U.S. patent application number 10/239463 was filed with the patent office on 2004-03-11 for method for modifying lignin composition and increasing in vivo digestibility of forages.
Invention is credited to Dixon, Richard A., Guo, Dianjing.
Application Number | 20040049802 10/239463 |
Document ID | / |
Family ID | 31991069 |
Filed Date | 2004-03-11 |
United States Patent
Application |
20040049802 |
Kind Code |
A1 |
Dixon, Richard A. ; et
al. |
March 11, 2004 |
Method for modifying lignin composition and increasing in vivo
digestibility of forages
Abstract
Methods for transforming forage legumes or woody plants with a
DNA construct comprising at least one open reading frame encoding
for a caffeoyl CoA 3-O-methyltransferase enzyme or a Medicago
sativa caffeic acid 3-O-methyltransferase enzyme or a fragment
thereof in either a sense or antisense orientation under a
lignification-associated tissue specific promoter have been found,
resulting in the down-regulation of the corresponding homologous
gene either through antisense inhibition or sense suppression, as
well as reduced lignin content and modified lignin composition in
the transgenic plants. The expression of the caffeoyl CoA
3-O-methyltransferase transgene produces an increased syringyl
lignin to guaiacyl lignin ratio in the transformed plant, and
greatly improved forage digestibility.
Inventors: |
Dixon, Richard A.; (Ardmore,
OK) ; Guo, Dianjing; (Montgomery, VA) |
Correspondence
Address: |
Eugenia S Hansen
Sidley Austin Brown & Wood
Suite 3400
717 N Hardwood
Dallas
TX
75201
US
|
Family ID: |
31991069 |
Appl. No.: |
10/239463 |
Filed: |
September 23, 2002 |
PCT Filed: |
March 23, 2001 |
PCT NO: |
PCT/US01/09398 |
Current U.S.
Class: |
800/278 ;
435/193; 435/320.1; 435/468; 800/313 |
Current CPC
Class: |
C12N 9/1007 20130101;
C12N 15/8255 20130101 |
Class at
Publication: |
800/278 ;
800/313; 435/468; 435/193; 435/320.1 |
International
Class: |
A01H 001/00; C12N
015/82; A01H 005/00; C12N 009/10 |
Claims
We claim:
1. A method for modulating the lignin content of a forage legume
comprising transforming a forage legume cell with a vector
comprising a lignification-associated tissue specific promoter
functionally linked to a DNA construct comprising at least one open
reading frame encoding for a caffeoyl CoA 3-O-methyltransferase
enzyme or a fragment thereof; and generating plants from said
transformed forage legume cell
2. The method of claim 1, wherein said forage legume cell is
co-transformed with a vector comprising a lignification-associated
tissue specific promoter functionally linked to a DNA construct
comprising at least one open reading frame encoding for a Medicago
sativa caffeic acid 3-O-methyltransferase enzyme or a fragment
thereof.
3. A method for modulating the lignin content of a forage legume
comprising transforming a forage legume cell with a vector
comprising a lignification-associated tissue specific promoter
functionally linked to a DNA construct comprising at least one open
reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or a fragment thereof; and generating
plants from said transformed forage legume cell.
4. A method for modulating the lignin content of a forage legume
comprising transforming a forage legume cell with a vector
comprising a DNA construct comprising in tandem at least one open
reading frame encoding for a caffeoyl CoA 3-O-methyltransferase
enzyme or a fragment thereof under expression control of a first
lignification-associated tissue specific promoter and at least one
open reading frame encoding for a caffeic acid
3-O-methyltransferase enzyme or a fragment thereof under expression
control of a second lignification-associated tissue specific
promoter, wherein said first and second lignification-associated
tissue specific promoter can be the same or different; and
generating plants from said transformed forage legume cell.
5. A method for producing a forage legume having altered lignin
composition comprising transforming a forage legume cell with a DNA
construct comprising at least one open reading frame encoding for a
caffeoyl CoA 3-O-methyltransferase enzyme or a fragment thereof
under expression control of a lignification-associated tissue
specific promoter to form a transgenic cell; and cultivating said
transgenic cell under conditions conducive to regeneration and
plant growth.
