Review Trends in Plant Science Vol.13 No.8 increase their below-ground biomass accumulation will be table sugars [55].Lignin biosynthesis in monocot species important for increased carbon storage [81. should be studied further so as to be able to modify lignin biosynthesis intelligently in perennial grass feedstocks Economic considerations Current biofuels were marginally profitable before recent Preprocessing in planta:expression of cellulases and petroleum prices spiked.Ethanol production from maize cellulosomes was US$0.48 per gasoline energy equivalent liter(EEL)in Plant cell walls can be degraded by individual cellulases or 2005.when the price of gasoline was US$0.46 per liter [291 in concert by cellulosomes.which consist of a suite of However,the recent spike in the price of global crude oil enzymes.In planta expression of cellulases and cellulo- has made maize-based ethanol production very profitable. somes could potentially reduce the cost of enzymatic sac- The situation is similar for biodiesel production:soybean- charification of lignocellulosic biomass at the biorefinery by based biodiesel production was US$0.55 per diesel EEL in providing the enzymes needed for cell wall degradation. 2005 [29].Despite these potential profit margins, The effectiveness of in planta expression of free cellulases traditional crop-based biofuel production has led to direct or cellulosomes is still controversial because digestion is competition between food and energy and,as a result,the complicated by the subcellular localization of enzyme(s), recent crude oil price increase has already led to the global glycosylation of extracellular proteins,effective enzyme infation of the cost of food,feed and associated products. combinations,and the requirement for inducible expres- There is an imminent need to move to lignocellulosic sion to avoid premature cell wall digestion. biomass-based platforms.However,lignocellulosic ethanol Regardless of the challenges,preliminary research has production using the current platforms is not profitable shown the successful apoplastic expression of active Acid- [361.The lignocellulosic ethanol price is still as high as othermus cellulolyticus cellulase El in maize and tobacco US$0.70 to US$1.0 per EEL.However,as processing tech- [56,57].Moreover,recent research has indicated that nologies mature and biomass crops are modified for higher maize plants showing in planta expression of cellulase yield and lower recalcitrance,the cost of lignocellulosic- had higher biomass conversion efficiency (58].Researchers based ethanol production is expected to decrease to rival have indicated that no detrimental effects were found by maize grain platforms.Indeed,based on break-even price apoplast targeting of El and that cellulase activity aided yield,recent analysis has already indicated that switch- biomass conversion when plant material was milled grass is a more profitable crop than traditional crops such [56,59].These differences result from the different sub- as sorghum and maize [10].The competitiveness of ligno- cellular localization and activation mechanisms of the cellulosic ethanol as a sustainable energy supply in the enzymes.For example,heat-activated cellulase enzymes USA will therefore heavily depend on biotechnology break- such as El have no detrimental effects to plants growing in throughs to reduce cost and improve processing efficiency. typical ambient temperatures [55.561. Further study is necessary to determine different strat- Plant biotechnology solutions for bioenergy egies for in planta cell wall digestion enzyme expression, Novel enabling biotechnologies are crucial for reducing the with high-throughput approaches for optimized sub-cellu- costs of bioenergy production,particularly of lignocellulo- lar localization and different combinations of enzymes.In sic ethanol.The key issues include rapid domestication, addition,fundamental research still needs to be performed overcoming recalcitrance,efficient breakdown of cellulose, to modify cellulases for improved catalytic efficiency,ther- and increasing biomass and lipid production for ethanol mal stability,performance under extreme conditions, and biodiesel,respectively [37].Although aspects of these along with protein modifications designed to lead to important areas will be discussed individually below,it is reduced apoplastic glycosylation.Many cell-wall degrading important to solve these problems in concert. bacteria use cellulosomes,a suite of enzymes for cell wall hydrolysis [60].Cellulosomes have not yet been expressed Modification of lignin biosynthesis in transgenic plants,but the correct assemblage of cellulo- Lignin might be the most crucial molecule in need of some components in the plant apoplast potentially have modification for lignocellulosic feedstocks.It has been the promise of decreasing recalcitrance and facilitating the established that reducing lignin biosynthesis can lead to post-harvest hydrolysis of cellulose,which in turn might lower recalcitrance and higher saccharification efficiency aid simultaneous saccharification and fermentation [13.38-54].Recent studies have indicated two important aspects for lignin modification.First,both lignin content Abiotic stress resistance and composition are important.Although it is codependent Suboptimal water and other abiotic stresses are limiting on efficient processes to fractionate lignin,a more uniform factors for biomass production;stress tolerance traits are lignin structure might facilitate more efficient cell-wall therefore important to enable feedstock to be produced on degradation for fuel production (John Ralph,personal marginal or sub-marginal lands not favorable for food communication).Second,the pretreatment of biomass crops.Drought-,metal-,salt-,cold-and heat-stress all might even be rendered unnecessary if lignin content falls induce some similar responses in plants,yet each of these below a critical threshold,which would enhance down- stresses will induce a different set of genes [61].The stream enzymatic saccharification and fermentation steps upstream pathways for salt and drought stresses have for improved efficiency [55].Therefore,switchgrass,Mis- been well-characterized in Arabidopsis thaliana [62-64], canthus or poplar feedstocks with modified lignin can but until recently have led to only limited success in improve the efficiency of biomass conversion into fermen- translational research to produce field crop abiotic stress 425increase their below-ground