Biogenesis of MiRNAs

Plant miRNAs are typically encoded by miRNA genes (or MIRNA locus). In most cases, they exist in inter-regions of protein-coding genes and transcribe independently (Voinnet 2009). Biogenesis of miRNA begins with transcription at a MIRNA locus by RNA Polymerase II (Pol II) and produce 5′-capped and 3′-polyadenylated primary microRNA transcripts (pri-miRNAs) (Aukerman and Sakai 2003; Kurihara and Watanabe 2004; Voinnet 2009).

The pri-miRNAs contain imperfect stem-loop structure from which precursors (pre-miRNAs) are excised through the RNase III enzyme DICER LIKE 1 (DCL1) and several other proteins (Park et al. 2002; Reinhart et al. 2002; Voinnet 2009). The factors involved in the initial processing of pri — miRNAs in model plant Arabidopsis thanlina include subunits of the nuclear cap-binding complex (CBC), CBP20 and CBP80; SERRATE (SE), a C2H2- type zinc finger domain-containing protein; HYPONASTIC LEAVES 1 (HYL1), a member of dsRNA-binding protein family, and other components (Bartel 2009; Chen 2009; Chuck et al. 2009; Poethig 2009; Voinnet 2009; Zhu et al. 2009). A key feature of a pri-miRNA is that it is self-complementary and capable of forming the characteristic fold-back hairpin-like structure recognized by DCL1. Processing of a pri-miRNA by the DCL1 complex releases the precursor miRNAs (pre-miRNAs) which contain the stem-loop structure (Voinnet 2009; Zhu et al. 2009).

The pre-miRNAs are further processed by the DCL1 protein complex to generate a small RNA duplex consisting of a miRNA and its passenger strand (miRNA*). The duplex is exported into the cytoplasm by HASTY and methylated at the 3′ end by HEN1 (Park et al. 2002; Yu et al. 2005). One strand functions as the mature miRNA and is incorporated into the RNA — Induced Silencing Complex (RISC) to target mRNAs. The other strand, miRNA* is usually degraded, although some miRNA*s have been reported to be functional under certain conditions (Zhang et al. 2011).

Plant miRNAs recognize their targets through near-perfect complementarity to direct RISC-mediated cleavage, although in some cases translational inhibition and DNA methylation can be the mode of action of miRNA-mediated gene silencing (Chen 2004; Brodersen et al. 2008; Wu et al. 2010).

Biomass may require preprocessing before fed into a thermochemical conversion reactor. Drying, densification, pelletization and torrefaction are three most commonly used pre-preprocessing methods. Drying is needed to reduce biomass moisture content. Densification and pelletization increase the bulk density and flow characteristics; whereas torrefaction increases energy density and improves grindability. The degree and type of preprocessing depend on the thermochemical conversion process to be used. For example, pyrolysis may demand feedstock with relatively small particle size and moisture content compared to gasification process. However, since lignin is utilized in the thermochemical conversion processes, unlike biological conversion processes, removal or separation of lignin is not needed for the thermochemical conversion processes.

Biological pretreatment may be performed using brown-rot, white-rot and soft-rot fungi. However, based on physiology, enzymology and molecular genetics these fungi degrade the lignocellulosic biomass differently. Brown — rot fungi are effective in the degradation of cellulose, whereas white-rot and soft-rot fungi degrade both lignin and cellulose. White-rot fungi belonging to Basidiomycetes are among the most effective fungi in degrading lignin. Lignin degrading enzymes (such as peroxidases and laccase) produced by white-rot fungi (such as Phanerochate chrysosporium) are regulated by the C/N (carbon/nitrogen) ratio of the lignocellulosic feedstock. The higher C/N ratio of 30:1 for the fungi compared to lower C/N ratio 10:1 for the bacteria, makes the fungi more capable in degrading lignocellulose biomass (Kirk and Farrell 1987; Kerem et al.1992; Kumar et al. 2009). The ability to perform biological pretreatment at low energy levels compared to chemical pretreatment could make this technology a more economically viable pretreatment. However, long processing times ranging from weeks to months and inconsistent utilization of substrate may limit its viability at industrial scales.

