value of the current seed oils is limited by their FA composition that is not well suited for the manufacture of specialty chemicals and polymers. Therefore, due to complex inheritance of oil content and composition in seeds, several metabolic engineering approaches have been employed. Although, several examples of transgenic plants with altered oil composition were obtained, none of these plants had a high level of an unusual FAs necessary for industrial application (≈ 90–95% of the total FAs) [48]. These results suggest that plants vary considerably in the ability of their background metabolic machinery to handle the newly synthesized FAs. It is probable that further research will uncover genes for specialized

while for gene required for lipids synthesis visit http://www.canr.msu.edu/lgc/.

*3.1.3.2. Genetic strategies to modify oil content and composition*

fatty acid synthase (Reproduced with permission from Napier et al., 2014, [46]).

112 Plants for the Future

The possibility of producing TAGs in leaves and other vegetative tissues has recently attracted considerable attention [46]. A schematic representation of metabolic engineering strategies for manipulation of oil content and composition in vegetative and seed tissues is given in Figure 6. A number of reports have documented that TAG accumulation can be increased by ectopic expression of individual biosynthetic enzymes, TFs that control seed development and maturation, or by mutating genes involved in TAGs and FAs turnover [46]. However, in the majority of these investigations enhancement of TAGs concentration in leaf was low and/or dependent on carbohydrates supply. Because key enzymes for both oil synthesis and break‐ down are expressed in vegetative tissues, it was suggested that attaining significant amounts of storage lipid in the leaf will be essential in the re-orientation of C flux into TAGs [46]. Nevertheless, a remarkable increase in TAG levels (exceeding 15% of dry weight in vegetative tissue) has only been realized by integrated metabolic strategies directed to improve FAs and TAGs synthesis while inhibiting lipolysis. Additionally, the detection of non-seed proteins affecting the binding and stabilization of lipid-rich molecules in the cytosol of plant cells has highlighted a new angle of the cellular machinery influencing TAGs packaging in plant vegetative tissue. It will be attractive to clarify if oil accumulation in green biomass can be further improved without severely impairing photosynthesis and plant development. A possibility for achieving this target is the use of senescence-induced promoters to engineer plants in which TAG accumulations will be initiated only after leaves have reached their maximum size [52]. Another strategy that might be devised is to directly connect C fixation to FAs biosynthesis by introducing a functional glycolytic pathway that is efficient to transform 3-phosphoglycerate to phosphenolpyruvate. Independently from the strategy that will be employed, the challenge of using photosynthetic cells to accumulate very high amounts of oil is an attractive objective. However, reaching levels of oil accumulation exceeding those currently found in seed oil crops, namely*,* superior to 35% of TAGs (% dw), remains a noteworthy metabolic engineering goal.

#### **3.2. Genetic strategies to improve biomass yield**

Plant breeding is driven by the need to continually increase sustainable yield and quality of crop plants and by meeting projected increases in global food demand. Targeted genetic improvement in yield for developing new varieties suitable as biorefinery feed-stocks will depend on identifying genetic variation in critical morphological, structural, and physiological traits affecting biomass production. This involves manipulating complex traits, such as those associated with plant growth and development. Biomass yield can also be enhanced by manipulation of additional pathways such abiotic and biotic stress. These topics are outside the scope of this chapter and have been reviewed elsewhere [e.g. 53, 54].

#### *3.2.1. Yield genes*

To accomplish maximal biomass yield in the development of new biomass crops, it is relevant to: i) identify genes and genetic pathways that are crucial to biomass production, ii) recognize the selective forces that have molded the frequencies of these genes in current varieties, and iii) establish which morphological and physiological traits may eventually lead to more efficient plants in yield performance. Although there is genetic variability in yield traits, many of them (yield, yield stability, nutrient, and water use) that are important in crop productivity are multigenic traits and are often difficult to breed for. Quantitative trait loci (QTLs) mapping approaches are common genomic tools to dissect the genetic architecture underlying complex traits and to identify QTLs [55]. Furthermore, the development of high-throughput sequencing and genotyping technologies has greatly improved the accuracy of QTL analysis. In this respect, biomass QTL mapping has been conducted in several crops with the purpose of identifying genomic regions and genetic loci underlying biomass feedstock yield (e.g., poplar, maize, switchgrass, perennial ryegrass) or sugar yield in sugarcane [56]. These studies carried out in different species and population types have manifested the prevalence of additive main effects, digenic epistasis, QTL x environment interactions, multiple minor effects, and QTL distributed over several genomic regions; moreover it was shown that both parents were contributing favorable and unfavorable alleles irrespective of their biomass yield potential.

