The ability of some endophytes to colonize the xylem provides the opportunity for their systemic spread throughout the rest of the plant, via the transpirational stream in the xylem lumen. However, not all endophytes are capable of colonizing the aerial parts of plants. This may reflect the inability of some to adapt and survive the different niches represented by aerial tissues and organs (Compant et al. 2010). In switchgrass, B. phytofirmans strain PsJN titers were higher in the root than in the leaves 7 days post­inoculation of the roots. However, by 14 days post-inoculation, titers were higher in leaves and sheaths than in the roots, indicating translocation to these tissues (Kim et al. 2012). Generally, bacterial endophyte titers in the aerial plant tissues are reported to be lower than in the root (Rosenbleuth and Martinez-Romero 2006; Compant et al. 2008). In addition, a fair amount of variation can be observed in these tissues. Compant et al. (2008) reported that PsJN could be found in only 10-60% of grape inflorescence stalks and grape berries following initial inoculation of roots. These were localized to xylem vessels, and only a single or few cells were observed. These results further indicated the importance of the xylem for systemic spread of endophytes, allowing them to reach as far as the reproductive tissues. However, this spread was very slow, taking 5 weeks to reach inflorescence tissues. The very low titers of PsJN that ended up in these tissues was attributed to competition with other co-localized endophytes, which can inhabit different tissues and organs, reflecting different niches of colonization (Compant et al. 2011). This report of endophytic bacteria being low or absent in flowers and fruits echoes other comments (Hallman 2001), suggesting low vertical transmission. Bacterial colonization, in general, varies from one cultivar to another and depends on many factors. For example, in soybean, plant genotype, tissue age, season of isolation, and herbicide application, all affected colonization (Kuklinsky-Sobral et al. 2004).

In grasses, the most abundant matrix polysaccharide (i. e., hemicellulose) is xylan. In the last five years, several GTs and other enzymes from Arabidopsis and grasses that likely function in xylan biosynthesis have been characterized (Fig. 2). A complete list of the proteins that function in and the mechanism of xylan synthesis in the Golgi and subsequent release into the cell wall is still being unraveled. Here, we will discuss recent progress with an emphasis on results in grasses. Table 2 lists enzymes implicated in synthesis of the xylan backbone, reducing end oligosaccharide, and sidechains. The reader is referred for additional information to other recent reviews on the topic (Buanafina 2009; Faik 2010; Scheller et al. 2010; Carpita

2012) . When it has been assayed, cell wall material from loss-of-function xylan mutants all exhibit improved digestibility (Mortimer et al. 2010; Brown et al. 2011; Chen et al. 2013; Chiniquy et al. 2012), consistent with the role of this polysaccharide in stabilizing cell walls.

At least two Carbohydrate-Active enZyme (CAZy) database GT families are implicated in the backbone synthesis of xylans, namely GT43s and GT47s (Table 2). Studies in Arabidopsis showed that GT43s are responsible for xylan backbone synthesis since irx9 and irx!4 have drastically shorter xylan chains and reduced ability to transfer Xyl from UDP-Xyl onto xylo-oligosaccharide acceptors (Brown et al. 2007; Pena et al. 2007). Similarly, double mutation of two GT47 genes, irxl0 and irxl0-L severely reduced xylan length, but without affecting the reducing end of xylan. The importance of GT47 family enzymes in xylan synthesis has recently been extended to rice. The Osirx10 mutant has greatly reduced xylan amounts in stems without showing a reduction in xylan chain length, suggesting a somewhat different xylan synthesis mechanism in grasses compared with dicots (Chen et al. 2012). A biochemical study in wheat (Triticum aestivum) suggested proteins from the GT43, GT47, and GT75 families are promising candidates for members of the gluronoarabinoxylan synthetase. Coimmunoprecipitation indicated these three GT proteins interact with each other to form a complex exhibiting xylan synthesis activity (Zeng et al. 2010).

Other studies in Arabidopsis have identified proteins that function in synthesis of the xylan reducing-end oligosaccharide (Fig. 2B), which has been found in several dicots and conifers but not detected in grasses (York et al. 2008; Scheller et al. 2010). The sequence of this reducing end oligosaccharide, or "primer", is 4-p-D-Xylp-(1—>4)-p-D-Xylp-(1—>3)-a- L-Rhap-(1—>2)-a-D-GalpA-(1—>4)-D-Xylp. As summarized in Table 2, the mutants irx7/fra8, irx8, and parvus are depleted for the reducing-end oligosaccharide (Pena et al. 2007). IRX8 and PARVUS, both GT8s, are implicated in adding the galacturonic acid and a a-xylose residue to the primer (Lee et al. 2007; Pena et al. 2007). IRX7, and its close homolog F8H, have been implicated as Rha-specific xylosyltransferases, because they act on a diversity of sugars (Rennie et al. 2012). Despite the absence of the reducing-end primer in experiments in grasses, enzymes with sequence similarity to those implicated in its synthesis have been retained in the rice genome (Scheller et al. 2010).

