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Title: Two-Year Field Analysis of Reduced Recalcitrance Transgenic Switchgrass

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
Southeastern Sun Grant Center and the BioEnergy Science Center, a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.
OSTI Identifier:
1163412
DOE Contract Number:
AC36-08GO28308
Resource Type:
Journal Article
Resource Relation:
Journal Name: Plant Biotechnology Journal; Journal Volume: 12; Journal Issue: 7, September 2014
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; Bioenergy

Citation Formats

Baxter, H. L., Mazarei, M., Labbe, N., Kline, L. M., Cheng, Q., Windham, M. T., Mann, D. G. J., Fu, C., Ziebell, A., Sykes, R. W., Rodriguez, M., Davis, M. F., Mielenz, J. R., Dixon, R. A., Wang, Z. Y., and Stewart, C. N. Two-Year Field Analysis of Reduced Recalcitrance Transgenic Switchgrass. United States: N. p., 2014. Web. doi:10.1111/pbi.12195.
Baxter, H. L., Mazarei, M., Labbe, N., Kline, L. M., Cheng, Q., Windham, M. T., Mann, D. G. J., Fu, C., Ziebell, A., Sykes, R. W., Rodriguez, M., Davis, M. F., Mielenz, J. R., Dixon, R. A., Wang, Z. Y., & Stewart, C. N. Two-Year Field Analysis of Reduced Recalcitrance Transgenic Switchgrass. United States. doi:10.1111/pbi.12195.
Baxter, H. L., Mazarei, M., Labbe, N., Kline, L. M., Cheng, Q., Windham, M. T., Mann, D. G. J., Fu, C., Ziebell, A., Sykes, R. W., Rodriguez, M., Davis, M. F., Mielenz, J. R., Dixon, R. A., Wang, Z. Y., and Stewart, C. N. Mon . "Two-Year Field Analysis of Reduced Recalcitrance Transgenic Switchgrass". United States. doi:10.1111/pbi.12195.
@article{osti_1163412,
title = {Two-Year Field Analysis of Reduced Recalcitrance Transgenic Switchgrass},
author = {Baxter, H. L. and Mazarei, M. and Labbe, N. and Kline, L. M. and Cheng, Q. and Windham, M. T. and Mann, D. G. J. and Fu, C. and Ziebell, A. and Sykes, R. W. and Rodriguez, M. and Davis, M. F. and Mielenz, J. R. and Dixon, R. A. and Wang, Z. Y. and Stewart, C. N.},
abstractNote = {},
doi = {10.1111/pbi.12195},
journal = {Plant Biotechnology Journal},
number = 7, September 2014,
volume = 12,
place = {United States},
year = {Mon Sep 01 00:00:00 EDT 2014},
month = {Mon Sep 01 00:00:00 EDT 2014}
}
  • Five different types of transgenic ( GAUT4, miRNA, MYB4, COMT and FPGS) Panicum virgatum L. (switchgrass) were grown in a field in Knoxville, Tenn., USA over two consecutive years between 2011 and 2015 in separate experiments. Clonal replicates were established (year-one) and produced much greater biomass during the second year. After each growing season the above ground biomass was analyzed for cell wall sugars and for recalcitrance to enzymatic digestibility, and biofuel using a separate hydrolysis and fermentation (SHF) screen. Here, each transgenic event and control had more glucan, xylan and less ethanol (g/g basis) from the second year ofmore » growth relative to the first year plants. There was no correlation between plant carbohydrate content and biofuel production. In each of cell wall-targeted transgenics, GAUT4, MYB4, COMT and FPGS, the second year of growth resulted in increased carbohydrate abundance (up to 12%) and reduced recalcitrance through higher ethanol yields (up to 21%) over the non-transgenic control plants.« less
  • High biomass yields and minimal agronomic input requirements have made switchgrass, Panicum virgatum L., a leading candidate lignocellulosic bioenergy crop. Large-scale lignocellulosic biofuel production from such crops is limited by the difficulty to deconstruct cell walls into fermentable sugars: the recalcitrance problem. In this study, we assessed the field performance of switchgrass plants overexpressing the switchgrass MYB 4 ( PvMYB4) transcription factor gene. PvMYB 4 transgenic switchgrass can have great lignin reduction, which commensurately increases sugar release and biofuel production. Our results over two growing seasons showed that one transgenic event (out of eight) had important gains in both biofuelmore » (32% more) and biomass (63% more) at the end of the second growing season relative to non-transgenic controls. These gains