### *3.2.2. Molecular biology approaches for increasing biomass yield*

Although genetic dissection of yield components, such as those affecting biomass productions, can help to elucidate the physiological route from gene to phenotype, current progresses in our knowledge of how plants function and develop can expand potential and efficiency of plant breeding programs devoted to yield improvement. Insights into gene and genome sequences, the regulation of gene expression and the molecular and cellular mechanisms and pathways underlying plant architecture, development, and function, may offer new options to plant geneticists to comprehensively devise novel breeding programs.

These strategies include molecular approaches to increasing biomass yield and transgenic research directed toward increasing biomass yield through genetic modification of different plant traits.

### *3.2.2.1. Photosynthesis*

highlighted a new angle of the cellular machinery influencing TAGs packaging in plant vegetative tissue. It will be attractive to clarify if oil accumulation in green biomass can be further improved without severely impairing photosynthesis and plant development. A possibility for achieving this target is the use of senescence-induced promoters to engineer plants in which TAG accumulations will be initiated only after leaves have reached their maximum size [52]. Another strategy that might be devised is to directly connect C fixation to FAs biosynthesis by introducing a functional glycolytic pathway that is efficient to transform 3-phosphoglycerate to phosphenolpyruvate. Independently from the strategy that will be employed, the challenge of using photosynthetic cells to accumulate very high amounts of oil is an attractive objective. However, reaching levels of oil accumulation exceeding those currently found in seed oil crops, namely*,* superior to 35% of TAGs (% dw), remains a

Plant breeding is driven by the need to continually increase sustainable yield and quality of crop plants and by meeting projected increases in global food demand. Targeted genetic improvement in yield for developing new varieties suitable as biorefinery feed-stocks will depend on identifying genetic variation in critical morphological, structural, and physiological traits affecting biomass production. This involves manipulating complex traits, such as those associated with plant growth and development. Biomass yield can also be enhanced by manipulation of additional pathways such abiotic and biotic stress. These topics are outside

To accomplish maximal biomass yield in the development of new biomass crops, it is relevant to: i) identify genes and genetic pathways that are crucial to biomass production, ii) recognize the selective forces that have molded the frequencies of these genes in current varieties, and iii) establish which morphological and physiological traits may eventually lead to more efficient plants in yield performance. Although there is genetic variability in yield traits, many of them (yield, yield stability, nutrient, and water use) that are important in crop productivity are multigenic traits and are often difficult to breed for. Quantitative trait loci (QTLs) mapping approaches are common genomic tools to dissect the genetic architecture underlying complex traits and to identify QTLs [55]. Furthermore, the development of high-throughput sequencing and genotyping technologies has greatly improved the accuracy of QTL analysis. In this respect, biomass QTL mapping has been conducted in several crops with the purpose of identifying genomic regions and genetic loci underlying biomass feedstock yield (e.g., poplar, maize, switchgrass, perennial ryegrass) or sugar yield in sugarcane [56]. These studies carried out in different species and population types have manifested the prevalence of additive main effects, digenic epistasis, QTL x environment interactions, multiple minor effects, and QTL distributed over several genomic regions; moreover it was shown that both parents were contributing favorable and unfavorable alleles irrespective of their biomass yield potential.

the scope of this chapter and have been reviewed elsewhere [e.g. 53, 54].

noteworthy metabolic engineering goal.

*3.2.1. Yield genes*

114 Plants for the Future

**3.2. Genetic strategies to improve biomass yield**

Photosynthesis provides the primary energy and C input for plant growth. Improving photosynthesis has been identified as a key strategy for the production of crop plants with higher biomass yield [57]. Molecular targets were identified by the study of bottlenecks of photosynthesis) and approaches to overcome these bottlenecks were mostly based on the upregulation or down-regulation of single genes [58]. In some instances, synthetic pathways were used to overcome limitations of the endogenous pathways. A list of potential targets to improve photosynthesis is given in Table 2.