Other recent work has revealed enzymes that likely function to attach the xylan side chains, glucuronic acid and, in grasses, arabinose. Mortimer et al. (2010) identified mutants in two GT8 family genes, gux1 and gux2. The proteins encoded by these genes are Golgi-localized and required for the addition of both glucuronic acid and 4-O-methylglucuronic acid branches to xylan in Arabidopsis stem cell walls (Mortimer et al. 2010). Recently, another double mutant, irx15 and irx15L, was also found to be involved in xylan synthesis (Brown et al. 2011). These two genes, which belong to the domain of unknown function 579 family, might also be glucuronic acid transferases because they exhibited similar mutant features to gux1 and gux2 (Brown et al. 2011). For addition of the side chains of grass xylan, studies have focused on GT61 family members, which are much more highly expressed in grasses than in dicots (Mitchell et al. 2007). Repression of expression of a GT61 encoding gene, TaXat1, in wheat endosperm and its heterologous overexpression in Arabidopsis provided strong evidence that TaXAT1 possesses a-(1,3)-arabinosyltransferase activity (Anders et al.

2012) . Disruption in rice of another GT61 encoding gene, called Xax1, has also been published recently (Chiniquy et al. 2012). This mutant is lacking a previously observed but poorly characterized xylan substitution, an arabinofuranose residue substituted at the O-2 position of a xylosyl residues, in the structure p-Xylp-(1^-2)-a-Araf-(1^-3) (Fig. 2A). Based on H1-NMR and glycosidic linkage analysis, XAX1 possess a xylosyl transferase activity that can attach (P-1,4-Xylp)4 onto the acceptor p-Xylp-(1^2)-a-Araf-(1^3) (Chiniquy et al. 2012).

Other work has provided important insight into synthesis of the arabinofuranose (Araf) nucleotide sugar precursor of grass cell wall glucuronoarabinoxylan. A recent study of reversibly glycosylated proteins (RGPs) in Arabidopsis showed that the conversion of UDP-p-L — arabinopyranose (UDP-Arap) to UDP-p-L-Araf is indispensable for cell wall synthesis (Rautengarten et al. 2011). The rGps are in the GT75 family. The knockout mutants, rgp1 and rgp2 significantly reduced the total L-Ara content relative to the wild type and showed reduced UDP-Ara mutase (UAM) activity. UAM activity has been identified in rice, as well (Konishi et al. 2007; Konishi et al. 2011). Three rice genes with close sequence similarity to RGP-encoding genes were predicted to be UAM candidates (Konishi et al.

2007) . Later, knock-down of one of these three genes suppressed the UAM activity and reduced UDP-Araf amounts in mutant rice plants (Konishi et al. 2011). The mutant also decreased the incorporation of ferulic acid and p-coumaric acid to the cell wall and presented dwarfed and infertile phenotypes (Konishi et al. 2011). One of the GT75 family members in wheat mentioned above has been inferred to have the UAM activity but needs to be further studied (Zeng et al. 2010).

The final set of recent advances in our understanding of xylan synthesis in grasses relates to acylation of grass xylan arabinose residues by the hydroxycinnamates, ferulic acid and p-coumaric acid (Buanafina 2009). Consistent with expectations, in two studies of rice mentioned above, mutants with reduced Araf substituted xylan had reduced cell wall content of ferulic acid and p-coumaric acid (Konishi et al. 2011; Chiniquy et al. 2012). Mitchell and colleagues developed the hypothesis that 12 members of the BAHD family of acyl-CoA acyltransferases that were much more highly expressed in grasses than dicots might act as arabinofuranose feruloyl transferases (Mitchell et al. 2007). Later, silencing of four members of this family in rice (LOC_Os05g08640, LOC_Os01g09010, LOC_Os06g39470 and LOC_Os06g39390) reduced the ferulic acid content in young leaves by about 20%, leading to the refined hypothesis that one or more of the targeted genes acts as the feruloyl transferase (Piston et al. 2010). Recently, the overexpression of one of the same genes examined by Piston et al., LOC_Os06g39390, dubbed OsAT10, has led to the hypothesis that this protein functions as a p-coumaroyl transferase, as the overexpression plants have increased levels of p-coumaroyl esters bound to arabinose in the cell wall (Bartley et al. 2013a). The plants also exhibit a reduction in the level of polysaccharide-linked ferulic acid and show a concomitant improvement in digestibility (Bartley et al. 2013a), consistent with the model that ferulate — mediated crosslinking is important for grass cell wall digestibility.