represent a doubling of biofuel production per hectare, which is the highest gain reported from any field-grown modified feedstock. In contrast to this transgenic event, which had relatively low ectopic overexpression of the transgene, five of the eight transgenic events planted did not survive the first field winter. The dead plants were all high-overexpressing events that performed well in the earlier greenhouse studies. Disease susceptibility was not compromised in any transgenic events over the field experiments. These results demonstrate the power of modifying the expression of an endogenous transcription factor to improve biofuel and biomass simultaneously, and also highlight the importance of field studies for "sorting" transgenic events. In conclusion, further research is needed to develop strategies for fine-tuning temporal-spatial transgene expression in feedstocks to optimize desired phenotypes.« less
  • Genetic engineering of switchgrass (Panicum virgatum L.) for reduced cell wall recalcitrance and improved biofuel production has been a long pursued goal. Up to now, constitutive promoters have been used to direct the expression of cell wall biosynthesis genes toward attaining that goal. While generally sufficient to gauge a transgene's effects in the heterologous host, constitutive overexpression often leads to undesirable plant phenotypic effects. Green tissue-specific promoters from switchgrass are potentially valuable to directly alter cell wall traits exclusively in harvestable aboveground biomass while not changing root phenotypes. We identified and functionally characterized three switchgrass green tissue-specific promoters and assessedmore » marker gene expression patterns and intensity in stably transformed rice (Oryza sativa L.), and then used them to direct the expression of the switchgrass MYB4 (PvMYB4) transcription factor gene in transgenic switchgrass to endow reduced recalcitrance in aboveground biomass. These promoters correspond to photosynthesis-related light-harvesting complex II chlorophyll-a/b binding gene (PvLhcb), phosphoenolpyruvate carboxylase (PvPEPC), and the photosystem II 10 kDa R subunit (PvPsbR). Real-time RT-PCR analysis detected their strong expression in the aboveground tissues including leaf blades, leaf sheaths, internodes, inflorescences, and nodes of switchgrass, which was tightly up-regulated by light. Stable transgenic rice expressing the GUS reporter under the control of each promoter (756-2005 bp in length) further confirmed their strong expression patterns in leaves and stems. With the exception of the serial promoter deletions of PvLhcb, all GUS marker patterns under the control of each 5'-end serial promoter deletion were not different from that conveyed by their respective promoters. All of the shortest promoter fragments (199-275 bp in length) conveyed strong green tissue-specific GUS expression in transgenic rice. PvMYB4 is a master repressor of lignin biosynthesis. The green tissue-specific expression of PvMYB4 via each promoter in transgenic switchgrass led to significant gains in saccharification efficiency, decreased lignin, and decreased S/G lignin ratios. In contrast to constitutive overexpression of PvMYB4, which negatively impacts switchgrass root growth, plant growth was not compromised in green tissue-expressed PvMYB4 switchgrass plants in the current study. Each of the newly described green tissue-specific promoters from switchgrass has utility to change cell wall biosynthesis exclusively in aboveground harvestable biomass without altering root systems. The truncated green tissue promoters are very short and should be useful for targeted expression in a number of monocots to improve shoot traits while restricting gene expression from roots. Green tissue-specific expression of PvMYB4 is an effective strategy for improvement of transgenic feedstocks.« less
  • Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate-expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156more » overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development.« less
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