biomass accumulation will be important for increased carbon storage [8]. Economic considerations Current biofuels were marginally profitable before recent petroleum prices spiked. Ethanol production from maize was US$0.48 per gasoline energy equivalent liter (EEL) in 2005, when the price of gasoline was US$0.46 per liter [29]. However, the recent spike in the price of global crude oil has made maize-based ethanol production very profitable. The situation is similar for biodiesel production: soybean￾based biodiesel production was US$0.55 per diesel EEL in 2005 [29]. Despite these potential profit margins, traditional crop-based biofuel production has led to direct competition between food and energy and, as a result, the recent crude oil price increase has already led to the global inflation of the cost of food, feed and associated products. There is an imminent need to move to lignocellulosic biomass-based platforms. However, lignocellulosic ethanol production using the current platforms is not profitable [36]. The lignocellulosic ethanol price is still as high as US$0.70 to US$1.0 per EEL. However, as processing tech￾nologies mature and biomass crops are modified for higher yield and lower recalcitrance, the cost of lignocellulosic￾based ethanol production is expected to decrease to rival maize grain platforms. Indeed, based on break-even price yield, recent analysis has already indicated that switch￾grass is a more profitable crop than traditional crops such as sorghum and maize [10]. The competitiveness of ligno￾cellulosic ethanol as a sustainable energy supply in the USA will therefore heavily depend on biotechnology break￾throughs to reduce cost and improve processing efficiency. Plant biotechnology solutions for bioenergy Novel enabling biotechnologies are crucial for reducing the costs of bioenergy production, particularly of lignocellulo￾sic ethanol. The key issues include rapid domestication, overcoming recalcitrance, efficient breakdown of cellulose, and increasing biomass and lipid production for ethanol and biodiesel, respectively [37]. Although aspects of these important areas will be discussed individually below, it is important to solve these problems in concert. Modification of lignin biosynthesis Lignin might be the most crucial molecule in need of modification for lignocellulosic feedstocks. It has been established that reducing lignin biosynthesis can lead to lower recalcitrance and higher saccharification efficiency [13,38–54]. Recent studies have indicated two important aspects for lignin modification. First, both lignin content and composition are important. Although it is codependent on efficient processes to fractionate lignin, a more uniform lignin structure might facilitate more efficient cell-wall degradation for fuel production (John Ralph, personal communication). Second, the pretreatment of biomass might even be rendered unnecessary if lignin content falls below a critical threshold, which would enhance down￾stream enzymatic saccharification and fermentation steps for improved efficiency [55]. Therefore, switchgrass, Mis￾canthus or poplar feedstocks with modified lignin can improve the efficiency of biomass conversion into fermen￾table sugars [55]. Lignin biosynthesis in monocot species should be studied further so as to be able to modify lignin biosynthesis intelligently in perennial grass feedstocks. Preprocessing in planta: expression of cellulases and cellulosomes Plant cell walls can be degraded by individual cellulases or in concert by cellulosomes, which consist of a suite of enzymes. In planta expression of cellulases and cellulo￾somes could potentially reduce the cost of enzymatic sac￾charification of lignocellulosic biomass at the biorefinery by providing the enzymes needed for cell wall degradation. The effectiveness of in planta expression of free cellulases or cellulosomes is still controversial because digestion is complicated by the subcellular localization of enzyme(s), glycosylation of extracellular proteins, effective enzyme combinations, and the requirement for inducible expres￾sion to avoid premature cell wall digestion. Regardless of the challenges, preliminary research has shown the successful apoplastic expression of active Acid￾othermus cellulolyticus cellulase E1 in maize and tobacco [56,57]. Moreover, recent research has indicated that maize plants showing in planta expression of cellulase had higher biomass conversion efficiency [58]. Researchers have indicated that no detrimental effects were found by apoplast targeting of E1 and that cellulase activity aided biomass conversion when plant material was milled [56,59]. These differences result from the different sub￾cellular localization and activation mechanisms of the enzymes. For example, heat-activated cellulase enzymes such as E1 have no detrimental effects to plants growing in typical ambient temperatures [55,56]. Further study is necessary to determine different strat￾egies for in planta cell wall digestion enzyme expression, with high-throughput approaches for optimized sub-cellu￾lar localization and different combinations of enzymes. In addition, fundamental research still needs to be performed to modify cellulases for improved catalytic efficiency, ther￾mal stability, performance under extreme conditions, along with protein modifications designed to lead to reduced apoplastic glycosylation. Many cell-wall degrading bacteria use cellulosomes, a suite of enzymes for cell wall hydrolysis [60]. Cellulosomes have not yet been expressed in transgenic plants, but the correct assemblage of cellulo￾some components in the plant apoplast potentially have the promise of decreasing recalcitrance and facilitating the post-harvest hydrolysis of cellulose, which in turn might aid simultaneous saccharification and fermentation. Abiotic stress resistance Suboptimal water and other abiotic stresses are limiting factors for biomass production; stress tolerance traits are therefore important to enable feedstock to be produced on marginal or sub-marginal lands not favorable for food crops. Drought-, metal-, salt-, cold- and heat-stress all induce some similar responses in plants, yet each of these stresses will induce a different set of genes [61]. The upstream pathways for salt and drought stresses have been well-characterized in Arabidopsis thaliana [62–64], but until recently have led to only limited success in translational research to produce field crop abiotic stress Review Trends in Plant Science Vol.13 No.8 425