With the per base cost of sequencing on the rapid decline, draft genome sequencing endeavors are becoming more and more feasible in complex eukaryotes, and a high quality sequence-based reconstruction of an organisms genome is an invaluable tool to deciphering the underlying biology and performing intergenome comparisons at the population level or between species. As of this writing, there are 41 plant genera that have a reference sequence deposited on the phytozome genome browser (www. phytozome. net) (Goodstein et al. 2012, Table 1), with only two reported attempts at polyploid genomes such as bread wheat (Brenchley et al. 2012) and switchgrass (unpublished). As of this writing, a Panicum virgatum version 0.0 preliminary release of genotype AP13 is available via phytozome (www. phytozome. net/panicumvirgatum. php). The draft genome dataset consists of 15-fold coverage of the estimated 1.6Gbp genome size as a contig only dataset (summarized in Table 2) that consists of approximately 1,358 Mbp arranged in ~410k contigs (N50 of 4.2kb-83,229 contigs). 65,878 protein coding loci were identified, with 4,193 suspected with splice variation. A subset of contigs that aligned to the Setaria italica (Foxtail millet) coding sequence were aligned to the foxtail millet genome and referenced as such on the phytozome site. These data represent the first de novo assembly of the switchgrass genome and certainly provide a conduit for gene discovery and analysis of the effective gene space of the switchgrass genome, yet underscore the need for new technologies and approaches for deciphering large and complex genomes. Recent reports of hybrid 2nd and 3rd generation sequencing technologies such as Illumina’s HiSeq and Pacific Biosciences RS molecule sequencer (PacBio correction and assembly) (Koren et al. 2012) suggest that longer, accurate reads are becoming possible and scaffolding and super-scaffolding efforts can be augmented by this approach. However, in a polyploid situation, short — reads resulting from the Illumina sequencer may not necessarily accurately correct a long read (5-10kb) from a PacBio molecule sequencer with the correct sub-genome placement in regions of the genome that share high sequence identity. The approach may not be sensitive enough to detect the difference between sequencing errors and subgenome specific SNPs. Another emerging technology, named Moleculo where genomic DNA is fragmented into 10kb segments, clonally amplified, sheared and marked with a unique barcode and sequenced with the Illumina technology (http: // www. illumina. com/technology/moleculo-technology. ilmn), and assembled with proprietary bioinformatics creates long, synthetic reads. This approach holds promise for accurate reconstruction of longer reads and better chance of proper subgenome placement. A more costly, but traditional approach is to pursue a physical mapping approach and minimal tile path sequencing

using the hierarchal BAC-by-BAC approach supplemented with a mix of 2nd generation sequencing. With this approach, it would be prudent to assess the ability to readily separate homeologous genomic segments. The future of the reference genome sequence for switchgrass is uncertain, but as sequencing and advanced capture technologies evolve, we will be better positioned to unravel and understand more about the composition and arrangement of the switchgrass genome.

Herbicides for Establishment

Switchgrass has small seed (~600,000 to 900,000 seeds kg-1) that often are reported to be slow to establish (Aiken and Springer 1995). This characteristic provides a competitive advantage to weeds, resulting in excessive competition during establishment (Masters et al. 2004; Boydston et al. 2010; Mitchell et al. 2010a). Controlling weeds during the establishment year improves establishment and increases biomass production in subsequent years (Schmer et al. 2006; Mitchell et al. 2010a). Using current agronomic recommendations, it is feasible to produce 50% of the yield potential of the cultivar to be available for harvest after a killing frost in the planting year, and produce and harvest 75-100% of the yield potential of the cultivar in the first full growing season after planting (Mitchell et al. 2010a; Mitchell et al. 2012).

Warm-season annual grass weeds are the most detrimental to successful switchgrass establishment, since broadleaf weeds can easily be controlled with 2,4-D amine 2,4-dichlorophenoxyacetic acid, when switchgrass reaches the 4-leaf stage (Vogel 2004; Anonomous 2008b). Cool-season weeds are relatively easy to control since they can be controlled with glyphosate prior to planting (Sanderson et al. 2012). Use of a pre-emergence herbicide is typically recommended as an aid in establishing warm-season grasses. For example, application of metolachlor and/or atrazine [6-chloro-N-ethyl- N-(1-methylethyl)-1,3,5-triazine-2,4-diamine] was reported to improve biomass yield in big bluestem (Andropogon gerardii Vittman) during the second year (Masters 1997). In three environments in the central and northern Great Plains, pre-emergence application of atrazine and quinclorac (3,7-Dichloro-8-quinolinecarboxylic acid) resulted in acceptable stands and high biomass yields (Mitchell et al. 2010a). No differences were detected among switchgrass lowland and upland ecotypes for tolerance to atrazine and quinclorac. The use of a pre-emergence herbicide to control such weeds needs evaluations in other environments.