The average custom rate charged to windrow, rake, and bale hay and straw into rectangular solid bales as reported by Doye and Sahs (2012) is used to compute the harvest costs reported in Tables 5 and 6. These rates provide a market estimate of the marginal cost of the baling activity for the region. These rates could be expected to be sensitive to the level of timeliness required and to an increase in the demand for custom baling.

One advantage of establishing switchgrass as a bioenergy crop in the U. S. Southern Plains is that it could be harvested once per year anytime between July and February of the following year (Epplin et al. 2007). A harvest season of this length may result in the development of harvest units that include an economically efficient set of machines and workers that can harvest and deliver feedstock in a standardized form. Harvest units could develop in a manner similar to custom grain harvesting firms that harvest a substantial quantity of the grain produced in the U. S. Great Plains. Cost economies are such that a moderate sized grain producer has difficulty justifying combine ownership. For many farms in the region, hiring a custom harvester is more economical than either owning or leasing a combine.

Custom grain harvest firms take advantage of the economies of size associated with ownership and operation of machines used to harvest grain. Kastens and Dhuyvetter (2011) find that a typical custom grain harvest company harvests thousands of hectares per year, with several combines and trucks, and a crew of workers. These harvest companies may begin their season in regions where the crops mature first and migrate as the harvest season progresses. For example, some wheat harvest firms begin harvesting wheat in Texas in May and travel north as the crop matures, eventually into Canada.

Thorsell et al. (2004) introduce the concept of an economically efficient harvest unit for switchgrass. They assume switchgrass harvest and field storage would require machines that could mow, rake, and bale switchgrass biomass and a machine that could collect, transport, and stack bales at a location near an all-weather road. The search was limited to established technology and available agricultural equipment that could travel quickly and legally on country roads and highways. Self-propelled bale transporters that can travel in a field and collect large rectangular solid bales, transport them within and beyond the field, and stack them for field storage are commercially available. Because of differences in weather requirements between mowing and baling, Hwang (2007) modified Thorsell et al.’s (2004) harvest unit concept by separating the mowing unit from the raking-baling — stacking unit.

Table 7 includes a list of harvest machines used to compile a mowing unit and a coordinated raking-baling-stacking unit. The mowing unit consists of a 140 kW self propelled windrower with a 4.9 m rotary header. The coordinated raking-baling-stacking unit includes one bale transporter stacker, three 7.3 m wheel rakes that are powered by three 40 kW tractors, and three balers powered by three 147 kW tractors that produce 1.22 m x 1.22 m x 2.44 m bales. If the material is sufficiently dry when cut, the windrower may be used to mow and place the cut biomass in a windrow

for baling. If the material is not sufficiently dry a rake may be used to turn the material to aid the drying process. The rake may also be used to merge two or more windrows produced by the windrower into a larger windrow for more efficient baling. The raking-baling-stacking unit is coordinated in the sense that the throughput capacity of the three balers is consistent with the collection capacity of one bale transporter stacker.

Most information on switchgrass plant structure and development is for plants from native prairies or cultivars released for forage. However, more botanical and anatomical information is forthcoming for bioenergy specific switchgrass strains. Switchgrass plants are strongly rhizomatous perennials that often form large clumps (Sutherland 1986). Tiller density is generally greatest in the spring and declines as the growing season progresses (Mitchell et al. 1998). The number of leaves per tiller varies by growth stage, genotype, and environment (Redfearn et al. 1997), with the maximum number of collared leaves present prior to advancing to the elongation stage for Trailblazer switchgrass ranging from 3 to 4 (Mitchell and Moser 1995). Ligules are 1.5 to 4 mm long consisting of a fringe of hairs from a membranous base (Sutherland 1986; Casler et al. 2012). Roots of established switchgrass plants have reached depths of 3 m (Weaver 1954).