6. The method of claim 5, wherein said forage legume cell is
co-transformed with a DNA construct comprising at least one open
reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or a fragment thereof under expression
control of a lignification-associated tissue specific promoter.
7. A method for producing a forage legume having altered lignin
composition comprising transforming a forage legume cell with a DNA
construct comprising at least one open reading frame encoding for a
Medicago sativa caffeic acid 3-O-methyltransferase enzyme or a
fragment thereof under expression control of a
lignification-associated tissue specific promoter to form a
transgenic cell; and cultivating said transgenic cell under
conditions conducive to regeneration and plant growth.
8. A method for producing a forage legume having altered lignin
composition comprising transforming a forage legume cell with a DNA
construct comprising in tandem at least one open reading frame
encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or a
fragment thereof under expression control of a first
lignification-associated tissue specific promoter and at least one
open reading frame encoding for a caffeic acid
3-O-methyltransferase enzyme or a fragment thereof under expression
control of a second lignification-associated tissue specific
promoter to form a transgenic cell, wherein said first and second
lignification-associated tissue specific promoter can be the same
or different; and cultivating said transgenic cell under conditions
conducive to regeneration and plant growth.
9. A method for improving the digestibility of forage legumes
comprising stably incorporating into the genome of said forage
legume a DNA construct comprising at least one open reading frame
encoding for a 3-O-methyltransferase enzyme or a fragment thereof
from the lignin biosynthetic pathway under expression control of a
lignification-associated tissue specific promoter, wherein
expression of said enzyme or fragment produces a change in lignin
composition in said forage legume.
10. The method of claim 9, wherein said enzyme is caffeoyl CoA
3-O-methyltransferase.
11. The method of claim 10, wherein said forage legume is
co-transformed with at least one open reading frame encoding for a
caffeic acid 3-O-methyltransferase enzyme or fragment thereof under
expression control of a lignification-associated tissue specific
promoter.
12. The method of claim 9, wherein said DNA construct comprising in
tandem at least one open reading frame encoding for a caffeoyl CoA
3-O-methyltransferase enzyme or a fragment thereof under expression
control of a first lignification-associated tissue specific
promoter and at least one open reading frame encoding for a caffeic
acid 3-O-methyltransferase enzyme or a fragment thereof under
expression control of a second lignification-associated tissue
specific promoter, wherein said first and second
lignification-associated tissue specific promoter can be the same
or different.
13. The method of claim 9, 10, 11 or 12, wherein guaiacyl lignin
content is reduced.
14. A method for producing a woody plant having altered lignin
composition comprising transforming a woody plant cell with a DNA
construct comprising at least one open reading frame encoding for a
Medicago sativa caffeoyl CoA 3-O-methyltransferase enzyme or
fragment thereof under expression control of a
lignification-associated tissue specific promoter to form a
transgenic cell; and cultivating said transgenic cell under
conditions conducive to regeneration and plant growth.
15. The method of claim 14, wherein said woody plant cell is
co-transformed with a DNA construct comprising at least one open
reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or a fragment thereof under expression
control of a lignification-associated tissue specific promoter.
16. A method for producing a woody plant having altered lignin
composition comprising transforming a woody plant cell with a DNA
construct comprising at least one open reading frame encoding for a
Medicago sativa caffeic acid 3-O-methyltransferase enzyme or
fragment thereof under expression control of a
lignification-associated tissue specific promoter to form a
transgenic cell; and cultivating said transgenic cell under
conditions conducive to regeneration and plant growth.
17. A method for producing a woody plant having altered lignin
composition comprising transforming a woody plant cell with a DNA
construct comprising in tandem at least one open reading frame
encoding for a Medicago sativa caffeoyl CoA 3-O-methyltransferase
enzyme or fragment thereof under expression control of a first
lignification-associated tissue specific promoter and at least one
open reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or fragment thereof under expression
control of a second lignification-associated tissue specific
promoter to form a transgenic cell, wherein said first and second
lignification-associated tissue specific promoter can be the same
or different; and cultivating said transgenic cell under conditions
conducive to regeneration and plant growth.
