skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass

Abstract

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 assessed 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 themore » 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

Authors:
; ; ; ; ; ; ; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE; USDOE Office of Science (SC), Office of Biological and Environmental Research
OSTI Identifier:
1618722
Alternate Identifier(s):
OSTI ID: 1437560
Report Number(s):
NREL/JA-2700-71538
Journal ID: ISSN 1754-6834; 122; PII: 1119
Grant/Contract Number:  
PS02-06ER64304; AC36-08GO28308
Resource Type:
Journal Article: Published Article
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Name: Biotechnology for Biofuels Journal Volume: 11 Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
Springer Science + Business Media
Country of Publication:
Netherlands
Language:
English
Subject:
09 BIOMASS FUELS; green tissue-specific promoter; transgenic rice; transgenic switchgrass

Citation Formats

Liu, Wusheng, Mazarei, Mitra, Ye, Rongjian, Peng, Yanhui, Shao, Yuanhua, Baxter, Holly L., Sykes, Robert W., Turner, Geoffrey B., Davis, Mark F., Wang, Zeng-Yu, Dixon, Richard A., and Stewart, Jr., C. Neal. Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass. Netherlands: N. p., 2018. Web. doi:10.1186/s13068-018-1119-7.
Liu, Wusheng, Mazarei, Mitra, Ye, Rongjian, Peng, Yanhui, Shao, Yuanhua, Baxter, Holly L., Sykes, Robert W., Turner, Geoffrey B., Davis, Mark F., Wang, Zeng-Yu, Dixon, Richard A., & Stewart, Jr., C. Neal. Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass. Netherlands. doi:10.1186/s13068-018-1119-7.
Liu, Wusheng, Mazarei, Mitra, Ye, Rongjian, Peng, Yanhui, Shao, Yuanhua, Baxter, Holly L., Sykes, Robert W., Turner, Geoffrey B., Davis, Mark F., Wang, Zeng-Yu, Dixon, Richard A., and Stewart, Jr., C. Neal. Tue . "Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass". Netherlands. doi:10.1186/s13068-018-1119-7.
@article{osti_1618722,
title = {Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass},
author = {Liu, Wusheng and Mazarei, Mitra and Ye, Rongjian and Peng, Yanhui and Shao, Yuanhua and Baxter, Holly L. and Sykes, Robert W. and Turner, Geoffrey B. and Davis, Mark F. and Wang, Zeng-Yu and Dixon, Richard A. and Stewart, Jr., C. Neal},
abstractNote = {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 assessed 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.},
doi = {10.1186/s13068-018-1119-7},
journal = {Biotechnology for Biofuels},
issn = {1754-6834},
number = 1,
volume = 11,
place = {Netherlands},
year = {2018},
month = {4}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1186/s13068-018-1119-7

Save / Share:

Works referenced in this record:

A nomenclature for the genes encoding the chlorophylla/b-binding proteins of higher plants
journal, August 1992

  • Jansson, Stefan; Pichersky, Eran; Bassi, Roberto
  • Plant Molecular Biology Reporter, Vol. 10, Issue 3
  • DOI: 10.1007/BF02668357

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis
journal, February 2018

  • Biswal, Ajaya K.; Atmodjo, Melani A.; Li, Mi
  • Nature Biotechnology, Vol. 36, Issue 3
  • DOI: 10.1038/nbt.4067

Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation
journal, March 2010

  • Masumoto, C.; Miyazawa, S. -I.; Ohkawa, H.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 11
  • DOI: 10.1073/pnas.0913127107

Clustal W and Clustal X version 2.0
journal, September 2007


Overexpression of AtLOV1 in Switchgrass Alters Plant Architecture, Lignin Content, and Flowering Time
journal, December 2012


Expression of a monocot LHCP promoter in transgenic rice.
journal, July 1991


Protoplast isolation and transient gene expression in switchgrass,Panicum virgatum L.
journal, March 2008

  • Mazarei, Mitra; Al-Ahmad, Hani; Rudis, Mary R.
  • Biotechnology Journal, Vol. 3, Issue 3
  • DOI: 10.1002/biot.200700189

How and Why Do Plants Inactivate Homologous (Trans)genes?
journal, March 1995


ORIGINAL RESEARCH: Lignocellulose recalcitrance screening by integrated high-throughput hydrothermal pretreatment and enzymatic saccharification
journal, April 2010

  • Selig, Michael J.; Tucker, Melvin P.; Sykes, Robert W.
  • Industrial Biotechnology, Vol. 6, Issue 2
  • DOI: 10.1089/ind.2010.0009

Expression of ZmGA20ox cDNA alters plant morphology and increases biomass production of switchgrass ( Panicum virgatum L.)
journal, January 2016

  • Do, Phat T.; De Tar, Joann R.; Lee, Hyeyoung
  • Plant Biotechnology Journal, Vol. 14, Issue 7
  • DOI: 10.1111/pbi.12514

Silencing of 4-coumarate:coenzyme A ligase in switchgrass leads to reduced lignin content and improved fermentable sugar yields for biofuel production
journal, July 2011


Construction of a GFP-BAR plasmid and its use for switchgrass transformation
journal, January 2001


The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)
journal, September 1994


Anapleurotic CO 2 Fixation by Phosphoenolpyruvate Carboxylase in C 3 Plants
journal, May 1987

  • Melzer, Eva; O'Leary, Marion H.
  • Plant Physiology, Vol. 84, Issue 1
  • DOI: 10.1104/pp.84.1.58

Downregulation of Cinnamyl Alcohol Dehydrogenase (CAD) Leads to Improved Saccharification Efficiency in Switchgrass
journal, January 2011


Light-Regulated and Cell-Specific Expression of Tomato rbcS-gusA and Rice rbcS-gusA Fusion Genes in Transgenic Rice
journal, July 1993

