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Title: BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8more » Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. Lastly, the identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.« less
Authors:
 [1] ;  [1] ;  [2] ;  [3] ;  [4] ;  [2] ;  [5] ;  [1]
  1. Illinois State Univ., Normal, IL (United States); Univ. of Wisconsin-Madison, Madison, WI (United States). DOE Great Lakes Bioenergy Research Center
  2. Univ. of Wisconsin-Madison, Madison, WI (United States). DOE Great Lakes Bioenergy Research Center
  3. Michigan State Univ., East Lansing, MI (United States). DOE Great Lakes Bioenergy Research Center
  4. DOE Joint Genome Institute, Walnut Creek, CA (United States)
  5. Univ. of Wisconsin-Madison, Madison, WI (United States). DOE Great Lakes Bioenergy Research Center; Univ. of Wisconsin-Madison, Madison, WI (United States). Dept.of Biochemistry
Publication Date:
Grant/Contract Number:
FC02–07ER64494; AC02-05CH11231
Type:
Accepted Manuscript
Journal Name:
Frontiers in Plant Science
Additional Journal Information:
Journal Volume: 7; Journal ID: ISSN 1664-462X
Publisher:
Frontiers Research Foundation
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; binary vectors; Brachypodium; cellulose; lignin; monocot; p-coumarate; tissue-specific expression
OSTI Identifier:
1257265
Alternate Identifier(s):
OSTI ID: 1379077

Petrik, Deborah L., Cass, Cynthia L., Padmakshan, Dharshana, Foster, Cliff E., Vogel, John P., Karlen, Steven D., Ralph, John, and Sedbrook, John C.. BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses. United States: N. p., Web. doi:10.3389/fpls.2016.00055.
Petrik, Deborah L., Cass, Cynthia L., Padmakshan, Dharshana, Foster, Cliff E., Vogel, John P., Karlen, Steven D., Ralph, John, & Sedbrook, John C.. BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses. United States. doi:10.3389/fpls.2016.00055.
Petrik, Deborah L., Cass, Cynthia L., Padmakshan, Dharshana, Foster, Cliff E., Vogel, John P., Karlen, Steven D., Ralph, John, and Sedbrook, John C.. 2016. "BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses". United States. doi:10.3389/fpls.2016.00055. https://www.osti.gov/servlets/purl/1257265.
@article{osti_1257265,
title = {BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses},
author = {Petrik, Deborah L. and Cass, Cynthia L. and Padmakshan, Dharshana and Foster, Cliff E. and Vogel, John P. and Karlen, Steven D. and Ralph, John and Sedbrook, John C.},
abstractNote = {Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. Lastly, the identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.},
doi = {10.3389/fpls.2016.00055},
journal = {Frontiers in Plant Science},
number = ,
volume = 7,
place = {United States},
year = {2016},
month = {2}
}

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