18. A method for modulating the lignin content of a woody plant
comprising transforming a woody plant cell with a DNA construct
comprising at least one open reading frame encoding for a Medicago
saliva caffeoyl CoA 3-O-methyltransferase enzyme or fragment
thereof under expression control of a lignification-associated
tissue specific promoter to form a transgenic cell; and cultivating
said transgenic cell under conditions conducive to regeneration and
plant growth.
19. The method of claim 18, wherein said woody plant cell is
co-transformed with a DNA construct comprising at least one open
reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or a fragment thereof under expression
control of a lignification-associated tissue specific promoter.
20. A method for modulating the lignin content of a woody plant
comprising transforming a woody plant cell with a DNA construct
comprising at least one open reading frame encoding for a Medicago
saliva caffeic acid 3-O-methyltransferase enzyme or fragment
thereof under expression control of a lignification-associated
tissue specific promoter to form a transgenic cell; and cultivating
said transgenic cell under conditions conducive to regeneration and
plant growth.
21. A method for modulating the lignin content of a woody plant
comprising transforming a woody plant cell with a DNA construct
comprising in tandem at least one open reading frame encoding for a
Medicago sativa caffeoyl CoA 3-O-methyltransferase enzyme or
fragment thereof under expression control of a first
lignification-associated tissue specific promoter or fragment
thereof and at least one open reading frame encoding for a Medicago
sativa caffeic acid 3-O-methyltransferase enzyme or fragment
thereof under expression control of a second
lignification-associated tissue specific promoter to form a
transgenic cell, wherein said first and second
lignification-associated tissue specific promoter can be the same
or different; and cultivating said transgenic cell under conditions
conducive to regeneration and plant growth.
22. A method for making lignins with altered dimer bonding patterns
comprising transforming a plant cell with a DNA construct
comprising at least one open reading frame encoding for a caffeoyl
CoA 3-O-methyltransferase enzyme or fragment thereof under
expression control of a lignification-associated tissue specific
promoter to form a transgenic cell; and cultivating said transgenic
cell under conditions conducive to regeneration and plant
growth.
23. The method of claim 22, wherein said plant cell is
co-transformed with a DNA construct comprising at least one open
reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or a fragment thereof under expression
control of a lignification-associated tissue specific promoter.
24. A method for making lignins with altered dimer bonding patterns
comprising transforming a plant cell with a DNA construct
comprising at least one open reading frame encoding for a caffeic
acid 3-O-methyltransferase enzyme or fragment thereof under
expression control of a lignification-associated tissue specific
promoter to form a transgenic cell; and cultivating said transgenic
cell under conditions conducive to regeneration and plant
growth.
25. A method for making lignins with altered dimer bonding patterns
comprising transforming a plant cell with a DNA construct
comprising in tandem at least one open reading frame encoding for a
caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof under
expression control of a first lignification-associated tissue
specific promoter and at least one open reading frame encoding for
a caffeic acid 3-O-methyltransferase enzyme or fragment thereof
under expression control of a second lignification-associated
tissue specific promoterto form a transgenic cell, wherein said
first and second lignification-associated tissue specific promoter
can be the same or different; and cultivating said transgenic cell
under conditions conducive to regeneration and plant growth.
26. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, wherein the open
reading frame is in a sense orientation.
27. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, wherein the open
reading frame is in an antisense orientation.
28. The method of claim 26, wherein said promoter is a bean PAL2
promoter.
29. The method of claim 27, wherein said promoter is a bean PAL2
promoter.
30. A plant transformed by the method of claim 26.
31. A plant transformed by the method of claim 27.
32. A plant transformed by the method of claim 28.
33. A plant transformed by the method of claim 29.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisonal
Application No. 60/192,086 filed Mar. 24, 2000.
TECHNICAL FIELD OF INVENTION
[0002] This invention relates to a method of transforming plants,
transformed plants and use thereof.