Switchgrass reproduces sexually by seeds and asexually by rhizomes. The panicle inflorescence is diffuse and 15 to 55 cm long (Sutherland 1986; Casler et al. 2012). Spikelets are 3 to 5 mm long, are two-flowered with the upper floret perfect and the lower floret empty or staminate, and disarticulate below the glumes (Hitchcock 1951; Gould 1975; Casler et al. 2012). Florets are glabrous and awnless and the lemma of the fertile floret is slick and smooth (Casler et al. 2012). The seed is comprised of a smooth lemma and palea that hold tightly to the caryopsis (Casler et al. 2012). Glumes are unequal, with the first shorter than the second (Stubbendieck et al. 1997), and both are mostly removed by combining and cleaning (Casler et al. 2012). Switchgrass has been reported to contain 850 seeds g-1 (Wheeler and Hill 1957). However, Mitchell and Vogel (2012) demonstrated that differences in switchgrass seed weight exist within and among cultivars, reporting the seed number for 19 seed lots of four cultivars across two years ranged from 473 to 702 seeds g-1. Advantages to switchgrass seed are that it is easily threshed and cleaned (Casler et al. 2012) and commercial planting equipment can be calibrated easily to account for differences in seed lot seed number (Mitchell and Vogel 2012a). Unlike other native warm-season grasses, switchgrass seed is smooth and flows readily through grass drills. Additionally, a switchgrass seed industry has existed for over 50 years and numerous private companies and public crop improvement associations are involved in seed production, distribution, and marketing (Casler et al. 2012). The recommended seeding rate for switchgrass in the Great Plains is 300 to 400 PLS m-2 (Mitchell et al. 2010b), but successful stands can be established with seeding rates as low as 100 PLS m-2 if quality seed is used (Vogel 1987).

Switchgrass, a warm-season grass native to North America, has received much attention in recent years given its high yield potential, broad adaptability, and tolerance of marginal sites. The more robust lowland switchgrass ecotypes typically are preferred for bioenergy production systems given their greater yield potential, but lowlands have less cold tolerance, hence lower survivability, at greater latitude.

Switchgrass can be established both with conventional tillage and no-till planting systems. For either planting method, it is critical to have a clean, firm seedbed and to minimize weed competition after planting. Seeding depth is a critical issue for establishment, and can be greatly dependent on the firmness of the seedbed. Seed generally should not be planted more than about one centimeter deep for best establishment, although deeper plantings may be needed on drier sites.

Seeding rates as low as 1 kg ha-1 have been successful, but recommendations typically range from 4 to 8 kg ha-1. Seed often have high levels of dormancy, and this can cause stand failures in late-spring plantings if this is not accounted for. Several methods have been used to break dormancy successfully, but aging seed—holding for a year or so—may be the simplest method for doing so. Some have used planting timing to naturally break dormancy, and planting sometime from late fall to early spring can work well for this. Recommendations to this effect will be unlikely in regions where weed control issues are a challenge, however.

Switchgrass is known to benefit from mycorrhizal associations, and new research is showing a role for bacterial interactions that increase seedling growth. Many of the microbes produce plant-active hormones, and some may play a role in low N requirements due to biological N fixation. This is likely to be an important and growing realm of research in the future.

Little fertility is required for new switchgrass stands, and N as a general rule should not be applied in the year of seeding if the planting faces strong weed competition. Although mature, productive stands can benefit from N fertilization, input requirements will vary based on soils and harvest management, among other factors. As a rule of thumb, matching nutrient inputs to removal is likely to be closest to optimum in terms of meeting economic considerations and minimizing environmental impacts.

Weeds are a major issue in switchgrass establishment and there are very few herbicides currently labeled for use during switchgrass establishment. A number of herbicides have been evaluated in pre — and post-emerge applications to new seedings, and experimental results often vary by region —and even by switchgrass strain. For example, pre-emergence applications of atrazine have been successful in many cases but have been observed to harm certain lowland ecotypes. Safener treatments that protect seedlings from herbicide damage have proven less successful for switchgrass to date, but this work is ongoing.

One of the most successful ways to reduce weed problems at establishment is to plant switchgrass into the stubble of a glyphosate- resistant crop (especially soybeans) and follow good agronomic practices. Use of companion crops, grazing, mowing, or tillage may all provide some positive effect in controlling weeds—or at least, holding back the competition — in the establishment year. Once this first-year hurdle is overcome, there is typically little need for herbicides in established stands.

