Review Trends in Plant Science Vol.13 No.8 tolerance.Improved cold and drought tolerance has been biosynthesis and modification enzymes:the overexpres- reported in tobacco and potato by transformation with the sion of cellulose synthase in poplar has led to higher gene encoding DREB1A(dehydration response element lignocellulosic biomass biosynthesis [78].Overall,the pro- B1A),which is driven by a promoter of a stress-responsive duction of biomass can be further increased with the water channel,RD29A [65,66].Rice plants with induced engineering of plant hormone response genes or genes expression of a NAC (for NAM,ATAF and CUC)-type involved in developmental processes [70-74,77,79,80]. transcriptional factor,OsNAC6,have been shown to enhance tolerance to both high salinity and plant patho- Male sterility and biocontainment gens 67]. Male sterility is another desirable feature for feedstock More research is required to understand the effects of development to prevent transgene escape from genetically abiotic stresses on bioenergy crops from two different modified feedstock [1].Induced male sterility is one perspectives.First,genetic variation among different cul- approach to limit transgene flow.Male sterility can be tivars should be explored both in the laboratory and in field induced in plants by either knocking out the expression studies,which will guide breeding and genetic engineering of genes important in pollen development or pollen-specific for feedstock improvements.Indeed,switchgrass shows silencing of major metabolic genes [81-83].Another large phenotypic variation for water and cold-stress toler- approach to prevent transgene flow is the excision of the ance even within cultivars [11,68,69]:the different culti- transgene in pollen through pollen-specific recombinase vars might be incorporated in breeding programs for activity [84,85].Most of the proposed bioenergy crops such better-adapted feedstocks.Second,basic science from as switchgrass have wild relatives,and transgene fow is model species needs to be translated into field crop im- expected to be a major issue limiting the application of provement,and many key stress-response genes identified genetic engineering in these species.Preventing transgene in model species should be explored for the genetic modi- flow is therefore an important issue for feedstock improve- fication of bioenergy feedstocks such as switchgrass and ment by genetic modification. poplar. Metabolic engineering Increasing biomass production and yield Metabolic engineering will play an important role in The importance of altering plant growth and development improving biodiesel,biomass and sugar production.The to increase the biomass production for bioenergy cannot be future of biodiesel will largely depend on metabolic engin- over-emphasized.Given that most lignocellulosic biomass eering to improve oil content and composition in seeds crop candidates are relatively undomesticated,rapid pro- [15,86-89].Previous oilseed research has focused mainly gress should be attainable.First of all,the molecular on changing fatty acid profiles,particularly for nutritional mechanisms controlling plant architecture need to be bet- purposes [87,89].Recent efforts have also led to an increase ter understood.Current knowledge in the field can be in lipid production via induced expression of key exogenous translated into developing bioenergy feedstocks with desir- lipid biosynthesis genes [88].Metabolic engineering can able architectural features such as dwarf stature and erect also help to increase the production of sugar and starch for leaves.It has been shown that these features can be ethanol production using current platforms 90].For achieved by modifying biosynthesis or signal transduction example,recent research has indicated that the overex- for key plant growth hormones including GA(gibberellic pression of a bacterial sucrose isomerase in vacuoles could acid),IAA(indole-3-acetic acid)and brassinosteroids [70- double the sucrose yield for sugarcane [90].Metabolic 74].Biotechnology could make rapid improvements in engineering will also become an important approach for bioenergy feedstocks using genomics-guided improve- increasing non-fuel bioproducts,and advanced bioproducts ments.For example,GA pathway genes such as Agai might be the greatest long-term benefit of the current (gibberellic acid-insensitive)could be introduced into biofuels research spike.Although it is possible that some switchgrass to dwarf the plants,which should produce a alternative,non-biobased,fuel could ultimately replace crop with an increased annual biomass that is easier to petroleum,plastics and other bioproducts will require harvest [75].In addition,dwarfing might also help to new feedstocks in the absence of petroleum feedstocks. change the lignin content of the overall biomass.Following Overall,plant biotechnology will play a central role in dwarfing,biomass allocation should shift to the leaves.The the next generation of bioenergy options to produce ligno- leaves of switchgrass have been shown to contain a lower cellulosic feedstocks with higher yield,better water-use proportion of lignin than that found in stems [76];dwarfing efficiency,greater net energy gain,lower recalcitrance, would increase the cellulosic content needed as feed,or for enhanced abiotic stress tolerance,and improved ecological saccharification and fermentation needed for ethanol pro- benefits,such as better carbon fixation and water and soil duction.One of the major goals of poplar domestication is conservation. to produce dwarf Trees-pathways that are controlled by auxin,GA and brassinosteroids could potentially be Fueling the future altered to achieve this goal [70-74]. The future of bioenergy will depend on breakthrough Second,developmental programming of feedstock needs technologies.However,the importance of basic research to be altered to increase biomass production.For example, on pathways and genes involved in cell wall biosynthesis, delaying the onset of flowering has been reported to result plant development,and metabolite production should not in increased biomass [77].Third,biomass production can be ignored.Translational systems biology is needed for also be increased by the genetic modification of cell wall biofuel applications (Figure 2)[91].The use of 'omics 426tolerance. Improved cold and drought