BACKGROUND OF THE INVENTION
[0003] Lignin is the major structural component of secondarily
thickened plant cell walls. It is a complex polymer of hydroxylated
and methoxylated phenylpropane units, linked via oxidative coupling
that is probably catalyzed by both peroxidases and laccases
(Boudet, et al. 1995. "Tansley review No. 80: Biochemistry and
molecular biology of lignification," New Phytologist 129:203-236).
Lignin imparts mechanical strength to stems and trunks, and
hydrophobicity to water-conducting vascular elements. Although the
basic enzymology of lignin biosynthesis is reasonably well
understood, the regulatory steps in lignin biosynthesis and
deposition remain to be defined (Davin, L. B. and Lewis, N. G.
1992. "Phenylpropanoid metabolism: biosynthesis of monolignols,
lignans and neolignans, lignins and suberins," Rec Adv Phytochem
26:325-375).
[0004] There is considerable interest in the potential for genetic
manipulation of lignin levels and/or composition to help improve
digestibility of forages and pulping properties of trees (Dixon, et
al. 1994. "Genetic manipulation of lignin and phenylpropanoid
compounds involved in interactions with microorganisms," Rec Adv
Phytochem 28:153178; Tabe, et al. 1993. "Genetic engineering of
grain and pasture legumes for improved nutritive value," Genetica
90:181-200; Whetten, R. and Sederoff, R. 1991. "Genetic engineering
of wood," Forest Ecology and Management 43:301-316). Small
decreases in lignin content have been reported to positively impact
the digestibility of forages (Casler, M. D. 1987. "In vitro
digestibility of dry matter and cell wall constituents of smooth
bromegrass forage," Crop Sci 27:931-934). By improving the
digestibility of forages, higher profitability can be achieved in
cattle and related industries. In forestry, chemical treatments
necessary for the removal of lignin from trees are costly and
potentially polluting.
[0005] Lignins contain three major monomer species, termed
p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S), produced by
reduction of CoA thioesters of coumaric, ferulic and sinapic acids,
respectively (see FIG. 1). In angiosperms, guaiacyl and syringyl
units predominate, and the S/G ratio affects the physical
properties of the lignin. The S and G units are linked through five
different dimer bonding patterns (Davin, L. B. and Lewis, N. G.
1992. Rec Adv Phytochem 26:325-375). The mechanisms that determine
the relative proportions of these linkage types in a particular
lignin polymer are currently unknown. Furthermore, there is
considerable debate as to whether lignin composition and structure
are tightly controlled, or are flexible depending upon monomer
availability (Lewis, N. G. 1999. "A 20th century roller coaster
ride: a short account of lignification," Current Opinion in Plant
Biology 2:153-162; Sederoff, et al. 1999, "Unexpected variation in
lignin," Current Opinion in Plant Biology 2:145-152).
[0006] Lignin levels increase with progressive maturity in stems of
forage crops, including legumes such as alfalfa (Jung, H. G. and
Vogel, K. P. 1986. "Influence of lignin on digestibility of forage
cell wall material," J Anim Sci 62:1703-1712) and in grasses such
as tall fescue (Buxton, D. R. and Russell, J. R. 1988. "Lignin
constituents and cell wall digestibility of grass and legume
stems," Crop Sci 28:553-558). In addition, lignin composition
changes with advanced maturity towards a progressively higher S/G
ratio (Buxton, D. R. and Russell, J. R. 1988. Crop Sci 28:553-558).
Both lignin concentration (Albrecht, et al. 1987. "Cell-wall
composition and digestibility of alfalfa stems and leaves," Crop
Sci 27:735-741; Casler, M. D. 1987. Crop Sci 27:931-934; Jung, H.
G. and Vogel, K. P. 1986. J Anim Sci 62:1703-1712) and lignin
methoxyl content, reflecting increased S/G ratio (Sewalt, et al.
1996. "Lignin impact on fiber degradation. 1. Quinone methide
intermediates formed from lignin during in vitro fermentation of
corn stover," J Sci Food Agric 71:195-203), have been reported to
negatively correlate with forage digestibility for ruminant
animals. Although a number of studies have linked decreased forage
digestibility to increased S/G ratio as a function of increased
maturity (Buxton, D. R. and Russell, J. R. 1988. Crop Sci
28:553-558; Grabber, et al. 1992. "Digestion kinetics of parenchyma
and sclerenchyma cell walls isolated from orchardgrass and
switchgrass," Crop Sci 32: 806-810), other studies have questioned
the effect of lignin composition on digestibility (Grabber, et al.