Diseases and insect pests may be a growing problem for switchgrass in the future as planted acres increase. The plant is host to a number of fungi, but pathogenicity is low for most species, and viral diseases may be an issue on the horizon. Accounts of yield reductions due to disease have increased in recent years, and breeding and selection for switchgrass as a biofuel crop needs to account for disease susceptibility to avoid potential disease vulnerability. Similarly, few insect pests have been a serious issue for switchgrass in the past, but several potential "species of interest" that could cause economic losses have been identified in recent years.

Harvest practices have potential to affect many parts of the supply chain in terms of fertility inputs, energy and CO2 balance, storage needs, and feedstock quality for processing. While most recommendations call for a single, end-of-season harvest, this may not account for the numerous feedbacks to the overall system. Although it makes great sense for minimizing costs on farm (i. e., by reducing nutrient losses), single, end-of — season harvests in a short harvest window may not account for the demands to the system in terms of meeting year-round processing needs. Rather, such a framework is likely to add to the equipment required to harvest, handle and move the material, as well as increase the demands for storage capacity. Thus, to be most effective, the development of switchgrass for biomass — to-bioenergy systems will need to consider the function of the system as a whole, and this is a different paradigm for much of agriculture.

Thermochemical conversion of biomass to biofuels is a highly accelerated form of the geologic processes that created petroleum fossil fuels. This mode of conversion consists of passing biomass through a heated reactor in the absence of oxygen at or above ambient pressures. Residence times within the reactor are dictated by the system type and vary from tenths of a second to up to an hour (NSF 2008). Within the reactor, most of the biomass is pyrolyzed into small molecules that flow out of the reactor as gasses. Depending on the severity of the reactor conditions, the major products are liquids or gasses, plus non-fuel by-products such as tars and mineral-rich char.

When the reactor temperatures are low (~100-750°C), most of the pyrolysis products condense when cooled to produce a liquid referred to as bio-oil. Bio-oil is a complex hydrocarbon mixture that includes water (~25%); 1- to 4-carbon alcohols, acids, and aldehydes (total ~45%); carbohydrates (~10%); and phenolics and other lignin derivatives (~20%) (NSF 2008). Additional heating in the presence of chemical catalysts upgrades and distills these products to form higher chain-length, less oxygenated hydrocarbons. Attractively, these upgraded mixtures are suitable for use in conventional combustion engines with or without blending with petroleum-derived fuels.

At higher reactor temperatures (750-1200°C) and in the presence of some oxygen, the most useful pyrolysis products are CO and H2, which are referred to as syngas. CO2, H2O, H2S, and other impurities also form (NSF

2008) . Of course, H2 in itself is a high-energy fuel molecule, though use as a transportation fuel is not yet technically feasible. For immediate needs, after a cleaning step, the CO and H2 can be recombined with heating and catalysts to form alkanes, via Fischer-Tropsch synthesis, or alcohols, especially

methanol. Another method of upgrading syngas to transportations fuel is known as indirect fermentation. In this process, anaerobic bacteria can utilize the CO to form ethanol, and in limited cases, butanol (Mohammadi et al. 2011). Bacteria can also couple the oxidation of CO to CO2 to produce H2 (Oelgeschlager et al. 2008).

Relative to current biochemical conversion, the short residence times within the reactor bed provide the possibility for distributed production of thermo-converted fuels, reducing the distance that low density biomass must be transported for biofuel production (NSF 2008). On the other hand, a major challenge for thermochemical processes is optimizing the energy efficiency and the fraction of carbon from the biomass that is incorporated into the final, useable fuel. A wide diversity of feedstocks, including switchgrass (Boateng et al. 2006), can be used for thermochemical conversion, and this technology has been seen as having the advantage of being largely feedstock-independent. However, scientists have recently begun to explore possible correlations between feedstock content and syngas and bio-oil formation (Boateng et al. 2006; Gan et al. 2012). Desirable biomass qualities for thermochemical biofuel production may rely on the details of the method of conversion. These qualities include higher content of reduced compounds (i. e., lignin) to maximize the starting material energy potential, lower nitrogen and mineral content to prevent these molecules from catalyzing oil degradation, and reduction of crosslinks between biomass components to allow staged conversion for the different biomass content fractions.