There are very few herbicides currently labeled for use during switchgrass establishment. The scientific literature provides limited information on the phytotoxicity and efficacy of the herbicides used in other warm-season grasses when used for weed control in switchgrass establishment. Currently only quinclorac (Paramount®; Anonymous 2008c,

2010) is labeled in the USA, while nicosufuron (Accent®; Anonymous 2008a) has a supplemental label in the state of Tennessee for weed control during switchgrass establishment once it reaches the 2-leaf stage. In non-crop areas and Conservation Reserve Program (CRP) sites, sulfosulfuron (Outrider®; Anonymous 2011a) controls johnsongrass, and nutsedge (Cyperus sp.) when applied to newly seeded switchgrass after the 3-leaf stage. Use of atrazine, which is labeled for corn (Zea mays L.) and CRP plantings of switchgrass, has led to successful establishment of upland switchgrass as a companion crop in corn fields (Hintz et al. 1998). Although atrazine can improve switchgrass establishment by controlling broadleaf weeds and cool-season grasses (Martin et al. 1982; Bahler et al. 1984), it does not control warm — season annual grass weeds (Boydston et al. 2010; Mitchell et al. 2010a). Injury to switchgrass is reported to differ with herbicide used, application rates, growth stage at application, and the ecotype of switchgrass being evaluated. Research results have varied and are sometimes contradictory. Mitchell et al. (2010a) reported that lowland and upland ecotypes had comparable tolerances to atrazine and quinclorac that effectively controlled weeds and resulted in acceptable plant stands in both switchgrass ecotypes. ‘Pathfinder’, an upland ecotype, is reported to have greater tolerance to pre-emergent applications of atrazine, and the use of atrazine aids its establishment (Martin et al. 1982; Vogel 1987; Masters et al. 1996; Hintz et al. 1998). However, despite this tolerance, there are reports of increasing injury in Pathfinder as atrazine application rate increases from 1.1 to 2.2 kg ha1 (Martin et al. 1982; Vogel 1987; McKenna et al. 1991; Masters et al. 1996; Hintz et al. 1998). Imazapic (2-[4,5-dihydro-4-methyl-4-(1-ethylethyl)- 5-oxo-1H-imidazol-2-yl]-5-methyl-3-pyridinecarboxylic acid) often reduced switchgrass stands and is not recommended for switchgrass establishment (Mitchell et al. 2010a).

Despite these successes for atrazine use in upland ecotypes (Bovey and Hussey 1991), recommended that atrazine should not be used when establishing Alamo, a lowland switchgrass ecotype, due to excessive injury. Furthermore, the phytoxicity of atrazine may also be site specific. Bahler et al. (1984) reported that atrazine application reduced switchgrass seedling density, with the degree of damage being greater in loamy sandy soil than in silty clay loam soil. The upland switchgrass cultivar ‘Cave-in­Rock’ tolerated atrazine (1.1 kg a. i. ha-1), while a lowland strain derived from Alamo was killed by atrazine (T. J. Butler, unpublished data). Time to rainfall after planting appears to mediate atrazine activity on lowland switchgrass. In an Oklahoma study, atrazine application followed by rainfall the succeeding day resulted in complete lowland switchgrass mortality. The second year, however, rainfall did not occur for two weeks upon atrazine application, and the lowland switchgrass had only transient injury (T. J. Butler, unpublished data).

Some alternatives to atrazine have been evaluated. For example, an application of 1.6 kg a. i. siduron ha-1 effectively controlled large crabgrass with no effect on ‘Caddo,’ an upland switchgrass ecotype (McMurphy 1969). However, subsequent work indicated that a pre-emergence application of 2.2 kg ai siduron ha-1 caused significant injury to Alamo, a lowland ecotype (Bovey and Hussey 1991). Although Mitchell et al. (2010a) reported that both upland and lowland switchgrass ecotypes tolerated 560 g a. i. ha-1 quinclorac applied pre-emergence in the central and northern Great Plains, similar quinclorac pre-emergence applications in the southern Great Plains have reduced lowland switchgrass emergence (T. J. Butler, personal comm.). Masters et al. (1996) reported that imazethapyr improved big bluestem establishment (77-94%) similar to atrazine (18-95%) and proved a suitable replacement for atrazine when establishing big bluestem; however imazethapyr generally reduced switchgrass establishment (stand frequency) of the upland ecotype ‘Trailblazer’. Although the concern for using atrazine is valid in some regions, atrazine has been used effectively in hundreds of small plot trials and production scale fields on all available upland and lowland switchgrass strains in the central and northern Great Plains.