1997. "p-hydroxyphenyl, guaiacyl, and syringyl lignins have similar
inhibitory effects on wall degradability," J Agric Food Chem
45:2530-2532). Further, the hardwood gymnosperm lignins are highly
condensed, essentially lacking S residues, and this makes them less
amenable to chemical pulping, in apparent contradiction to the
concept that reducing S/G ratio would be beneficial for forage
digestibility. The reported lack of agreement in the relationship
of lignin composition to forage digestibility and chemical pulping
is partly due to the fact that the studies to date either have been
in vitro, or have compared plant materials at different
developmental stages, different varieties or even different
species. Therefore, the development of isogenic lines that can be
directly compared to reveal the effects of altered S/G ratio on
forage digestibility would be beneficial.
[0007] The formation of the G and S units of lignin requires the
activity of O-methyl-transferase enzymes. In angiosperms, the
caffeic acid 3-O-methyltransferase (COMT) of lignin biosynthesis
was originally described as being bifunctional, converting caffeic
acid to ferulic acid and converting 5-hydroxyferulic acid to
sinapic acid (Davin, L. B. and Lewis, N. G. 1992. Rec Adv Phytochem
26:325-375), as shown in FIG. 1. Methylation of the caffeate moiety
also occurs at the level of the CoA thioester, catalyzed by
caffeoyl CoA 3-O-methyltransferase (CCOMT) (Pakusch, et al., 1989,
"S-adenosyl-L-methionine: trans-caffeoyl-coenzyme A
3-O-methyltransferase from elicitor-treated parsley cell suspension
cultures," Arch Biochem Biophys 271:488-494). The involvement of
the CCOMT enzyme in a parallel pathway to lignin monomer formation
has been proposed (Ye, et al. 1994. "An alternative methylation
pathway in lignin biosynthesis in Zinnia," Plant Cell 6:1427-1439;
Zhong, et al. 1998. "Dual methylation pathways in lignin
biosynthesis," Plant Cell 10:2033-2045). In vivo labeling studies
in Magnolia kobus have shown that the methylation status of lignin
monomers can also be determined at the level of the aldehyde or
alcohol (Chen, et al. 1999. "Evidence for a novel biosynthetic
pathway that regulates the ratio of syringyl to guaiacyl residues
in lignin in the differentiating xylem of Magnolia kobus DC,"
Planta 207:597-603). This is supported by the observation that the
enzyme designated as ferulate 5-hydroxylase has a higher affinity
for feruloyl aldehyde than for ferulic acid, at least in sweet gum
(Osakabe, et al. 1999. "Coniferyl aldehyde 5-hydroxylation and
methylation direct syringyl lignin biosynthesis in angiosperms,"
Proc Natl Acad Sci USA 96:8955-8960) and Arabidopsis (Humphreys, et
al. 1999. "New routes for lignin biosynthesis defined by
biochemical characterization of recombinant ferulate 5-hydroxylase,
a multifunctional cytochrome P450-dependent monooxygenase," Proc
Natl Acad Sci USA 96:10045-10050). Furthermore, 5-hydroxyconiferyl
aldehyde has recently been shown to be a good substrate for COMT
from various tree species (Li, et al. 2000. "5-Hydroxyconiferyl
aldehyde modulates enzymatic methylation for syringyl monolignol
formation, a new view of monolignol biosynthesis in angiosperms," J
Biol Chem 275:6537-6545). It has been reported that the inhibitory
effect of 5-hydroxyconiferyl aldehyde on methylation of caffeate by
COMT might prevent COMT from carrying out the first methylation
step in the biosynthesis of S lignin (Li, et al. 2000. J Biol Chem
275:6537-6545). Thus, although studies of enzyme substrate
specificities in vitro suggest that lignin monomers can be formed
via the operation of a complex metabolic grid, involving
O-methylation at multiple stages as shown in FIG. 1, whether this
occurs in vivo has yet to be determined.