Genetic maps serve many practical biological purposes and are a key tool in both classical genetic and modern genomic research. Generally, two factors need to be considered in selecting appreciate parental lines to begin the development of mapping populations: 1) DNA polymorphism; 2) hybrid fertility (Xu 2010). A good segregating population is required for the construction of a genetic map. Being a species of wind-facilitated cross-pollination and strong genetic self-incompatibility, switchgrass is an allogamous species (Taliaferro and Hopkins 1996; Casler et al. 2011). Theoretically, every plant in switchgrass is heterozygous in many loci and homozygous in other loci. Switchgrass homozygous inbreds are unavailable in nature. Therefore, crossing of two heterozygous parents would produce a pseudo-F1 population, which often displays substantial segregations. Some loci may have four different alleles between the crossing parents, generating four genotype classes in the progeny. Many others may either follow the F2 pattern in a 1:2:1 ratio (called intercross loci) or the backcross pattern in a 1:1 ratio (called testcross loci) (Lu et al. 2004). Using the testcross markers, i. e., those that are segregating in one parent but not in the other, a

so-called "pseudo-testcross" strategy was proposed for linkage mapping in a controlled cross between two outbred parents (Grattapaglia and Sederoff 1994).

Although it only makes use of a portion of markers from the genome, "pseudo-testcross" strategy provides a simple way for genetic mapping of outcrossing species and has been utilized in practical mapping projects for switchgrass (Missaoui et al. 2005b; Okada et al. 2010). The population of Missaoui et al. (2005b) was composed of 85 full-sib progeny from a cross of ‘Alamo’ genotype AP13 (seed parent) and ‘Summer’ VS 16, whereas the population of Okada et al. (2010) consisted of 238 full-sib plants derived from crossing one genotype (seed parent) of ‘Kanlow’ with a selection of ‘Alamo’. Because male and female meioses in the full-sib populations were distinct and independent processes, two separate parental maps were constructed, one map for the male parent and another for the female parent (Okada et al. 2010).

Recently a self-compatible lowland switchgrass genotype ‘NL 94 LYE 16 x 13’ was identified. A first (S1) generation inbred population from selfing ‘NL 94 LYE 16 x 13’ was developed with the assistance of marker — based identification (Liu and Wu 2012a). This S1 population is similar to an F2 population derived from selfing a F1 hybrid of a cross between two different inbred lines; therefore, only one map was constructed instead of two separate (male and female) maps (Liu et al. 2012).

Although genetic manipulation of miRNAs in transgenic plants for switchgrass improvement is still in its infancy, there have already been excellent examples demonstrating the effectiveness of this approach in genetically modifying switchgrass (Chuck et al. 2011; Fu et al. 2012). The miR156 family is one of the most ancient miRNA families found in a large number of plants, from moss to flowering plants (Zhang et al. 2006a; Xie et al. 2006; Axtell and Brown 2008). The miR156 has been reported to target SQUAMOSA promoter-binding-like (SPL) genes, which encode plant-specific transcription factors (Klein et al. 1996; Cardon et al. 1999; Xie et al. 2006; Schwarz et al. 2008; Yang et al. 2008; Yamaguchi et al. 2009). Several studies have suggested the important roles the miR156 genes and their targets play in various plant developmental processes, especially in the transition from juvenile to adult development and floral induction (Schwab et al. 2005; Xie et al. 2006; Chuck et al. 2007; Poethig 2009; Wang et al. 2009; Wu et al. 2009; Yamaguchi et al. 2009). The Arabidopsis miR156, when overexpressed in transgenic plants, dramatically impacted plant morphology, resulting in accelerated leaf growth, greatly enhanced branching and biomass, and delayed flowering (Schwab et al. 2005; Wu and Poethig 2006; Wang et al. 2009; Wu et al. 2009). In maize (Zea mays L.), a classic dominant mutant Corngrass1 (Cg1) which was found about 80 years ago, exhibits phenotypes of dwarfism, multiple-tillers and prolonged vegetative phase (Chuck et al. 2007). In 2007, Chuck et al. successfully cloned this gene and found it encodes two tandem miR156 precursors (zma-miR156b and zma-miR156c) (Chuck et al. 2007). The phenotypes of Cg1 result from the overexpression of miR156s, which impacts the expression of a couple of targets of the SPL family of transcription factors, and the level of miR172 whose targets are involved in juvenile development (Chuck et al. 2007). Interaction of miR156 and its targets also has been studied extensively in rice (Xie et al. 2006, 2012). MiR156s play important roles in rice development (Xie et al. 2006, 2012; Jiao et al. 2010; Miura et al. 2010). Xie et al. (2006) reported that overexpression of two different rice miR156 precursors (stem-loop structures) in transgenic rice plants resulted in reduced plant height, delayed flowering and increased tiller number (Xie et al. 2006), similar to that observed in the maize Cg1 mutant (Chuck et al. 2007) and transgenic Arabidopsis plants overexpressing miR156 (Schwab et al. 2005).