Post-emergence herbicides have also demonstrated mixed results. However, in well-managed established stands, such herbicide application is seldom needed (Mitchell et al. 2010a). Applications of quinclorac at 0.56 kg a. i. ha-1 or pendimethalin at 1.1 kg a. i. ha-1 at the 1-2 leaf stage have been shown to improve weed control but reduce switchgrass in irrigated stands in the arid west (Boydston et al. 2010). Post-emerge quinclorac applications reduced switchgrass biomass at establishment by 33% compared to a control receiving pre-mergence atrazine only, but this effect was less than an yield 89% reduction with post-emerge pendimethalin application (Boydston et al. 2010). However, in rain-fed production in the central and northern Great Plains, the application of quinclorac to established upland and lowland switchgrass strains has not been observed to reduce stands. Work by Curran et al. (2011) showed that quinclorac applied 4 wk after planting achieved better weed control in Cave-in-Rock switchgrass than when applied 6 wk after planting. Additionally, application of 2.2 kg a. i. ha-1 MSMA to greenhouse grown lowland switchgrass at the 3-to-4-leaf stage did not cause significant injury compared to the control (Bovey and Hussey 1991). Kering et al. (2012b) evaluated lowland switchgrass establishment with competition from large crabgrass [Digitaria sanguinalis (L.) Scop.], broadleaf signalgrass [Urochloa platyphylla (Munro ex C. Wright) R. D. Webster], Johnsongrass [Sorghum halepense (L.) Pers.], and Texas panicum [Urochloa texana (Buckley) R. Webster] and reported that switchgrass establishment was improved with a combination or quinclorac + foramsulfuron + pendimethalin at the 1-2 leaf stage (13-26% stand) and MSMA at the 3-4-leaf stage (7-35% stand) compared to an untreated control (0-3% stand). However such results are less than satisfactory based on a minimum goal of 40% coverage at the end of the first season (Vogel 1987; Masters 1997).

The best outcomes for switchgrass establishment result from using sound agronomic practices for weed control. For example, when grass weeds are controlled the previous season, especially where glyphosate — tolerant soybeans, corn, or cotton are grown, switchgrass has a much greater chance of successful establishment (Christensen and Koppenjan 2010; Mitchell et al. 2010a). Mitchell et al. (2010a, 2012) provided recommendations that produce harvestable yields after a killing frost in the planting year if precipitation is adequate: 1) develop a good seedbed (no-till seed into soybean stubble or clean till and pack to leave a faint footprint); 2) plant within 3 weeks before or after the optimum maize planting date; 3) use high quality certified seed of adapted material; 4) plant at least 300 PLS m-2; 5) use a planter that controls depth and plant seeds 0.6 to 1.2 cm deep; 6) manage weeds with a pre-emergent application of 1.1 kg ha-1 of atrazine plus 560 g ha-1 of quinclorac then mow or spray broadleaf weeds with

Another mechanism of plant growth promotion by endophytes is bio­control of pathogens. Endophytes have evolved a diverse range of bio­control mechanisms including production of antibiotics, both antifungal and antibacterial, siderophore secretion, and enzyme production (reviewed by Compant et al. 2005b). Together, these bio-control properties enable endophytes to outcompete pathogens for their niche and limit damages caused by plant pathogens as well as protect their host plant, resulting in increased survival and growth.

Fungal endophytic colonization confers a positive impact on resistance to pests, mites, and nematodes in grasses (Schardl et al. 2004). Perennial ryegrass (L. perenne) plants colonized by N. lolii reduced aphid populations, adult life span and fecundity (Meister et al. 2006). Neotyphodium spp. form mutualistic associations with several grass genera and produce a range of bio-control agents, some of which have insecticidal properties whereas others are associated with health and welfare issues for grazing animals. Through selection, several novel endophytes that produce predominantly insecticidal bio-control agents have now been successfully commercialized in many temperate grassland areas in New Zealand, Australia, USA, and South America (Easton 2007).

One of the most commonly recognized bio-control mechanisms associated with endophytic plant growth promoting bacteria and fungi is the production of antibiotics. Agents produced include but are not limited to pyrrolnitrin, phenazines, herbicolin, and oomycin. Furthermore, many endophytic organisms are able to produce multiple agents, which have bio-cidal properties towards various organisms. Pyrrolnitrin, a secondary metabolite isolated from B. cepacia, was shown to have activities against both phytopathogenic fungi and bacteria (El-Banna and Winkelmann 1998). The gene cluster regulating the production of pyrrolnitrin is similar to the gene cluster in Pseudomonas and was suggested to have been acquired by horizontal gene transfer (de Souza and Raaijmakers 2003). Other strains of Burkholderia were reported to produce a large variety of anti-fungal agents such as occidiofungin and burkholdinesn (Lu et al. 2009; Tawfik et al. 2010). Burkholderia MP-1 produces at least four anti-fungal compounds including phenylacetic acid, hydrocinnamic acid, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetate methyl ester (Mao et al. 2006). The small size of genes encoding antibacterial agents and the relatively small number of genes in bacteria and fungi may allow genes encoding antibiotic agents to be transformed to various growth promoting endophytes.