[0008] Several studies have addressed the properties of the
O-methyltransferases involved in lignin biosynthesis in the world's
major forage legume, alfalfa (Medicago sativa L.) (Gowri, et al.
1991. "Stress responses in alfalfa (Medicago sativa L.) X.
Molecular cloning and expression of S-adenosyl-L-methionine:
caffeic acid 3-O-methyltransferase, a key enzyme of lignin
biosynthesis," Plant Physiol 97:7-14; Inoue, et al. 1998.
"Developmental expression and substrate specificities of alfalfa
caffeic acid 3-O-methyltransferase and caffeoyl CoA
3-O-methyltransferase in relation to lignification," Plant Physiol
117:761-770; Kersey, et al. 1999. "Immunolocalization of two lignin
O-methyltransferases in stems of alfalfa (Medicago sativa L.),"
Protoplasma 209:46-57). COMT from alfalfa expressed in E. coli
shows preference (approximately 2:1) for 5-hydroxyferulic acid over
caffeic acid, whereas CCOMT shows a similar degree of preference
for caffeoyl CoA compared to 5-hydroxyferuolyl CoA (Inoue, et al.
1998. Plant Physiol 117:761-770). These studies suggest, but do not
prove, that COMT may be involved preferentially in the formation of
S lignin in alfalfa, and CCOMT in the formation of G lignin.
[0009] The substrate preference of COMT in crude alfalfa stem
extracts changes with increasing internode maturity, in a manner
consistent with the increase in lignin methoxyl group content with
increasing maturity (Inoue, et al. 1998. Plant Physiol 117:761-770;
Inoue, et al. 2000. "Substrate preferences of caffeic
acid/5-hydroxyferulic acid 3-O-methyltransferases in developing
stems of alfalfa (Medicago sativa L.)," Arch Biochem Biophys
375:175-182). Thus, in young internodes, the activity shows a
preference for caffeic acid over 5-hydroxyferulic acid, whereas the
opposite is true in the older internodes. An O-methyltransferase
with preference for caffeic acid (COMT II) has recently been
separated from the previously characterized COMT, and does not
react with antisera recognizing the products of the previously
characterized alfalfa COMT or CCOMT genes. This enzyme is most
active against caffeic acid, for which it has a very low Km value
(approximately 40-fold lower than lignification-associated COMT),
but also methylates 5-hydroxyferulic acid, caffeoyl CoA,
5-hydroxyferuolyl CoA, quercetin and catechol (Inoue, et al. 2000.
Arch Biochem Biophys 375:175-182). It is only present in young
internodes and has disappeared by the fifth internode.
[0010] Tissue print hybridization analysis indicates that both COMT
and CCOMT transcripts are localized to developing xylem elements in
alfalfa stems, whereas CCOMT transcripts are also found in phloem
(Inoue, et al. 1998. Plant Physiology 117:761-770).
Immunolocalization studies at the light and electron microscope
levels demonstrated expression of both COMT and CCOMT in the
cytoplasm of alfalfa xylem parenchyma cells (Kersey, et al. 1999
Protoplasma 209:46-57). The presence of both enzymes in the same
cells is consistent with the "metabolic grid" hypothesis for lignin
monomer formation.
[0011] There have been several reports on the effects of
down-regulation of COMT activity on lignin content and composition
in transgenic tobacco and poplar (Ni, et al. 1994. "Reduced lignin
in transgenic plants containing an engineered caffeic acid
O-methyltransferase antisense gene," Transgenic Res 3:120-126;
Atanassova, et al. 1995. "Altered lignin composition in transgenic
tobacco expressing O-methyltransferase sequences in sense and
antisense orientation," Plant J 8:465-477; Van Doorsselaere, et al.