The facts that overexpression of the miR156 genes could increase biomass and "hold plants in the juvenile phase of development" in many plant species (Schwab et al. 2005; Xie et al. 2006; Chuck et al. 2007, 2011; Li et al. unpublished) suggest that they would be potential candidates to improve biomass yield and feedstock quality in switchgrass. To test this hypothesis, two research groups overexpressed the miR156 gene in switchgrass, independently (Chuck et al. 2011; Fu et al. 2012). Chuck et al.

(2011) introduced the Cg1 cDNA fused to the maize Ubiquitin (Ubi) promoter into switchgrass. As expected, overexpression of Cg1 causes pleiotropic morphological and developmental changes in transgenic plants. The vegetative phase was prolonged and flowering time was delayed. In this case, flowering was not observed in transgenic plants, even after having been grown for two years both in the field and in the greenhouse. Total sterility with no flowering is a favorable trait for preventing transgene escape. Although in this case, the production of biomass, one of the important target traits for switchgrass breeding, was not improved in transgenics compared to wild type controls, the amounts of lignin were decreased and the levels of glucose and other sugars were increased in transgenic plants relative to wild type controls. Later, Fu et al. (2012) reported results in overexpressing the precursor of rice Osa-miR156b gene, also driven by the maize Ubi promoter, in transgenic switchgrass. The authors found that biomass yield was related to the expression levels of the exogenous rice miR156. Higher levels of miR156 in transgenic plants resulted in severely stunted plant growth, whereas moderate levels of miR156 expression led to improved biomass production and loss of the ability in flowering. Transgenic plants with low levels of rice miR156 expression flowered normally and their biomass yield was increased. The latter two groups of transgenic plants produced 58-101% more biomass than wild type controls. The authors also found that overexpression of miR156 could improve the solubilized sugar yield and forage digestibility in transgenic plants (Fu et al. 2012).

Recently, we found that overexpression of rice miR156b/c and miR156d genes led to enhanced drought tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.), which is associated with less water consumption and increased water retention capacity (Li et al. unpublished data). These results point to the potential of manipulating miR156 genes in transgenic switchgrass for enhanced stress tolerance. Moreover, Sun et al. (2012) found that the expression level of miR156 increased in switchgrass when subjected to drought stress, suggesting that miR156 may be involved in plant stress responses in this bioenergy crop, and could be a good candidate for manipulation using transgenic approach to produce new switchgrass cultivars with enhanced stress tolerance.

Biomass pyrolysis is the thermal breakdown of biomass using high temperature in the absence of oxygen. Pyrolysis, similar to other thermochemical conversion technologies, results in three products: solid (biochar), liquid (bio-oil), and gas (syngas/producer gas). For pyrolysis, the target product is usually either bio-oil (using fast pyrolysis) or biochar (using slow pyrolysis). Slow pyrolysis has been used for centuries to produce solid, cleaner burning fuels. Only recently (1980s) has fast pyrolysis been recognized as an alternative to produce liquid fuel (Meier and Faix 1999). The main differences between the fast and slow pyrolysis are summarized in Table 4.

Slow pyrolysis was discovered and used many centuries ago, when charcoal and coal-tar were produced using slow pyrolysis of wood and coal. Charcoal was used as a fuel to create a smokeless flame and increase the combustion temperature. The coking process is also used in manufacture of steel. Recent interest in liquid fuels has changed the focus to fast pyrolysis, which results in much higher liquid yield.