  • Kyozuka, J.; McElroy, D.; Hayakawa, T.
  • Plant Physiology, Vol. 102, Issue 3, p. 991-1000
  • DOI: 10.1104/pp.102.3.991

A simple and reliable multi-gene transformation method for switchgrass
journal, April 2014


Constitutive and tissue-specific differential expression of the cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest
journal, July 1998

  • Datta, K.; Vasquez, A.; Tu, J.
  • Theoretical and Applied Genetics, Vol. 97, Issue 1-2
  • DOI: 10.1007/s001220050862

Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development.
journal, August 1996

  • Finnegan, E. J.; Peacock, W. J.; Dennis, E. S.
  • Proceedings of the National Academy of Sciences, Vol. 93, Issue 16
  • DOI: 10.1073/pnas.93.16.8449

Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time
journal, March 2001

  • Peña, Leandro; Martín-Trillo, Mar; Juárez, José
  • Nature Biotechnology, Vol. 19, Issue 3
  • DOI: 10.1038/85719

Development of an integrated transcript sequence database and a gene expression atlas for gene discovery and analysis in switchgrass ( Panicum virgatum L.)
journal, February 2013

  • Zhang, Ji-Yi; Lee, Yi-Ching; Torres-Jerez, Ivone
  • The Plant Journal, Vol. 74, Issue 1
  • DOI: 10.1111/tpj.12104

LEAFY controls floral meristem identity in Arabidopsis
journal, May 1992


Nucleotide sequences of two genes encoding the light harvesting chlorophyll a/b binding protein of rice
journal, January 1989


Production of polyhydroxybutyrate in switchgrass, a value-added co-product in an important lignocellulosic biomass crop
journal, September 2008


Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks
journal, October 2011


Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass
journal, February 2011

  • Fu, Chunxiang; Mielenz, Jonathan R.; Xiao, Xirong
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 9, p. 3803-3808
  • DOI: 10.1073/pnas.1100310108

Evaluating environmental consequences of producing herbaceous crops for bioenergy
journal, April 1998


GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
journal, December 1987


Structure and Characterization of a Gene for Light-Harvesting Chi a/b Binding Protein from Rice
journal, April 1991


The LP2 leucine-rich repeat receptor kinase gene promoter directs organ-specific, light-responsive expression in transgenic rice
journal, December 2009


High throughput Agrobacterium-mediated switchgrass transformation
journal, March 2011


Describing and Quantifying Growth Stages of Perennial Forage Grasses
journal, January 1991


Development of switchgrass (Panicum virgatum) as a bioenergy feedstock in the United States
journal, June 2005


Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation
journal, July 2011


A protocol for consistent, large-scale production of fertile transgenic rice plants
journal, November 1998


Targeted mutagenesis in tetraploid switchgrass ( Panicum virgatum L.) using CRISPR/Cas9
journal, August 2017

  • Liu, Yang; Merrick, Paul; Zhang, Zhengzhi
  • Plant Biotechnology Journal, Vol. 16, Issue 2
  • DOI: 10.1111/pbi.12778

Novel green tissue-specific synthetic promoters and cis-regulatory elements in rice
journal, December 2015

  • Wang, Rui; Zhu, Menglin; Ye, Rongjian
  • Scientific Reports, Vol. 5, Issue 1
  • DOI: 10.1038/srep18256

Plant synthetic biology
journal, May 2015


The Promoter for the Maize C4 Pyruvate,orthophosphate Dikinase Gene Directs Cell- and Tissue-Specific Transcription in Transgenic Maize Plants
journal, January 2000

  • Taniguchi, M.; Izawa, K.; Ku, M. S. B.
  • Plant and Cell Physiology, Vol. 41, Issue 1
  • DOI: 10.1093/pcp/41.1.42

Field-grown miR156 transgenic switchgrass reproduction, yield, global gene expression analysis, and bioconfinement
journal, November 2017

  • Johnson, Chelsea R.; Millwood, Reginald J.; Tang, Yuhong
  • Biotechnology for Biofuels, Vol. 10, Issue 1
  • DOI: 10.1186/s13068-017-0939-1

Agrobacterium-Mediated Transformation of Switchgrass and Inheritance of the Transgenes
journal, October 2009


Transgenic switchgrass ( Panicum virgatum L.) biomass is increased by overexpression of switchgrass sucrose synthase ( PvSUS1 )
journal, November 2014

  • Poovaiah, Charleson R.; Mazarei, Mitra; Decker, Stephen R.
  • Biotechnology Journal, Vol. 10, Issue 4
  • DOI: 10.1002/biot.201400499

Field Evaluation of Transgenic Switchgrass Plants Overexpressing PvMYB4 for Reduced Biomass Recalcitrance
journal, January 2015

  • Baxter, Holly L.; Poovaiah, Charleson R.; Yee, Kelsey L.
  • BioEnergy Research, Vol. 8, Issue 3
  • DOI: 10.1007/s12155-014-9570-1

Altered morphology in transgenic tobacco plants that overproduce cytokinins in specific tissues and organs
journal, October 1992


Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.)
journal, March 2012


Plant synthetic promoters and transcription factors
journal, February 2016


PvNAC1 and PvNAC2 Are Associated with Leaf Senescence and Nitrogen Use Efficiency in Switchgrass
journal, December 2014


Molecular characterization and functional analysis of the OsPsbR gene family in rice
journal, November 2016


Advanced genetic tools for plant biotechnology
journal, October 2013

  • Liu, Wusheng; Yuan, Joshua S.; Stewart Jr, C. Neal
  • Nature Reviews Genetics, Vol. 14, Issue 11
  • DOI: 10.1038/nrg3583