1995. "A novel lignin in poplar trees with a reduced caffeic
acid/5-hydroxyferulic acid O-methyltransferase activity," Plant J
8:855-864; Zhong, et al. 1998. Plant Cell 10:2033-2045). The
results of these studies have been somewhat contradictory, possibly
due to unspecified differences in tissue maturity, use of
homologous versus heterologous transgenes, and use of different
methods for lignin analysis. However, in cases where COMT has been
reduced to levels below approximately 20% of wild-type by
expression of a homologous transgene, a strong reduction in S/G
ratio is accompanied by no apparent change in lignin content
(Atanassova, et al. 1995. Plant J 8:465-477; Van Doorsselaere, et
al. 1995. Plant J 8:855-864). In tobacco, down-regulation of CCOMT
leads to a corresponding decrease in Klason lignin levels
accompanied by decreases in the absolute levels of both S and G
units (Zhong, et al. 1998. Plant Cell 10:2033-2045).
[0012] Most studies on genetic modification of lignin biosynthesis
in transgenic plants have utilized the cauliflower mosaic virus 35S
promoter to drive expression of sense or antisense
lignification-associated genes (Halpin, et al. 1994. "Manipulation
of lignin quality by down-regulation of cinnamyl alcohol
dehydrogenase," Plant J 6:339-350; Ni, et al. 1994. Transgenic Res
3:120-126; Atanassova, et al. 1995. Plant J 8:465-477; Van
Doorsselaere, et al. 1995. Plant J 8:855-864; Piquemal, et al.
1998. "Down-regulation of cinnamoyl-CoA reductase induces
significant changes of lignin profiles in transgenic tobacco
plants," Plant J 13:71-83; Zhong, et al. 1998. Plant Cell
10:2033-2045; Baucher, et al. 1999, "Down-regulation of cinnamyl
alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.)
and the effect on lignin composition and digestibility," Plant Mol
Biol 39:437-447). However, more effective down-regulation may be
obtained by driving expression of the transgene by a
vascular-tissue specific promoter. For example, modification of
lignin composition by overexpression of ferulate 5-hydroxylase in
transgenic Arabidopsis was more effective if the transgene was
driven by the lignification-associated Arabidopsis cinnamate
4-hydroxylase promoter than by the constitutive 35S promoter
(Meyer, et al. 1998. "Lignin monomer composition is determined by
the expression of a cytochrome P450-dependent monooxygenase in
Arabidopsis," Proc Natl Acad Sci USA 95:6619-6623).
[0013] To date, there have been very few published reports on the
genetic modification of lignin in forage crops, and most studies
having concentrated on model systems such as Arabidopsis and
tobacco, or tree species such a poplar. In one study,
down-regulation of cinnamnyl alcohol dehydrogenase, an enzyme later
in the monolignol pathway than COMT or CCOMT, led to a small but
significant improvement in in vitro dry matter digestibility in
transgenic alfalfa (Baucher, et al. 1999. Plant Mol Biol
39:437-447). U.S. Pat. No. 5,451,514 discloses a method of altering
the content or composition of lignin in a plant by stably
incorporating into the genome of the plant a recombinant DNA
encoding an mRNA having sequence similarity to cinnamyl alcohol
dehydrogenase. U.S. Pat. No. 5,850,020 discloses a method for
modulating lignin content or composition by transforming a plant
cell with a DNA construct with at least one open reading frame
coding for a functional portion of one of several enzymes isolated
from Pinus radiata (pine) or a sequence having 99% homology to the
isolated gene: cinnamate 4-hydroxylase (C4H), coumarate
3-hydroxylase (C3H), phenolase (PNL), O-methyltransferase (OMT),
cinnamoyl-CoA reductase (CCR), phenylalanine ammonia-lyase (PAL),
4-coumarate:CoA ligase (4CL), and peroxidase (POX). U.S. Pat. No.
5,922,928 discloses a method of transforming and regenerating
Populus species to alter the lignin content and composition using
an O-methyltransferase gene. The question of how altering S/G ratio
might affect digestibility of forage species is still
unanswered.
[0014] It has now been found that transformation of plants with the
lignin biosynthetic enzyme genes COMT or CCOMT in either a sense or
antisense orientation under a lignification-associated tissue
specific promoter, leading to the down-regulation of the
corresponding homologous gene as well as reduced lignin content and
modified lignin composition in the transgenic plants, results in
significant improvements in forage digestibility, particularly in
the case of CCOMT down-regulation.