Though thousands of diverse GHs have been discovered and hundreds characterized, their enzymatic activities are still too low to meet our demands. Scientists are employing rational, random, and semi-random methods to enhance GH catalytic efficiency and optimize substrate specificity. Site-directed mutagenesis is a common strategy of protein engineering in which mutations are purposefully introduced into the polypeptide. It has been successfully applied to increase the pH stability and range of the catalytic activity of Cel6A from T. reesei (Wohlfahrt et al. 2003), and to improve the thermostability of cellulase C and Xyn II from C. thermocellum and T. reesei, respectively (Nemeth et al. 2002; Fenel et al. 2004). To engineer novel enzymatic properties, researchers have also empolyed directed evolution, which consists of random mutagenesis followed by screens or selections to identify enzymatic features of interest. Error prone PCR-based mutagenesis produced modified Cel12A from T. reesei, and Cel6F from Orpinomyces PC-2, both of which showed changes in their pH optima (Wang et al. 2005; Hughes et al. 2006). Another powerful approach used for creating diversity is called in vitro recombination or DNA shuffling, which is based on random or directed recombination among different DNA fragments or conserved motifs (Coco et al. 2001). In this way, Heinzelman et al. (2009) identified highly thermostable class II cellobiohydrolase chimeras (Heinzelman et al. 2009). Similarly, a fusion of the Thermotoga maritima Cel5A with the CBM1 from T. reesei and CBM6 from C. stercorarium xylanase A generated a CBM-engineered Cel5A with 14- to 18-fold higher hydrolytic activity towards Avicel (Mahadevan et al.

2008) . In another example, a chimeric bifunctional enzyme endo5A-GS — Xyl11D, in which a glycine-serine (GS) linker separated the two enzymes, demonstrated 1.6-fold and 2.3-fold higher activity than the parent enzymes, respectively (Adlakha et al. 2011).

Researchers have also been inspired by the apparent modularity of cellulosome architecture to create artificial mini-cellulosomes in vitro and in heterologous bacterial systems. In a recent study, synthetic cellulosomes significantly enhanced cellulose hydrolysis, especially in the case of highly recalcitrant cellulose at low enzyme loading rates (You et al. 2012). Toward enabling industrial usage of the powerful cellulosome biochemical machinery, heterologous surface assembly of cellulosomes has been realized in yeast and Bacillus subtilis (Wen et al. 2010; Goyal et al. 2011; Fan et al.

2012) . The engineered yeast are capable of simultaneous saccharification and fermentation of crystalline cellulose to ethanol (Fan et al. 2012). The cellulosome engineering processes would be further enhanced by additional structural and biochemical characterization of component GHs and other enzymes.

Bioinformatics prediction of miRNAs that mainly relies on comparative genome-based EST analysis using known miRNAs in certain species is a well-established approach to discover conserved miRNAs in target species lacking genomic resources (Zhang et al. 2005). This method has been widely used for miRNA discovery in many plant species such as Arabidopsis (Wang et al. 2004; Adai et al. 2005), rice (Bonnet et al. 2004; Jones-Rhoades and Bartel 2004), cotton (Zhang et al. 2007), soybean (Zhang et al. 2008), tomato (Luan et al. 2010), brachypodium (Unver and Budak 2009), apple (Gleave et al. 2008), and other species.

Different known miRNA sequences as a query are used to search against NCBI’s switchgrass EST database. Matts et al. (2010) used miRNA sequences obtained from Arabidopsis (miRBase) as a query for general identification, and miRNA sequences from rice for identification of monocot-specific miRNAs (Matts et al. 2010). Xie et al. (2010) used 1699 known miRNAs from 29 plant species for switchgrass miRNA indetification (Xie et al. 2010). While Matts et al. (2010) used NCBI BLASTN as the search tool to find homologous miRNAs in switchgrass with the criteria of at least 18 nt and left 3 nt match (Matts et al. 2010), Xie et al. argued that BLASTN is not an ideal tool for miRNA discovery and might miss a lot of potential miRNA predictions (Xie et al. 2010). Instead, they adopted WATER to search against the EST database with the criteria of no >2 nt substitution (Xie et al. 2010).

The search by either BLASTN or WATER led to numerous hits among the ESTs, which were then subjected to a more strict screening by using different criteria. Matts et al. extracted the flanking region of the mature miRNA sequences and used a fold-back structure prediction software mFOLD to predict its secondary structures (Matts et al. 2010). These predicted secondary structures were then compared with those deposited in miRBase for verification (Matts et al. 2010; Xie et al. 2010). Xie et al. first removed repeated and protein-coding sequence hits, and then screened the rest of the hits by using 6 standards based on sequence complementarity between EST hits and query miRNA sequences, minimum length of pre — miRNA, secondary structure of predicted pre-miRNA, and sequence complementarity and structure of miRNA: miRNA* (Xie et al. 2010). Application of these criteria reduced some false positives and generated potential candidates for conserved microRNAs in switchgrass.