**Table 2.** Potential targets for improving plant photosynthesis.

<sup>1.</sup> Improving Rubisco function

i. Improving Rubisco catalytic activity

ii. Altering Rubisco amount per leaf area

<sup>2.</sup> Increasing thermostability of Rubisco activase to sustain Rubisco activity at high temperature

<sup>3.</sup> Enhancing CO2 concentration around Rubisco to maximize catalytic rate and minimize photorespiration

i. Turning C3 plants into C4 plants

An obvious target for increasing the source strength is the production of photoassimilates during photosynthesis. In this respect, research has been undertaken to introduce a more efficient, C4-like photosynthesis in C3 plants [58]. These last workers indicated that the introduction of single C4 enzymes (i.e., phosphoenol pyruvate carboxylase, PEPC, and pyruvate orthophosphate dikinase, PPDK) in C3 plants has until now not generated improve‐ ment in photoassimilate accumulation. This is probably attributable to disturbances in the fluxes of C4 intermediates for metabolic pathways excluding the C4 cycle. Notably in rice, the joined expression of two C4 cycle enzymes was found to raise photosynthetic ability up to 35% and grain yield up to 22%. In this research, the maize genes were transferred to the rice genome together with their corresponding promoters, which might have turned out in a superior spatial and temporal expression of the C4 cycle enzymes than the more expected expression of transgenes by using constitutive promoters. Additionally, work in transgenic tobacco has also showed that increased levels of fructose-1, 6-bisphosphatase (FBPase) and sedoheptu‐ lose-1,7-bisphosphatase (SBPase), two Calvin cycle enzymes, significantly increased dry weight [59].

Besides the above strategies based on C4 photosynthesis, other approaches have been taken to improve the efficiency of photosynthetic C assimilation. One of these strategies is focused on the enzyme RUBISCO ACTIVASE, a key regulator of RUBISCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase essential component of the photosynthetic process of fixing CO2 into organic C) activity [60]. Transgenic Arabidopsis plants expressing a heat-tolerant version of RUBISCO ACTIVASE showed a significant improvement in photosynthesis and leaf growth when exposed to heat. Other efforts to improve the photosynthetic efficiency of plants have been focused on: i) increasing electron flow by overexpressed CYTOCHROME C6 (CYTC6), a protein involved in the photosynthetic electron transport chain [61]; ii) engineering new pathways into the chloroplast that bypass photorespiration and release CO2 directly into the chloroplast stroma [62] ; and iii) using various 'add-ons' or CO2-concentrating mechanisms (CCMs) to elevate CO2 levels in the vicinity of RUBISCO. These CCMs were pursued by improving mesophyll CO2 conductance via overexpressing aquaporin [63] or stomatal CO2 conductance via manipulating stomatal characteristics [64]. In this respect, more recently Lin and co-workers [65] have successfully engineered tobacco plants containing a functioning RUBISCO from a cyanobacterium. The cyanobacterial enzyme has a greater catalytic rate than any 'C3' plant. The lines generated in this research open the way for future addition of the remaining components of the cyanobacterial CCM, an important step towards enhancing photosynthetic efficiency and improving crop yields. Furthermore, it has been shown that the triose-phosphate/phosphate translocator (TPT) strongly limits photosynthesis under high CO2 conditions [65]. The TPT provides a regulatory link between CO2 assimilation and cytosolic C metabolism. In this context an approach for increasing plant yield was performed by overexpressing sucrose transporters in sink cells, thereby enhancing sink demand and inducing an increase in photosynthesis and assimilates export. When overexpresssing a potato sucrose symporter (*StSUT1*) in storage parenchyma cells of pea seeds, there was enhanced sucrose influx into cotyledons and greater cotyledon growth rates [67]. In addition, it has been shown that enhancement of sucrose synthase activity represents a useful strategy for increas‐ ing starch accumulation and yield in potato tubers. In a future world of higher CO2 concen‐ tration, enhancing the capacity for sucrose export and carbon utilization would be an important component for maximizing photosynthesis and yield. While altering transport capacity alone is unlikely to change photosynthetic capacity, enhancing photosynthetic capacity as well as transport capacity could lead to improved plant growth and yield.

Technology for nucleus or chloroplast genome transformation has been advancing and it would enable easier and more precise manipulation of the photosynthesis process. It is expected that such plants could exhibit more efficient photosynthesis under controlled conditions; the plant factory in which plants are produced in an optimized growth environ‐ ment would have potential advantages of high productivity. In the future, the combined uses of several strategies would greatly help to improve photosynthetic capacity and thus plant growth and ultimately yield.