tolerance has been reported in tobacco and potato by transformation with the gene encoding DREB1A (dehydration response element B1A), which is driven by a promoter of a stress-responsive water channel, RD29A [65,66]. Rice plants with induced expression of a NAC (for NAM, ATAF and CUC)-type transcriptional factor, OsNAC6, have been shown to enhance tolerance to both high salinity and plant patho￾gens [67]. More research is required to understand the effects of abiotic stresses on bioenergy crops from two different perspectives. First, genetic variation among different cul￾tivars should be explored both in the laboratory and in field studies, which will guide breeding and genetic engineering for feedstock improvements. Indeed, switchgrass shows large phenotypic variation for water and cold-stress toler￾ance even within cultivars [11,68,69]; the different culti￾vars might be incorporated in breeding programs for better-adapted feedstocks. Second, basic science from model species needs to be translated into field crop im￾provement, and many key stress-response genes identified in model species should be explored for the genetic modi- fication of bioenergy feedstocks such as switchgrass and poplar. Increasing biomass production and yield The importance of altering plant growth and development to increase the biomass production for bioenergy cannot be over-emphasized. Given that most lignocellulosic biomass crop candidates are relatively undomesticated, rapid pro￾gress should be attainable. First of all, the molecular mechanisms controlling plant architecture need to be bet￾ter understood. Current knowledge in the field can be translated into developing bioenergy feedstocks with desir￾able architectural features such as dwarf stature and erect leaves. It has been shown that these features can be achieved by modifying biosynthesis or signal transduction for key plant growth hormones including GA (gibberellic acid), IAA (indole-3-acetic acid) and brassinosteroids [70– 74]. Biotechnology could make rapid improvements in bioenergy feedstocks using genomics-guided improve￾ments. For example, GA pathway genes such as Dgai (gibberellic acid-insensitive) could be introduced into switchgrass to dwarf the plants, which should produce a crop with an increased annual biomass that is easier to harvest [75]. In addition, dwarfing might also help to change the lignin content of the overall biomass. Following dwarfing, biomass allocation should shift to the leaves. The leaves of switchgrass have been shown to contain a lower proportion of lignin than that found in stems [76]; dwarfing would increase the cellulosic content needed as feed, or for saccharification and fermentation needed for ethanol pro￾duction. One of the major goals of poplar domestication is to produce dwarf Trees – pathways that are controlled by auxin, GA and brassinosteroids could potentially be altered to achieve this goal [70–74]. Second, developmental programming of feedstock needs to be altered to increase biomass production. For example, delaying the onset of flowering has been reported to result in increased biomass [77]. Third, biomass production can also be increased by the genetic modification of cell wall biosynthesis and modification enzymes: the overexpres￾sion of cellulose synthase in poplar has led to higher lignocellulosic biomass biosynthesis [78]. Overall, the pro￾duction of biomass can be further increased with the engineering of plant hormone response genes or genes involved in developmental processes [70–74,77,79,80]. Male sterility and biocontainment Male sterility is another desirable feature for feedstock development to prevent transgene escape from genetically modified feedstock [1]. Induced male sterility is one approach to limit transgene flow. Male sterility can be induced in plants by either knocking out the expression of genes important in pollen development or pollen-specific silencing of major metabolic genes [81–83]. Another approach to prevent transgene flow is the excision of the transgene in pollen through pollen-specific recombinase activity [84,85]. Most of the proposed bioenergy crops such as switchgrass have wild relatives, and transgene flow is expected to be a major issue limiting the application of genetic engineering in these species. Preventing transgene flow is therefore an important issue for feedstock improve￾ment by genetic modification. Metabolic engineering Metabolic engineering will play an important role in improving biodiesel, biomass and sugar production. The future of biodiesel will largely depend on metabolic engin￾eering to improve oil content and composition in seeds [15,86–89]. Previous oilseed research has focused mainly on changing fatty acid profiles, particularly for nutritional purposes [87,89]. Recent efforts have also led to an increase in lipid production via induced expression of key exogenous lipid biosynthesis genes [88]. Metabolic engineering can also help to increase the production of sugar and starch for ethanol production using current platforms [90]. For example, recent research has indicated that the overex￾pression of a bacterial sucrose isomerase in vacuoles could double the sucrose yield for sugarcane [90]. Metabolic engineering will also become an important approach for increasing non-fuel bioproducts, and advanced bioproducts might be the greatest long-term benefit of the current biofuels research spike. Although it is possible that some alternative, non-biobased, fuel could ultimately replace petroleum, plastics and other bioproducts will require new feedstocks in the absence of petroleum feedstocks. Overall, plant biotechnology will play a central role in the next generation of bioenergy options to produce ligno￾cellulosic feedstocks with higher yield, better water-use efficiency, greater net energy gain, lower recalcitrance, enhanced abiotic stress tolerance, and improved ecological benefits, such as better carbon fixation and water and soil conservation. Fueling the future The future of bioenergy will depend on breakthrough technologies. However, the importance of basic research on pathways and genes involved in cell wall biosynthesis, plant development, and metabolite production should not be ignored. Translational systems biology is needed for biofuel applications (Figure 2) [91]. The use of ‘omics’ Review Trends in Plant Science Vol.13 No.8 426