The need for sustainable energy is growing at an increasing rate with the alarmingly high rate of increase in population coupled with the fast growth of urbanization. By 2050 the world population is estimated to be seven billion computed at a conservative rate of growth. By 2100, the number is projected to be over ten billion by another estimate. The source of fossil fuels being predominantly used over time will face depletion around the end of this century unless non-conventional energy sources are put in place. Besides depletion, fossil fuel use is constrained by geo-political issues and threat of greenhouse gas emission. Among the alternative energy sources, bioenergy is emerging as the most promising as compared to atomic, solar, and wind. Bioenergy including bioethanol and biodiesel can be produced from cellular biomass, starch, sugar, and oil derived from several plants and plant products in huge amounts once the required strategies and technologies are formulated and validated for commercialization in cost-effective ways.

Scientific exploratory research conducted during the last few years has identified a large number of plants as potential sources of bioenergy. These include maize, sorghum, switchgrass, canola, soybean, and sugarcane among field crops; eucalyptus and poplar among forest trees; and jatropha, oil palm and cassava among plantation crops. Several other promising field crops including Brachypodium, minor oilseeds, sugarbeet, sunflower, and sweetpotato; forest trees including diesel trees and shrub willow; plantation crops such as Paulownia; many lower plants; and even vegetable oils, organic farm waste and municipal sludge have been found to be promising. Therefore, ‘fuel’ has made its place in the list of principal agricultural commodities along with food and fiber.

Significant studies have also been conducted in natural and social sciences to facilitate utilization of plants and plant products as the most potential source of bioenergy. In bioenergy crops, research has been carried out on genetics, genomics and breeding for relevant traits employing traditional and molecular breeding, genomics-assisted breeding, and genetic engineering. Physiological works have been done for in planta production of cell-degrading enzymes and enzymatic conversion of cell walls into biofuels. Significant advancement has been made on the works on post-harvest technologies and chemical engineering, fuel quality, and

vi Compendium of Bioenergy Plants: Switchgrass

greenhouse gas impacts of bioenergy. Most importantly, economics, public policies, and perceptions have also been critically examined.

There are, at present, only a few books on bioenergy crop plants available. I have myself edited a book recently with two other co-editors for the CRC Press of the Taylor and Francis Group. This book entitled ‘Handbook of Bioenergy Crop Plants’ elucidates on the general concepts of and concerns about bioenergy crop production, genetics, genomics and breeding of commercialized bioenergy crop plants, and emerging bioenergy crops or their groups besides deliberations on unconventional biomass resources such as vegetable oils, organic waste and municipal sludge.

As expected, there is also an array of research and review articles on the basic concepts, strategies and means of utilization of bioenergy crop plants and their products in scientific journals, web sites, newsletters, newspapers, etc. However, there is no endeavor to present any compilation about all the relevant aspects related to particular bioenergy crop plants already commercialized or having potential to be commercialized in near future. The present book series will hopefully fill up that vacuum. This is particularly important as the subject of bioenergy has already occupied its place in academia, research labs, and public life. This was the underlying force behind conception of a book series on ‘Compendium of Bioenergy Crop Plants’.

At the outset, I formulated the tentative outline for 15 chapters to maintain more or less uniformity throughout the volumes of the compendium. These included basic information on the crops; anatomical and physiological researches relevant to feedstock; special requirements related to agricultural and industrial infrastructure; elucidation on genetics, genomics and breeding of bioenergy traits; public platforms for sharing results and building initiatives; role of public and private agencies in fostering research and commercialization; regulatory, legal, social and economic issues; general concerns and their compliance; and also future prospects and recommendations. However, the volumes of this compendium are devoted to various crop plants and obviously the concerned volume editors had to improvize on the contents of the respective volumes based on the unique information available and specific requirements. Thus, each volume of this compendium has the ‘stand-alone’ potential at the same time, thanks to the excellent balancing job performed by the volume editors. Fortunately most, if not all, of the volume editors have long standing association with me as an author of a chapter in some other book, or volume editor of another book series or colleague in a research platform. Therefore, it has been highly comfortable and enriching for me to work with them again for this compendium. I take this opportunity to express my heartiest gratitude to them for offering me this opportunity. The authors of the chapters for each of the volumes have produced high

quality deliberations both in terms of comprehensive contents and lucid write-ups. As the series editor, I must join with my volume editors to extend our thanks to the authors of the chapters for their elegant contributions as well as sincere cooperation all along.

This compendium was originally conceptualized by my wife and colleague, Phullara. She had meticulously reviewed the relative importance and quantum of works accomplished in the commercialized and promising bioenergy crops plants and had eventually identified the leading bioenergy crop plants to which the individual volumes of this compendium are devoted to. She was always there for help in editing this compendium similar to several other book series containing over sixty books published or in press. Expressing just thanks will not do justice to her contribution to this book project. I have, therefore, dedicated this compendium to her in recognition of her contributions to this book project and also for all her support, advice and inspiration for all my academic activities besides shouldering most of our domestic loads, taking the major responsibility to navigate our family and nourish our three growing kids, Papai, Titai and Kinai, as that provided me with enough extra time for my book editing jobs in addition to my professional duties.

Chittaranjan Kole

Harvesting biomass for bioenergy removes large quantities of nutrients from the system (Vogel et al. 2002; Fike et al. 2006a, b; Mitchell et al. 2008; Guretzky et al. 2011). Typically, N is the most limiting nutrient for switchgrass production and is the most expensive annual input. Consequently, reducing N removal from the switchgrass systems has a positive effect on the economic and environmental sustainability. In switchgrass production systems, N removal is a function of biomass yield and N concentration, with biomass N concentration increasing as N fertilization rates increase (Vogel et al. 2002). In general, harvesting 10 Mg ha-1 of switchgrass DM with whole-plant N concentration of 1% will remove 100 kg of N ha-1, whereas if harvest is delayed until after senescence, N concentration can decline to 0.6% or less, resulting in the removal of only about 60 kg of N ha-1 (Mitchell and Schmer 2012). From a producer’s perspective, this 40 kg of N ha-1 reduction in N removal may be an acceptable trade-off for the yield losses associated with delaying harvest.

In a multi-environment study evaluating numerous N rates and harvest dates, biomass yield was optimized when switchgrass was harvested at the boot to post-anthesis stage and fertilized with 120 kg N ha-1 (Vogel et al. 2002). At this harvest date and fertility level, the amount of N removed at harvest was similar to the amount of N applied. Soil NO3-N did not increase throughout the study (Vogel et al. 2002), suggesting N losses from the system (and consequent environmental impact of such management) would be minimal.

Information on total nutrient removal in switchgrass production systems is lacking. Collins et al. (2008) reported irrigated switchgrass in the Pacific Northwest yielded from 14.5 to 20.4 Mg dry matter ha-1 y-1 and each kg of N produced 83 kg of biomass. Macronutrient export averaged 214 kg N ha-1, 40 kg P ha-1, 350 kg K ha-1, 15 kg S ha-1, 60 kg Ca ha-1, 38 kg Mg ha-1, and 6 kg Fe ha-1. Averaged across cultivars, switchgrass removed less than 1 kg ha-1 of B, Mn, Cu, and Zn. In the eastern USA, delaying harvest until spring reduced ash content and leached nutrients from the vegetation (Adler et al. 2006). Although management of all nutrients in the system is important, N is the most expensive, has the greatest potential for environmental contamination, and has the greatest influence on life cycle assessment (Mitchell and Schmer 2012). Consequently, given the interaction of N rate and harvest date, it is important to only replace the N needed for the production system to prevent over-fertilization and soil N accumulation.

Total DNA was extracted from about 20-50 mg of a trap culture of mycorrhizal fungal mycelium using the DNeasy PlantMini Kit (Qiagen). Partial ribosomal SSU DNA fragments were then amplified using a universal eukaryotic primer NS31 (5′-TTG GAG GGC AAG TCT GGT GCC-3′) (Simon et al. 1992) and the primer AM1 (5′-GTT TCC CGT AAG GCG CCG AA-3′), which only amplifies AM fungal SSU sequences but not plant sequences (Helgason et al. 1998). Basically, the PCR reaction follows the protocol described above. PCR products were run on agarose gel to ensure that only one band amplified, and then they were purified with Qiagen PCR purification kit for direct sequencing with either NS31 or AM1 primer. Also, PCR products can be cloned to the pGEM-T vector and then sequenced with T7 and /or SP6 primers.

Visual identification can be carried out on AM fungal spores, as they are larger than other fungal spores; most spores are between 100 to 200 pm in diameter and can be easily observed under a dissecting microscope (Jarstfer and Sylvia 2002). AM fungi in roots can also be observed after chemical staining by using the staining agent 0.05% trypan blue in lactophenol reported by Phillips and Hayman (1970). The chemical trypan blue is considered a carcinogen but is still used in some laboratories (Utobo et al.

2011) . Alternatively, a simple and inexpensive method has been developed (Vierheilig et al. 1998) with ink and vinegar as the staining agent, which is not toxic. Although it is very easy to use, cheap, quick, and can be used for large number of samples, not all inks stain all AM fungi. In general, almost all black inks give good staining, and the structures are clearest under a dark field illumination with a stereomicroscope (Vierheilig et al. 2005).