SUMMARY OF THE INVENTION
[0015] In one aspect, the present invention is a method for
modulating the lignin content of a forage legume comprising
transforming a forage legume cell with a vector comprising a
lignification-associated tissue specific promoter functionally
linked to a DNA construct comprising at least one open reading
frame encoding for either a caffeoyl CoA 3-O-methyltransferase
enzyme or fragment thereof or a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or fragment thereof; and generating
plants from the transformed forage legume cell. In another
embodiment, the forage legume cell is co-transformed with one
vector comprising a lignification-associated tissue specific
promoter functionally linked to a DNA construct comprising at least
one open reading frame encoding for a caffeoyl CoA
3-O-methyltransferase enzyme or fragment thereof and another vector
comprising a lignification-associated tissue specific promoter
functionally linked to a DNA construct comprising at least one open
reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or fragment thereof. In yet another
embodiment, a DNA construct comprising in tandem at least one open
reading frame encoding for a caffeoyl CoA 3-O-methyltransferase
enzyme or fragment thereof under expression control of a first
lignification-associated tissue specific promoter and at least one
open reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or fragment thereof under expression
control of a second lignification-associated tissue specific
promoter, wherein said first and second lignification-associated
tissue specific promoter can be the same or different, can be used
in this method. The open reading frame can be in either a sense
orientation or an antisense orientation. An exemplary
lignification-associated tissue specific promoter is a bean PAL2
promoter.
[0016] In another aspect, the present invention is a method for
producing a forage legume having altered lignin composition
comprising transforming a forage legume cell with a DNA construct
comprising either at least one open reading frame encoding for a
caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof or a
Medicago sativa caffeic acid 3-O-methyltransferase enzyme or
fragment thereof under expression control of a
lignification-associated tissue specific promoter to form a
transgenic cell; and cultivating said transgenic cell under
conditions conducive to regeneration and plant growth. In another
embodiment, the forage legume cell is co-transformed with one DNA
construct comprising at least one open reading frame encoding for a
caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof under
expression control of a lignification-associated tissue specific
promoter and another DNA construct comprising at least one open
reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or fragment thereof under expression
control of a lignification-associated tissue specific promoter. In
yet another embodiment, a DNA construct comprising in tandem at
least one open reading frame encoding for a caffeoyl CoA
3-O-methyltransferase enzyme or fragment thereof under expression
control of a first lignification-associated tissue specific
promoter and at least one open reading frame encoding for a
Medicago sativa caffeic acid 3-O-methyltransferase enzyme or
fragment thereof under expression control of a second
lignification-associated tissue specific promoter, wherein said
first and second lignification-associated tissue specific promoter
can be the same or different, can be used in this method. The open
reading frame can be in either a sense orientation or an antisense
orientation. An exemplary lignification-associated tissue specific
promoter is a bean PAL2 promoter.
[0017] In another aspect, the present invention is a method for
improving the digestibility of forage legumes comprising stably
incorporating into the genome of said forage legume a DNA construct
comprising at least one open reading frame encoding for a
3-O-methyltransferase enzyme or a fragment thereof from the lignin
biosynthetic pathway under expression control of a
lignification-associated tissue specific promoter, wherein
expression of the enzyme or enzyme fragment produces a change in
lignin composition in the forage legume. One enzyme useful in this
method is caffeoyl CoA 3-O-methyltransferase, which preferably
causes a reduction in guaiacyl lignin content. In another
embodiment, the forage legume is co-transformed with one DNA
construct comprising at least one open reading frame encoding for a
caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof under
expression control of a lignification-associated tissue specific
promoter and another DNA construct comprising at least one open
reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or fragment thereof under expression
control of a lignification-associated tissue specific promoter. In
yet another embodiment, a DNA construct comprising in tandem at
least one open reading frame encoding for a caffeoyl CoA
3-O-methyltransferase enzyme or fragment thereof under expression
control of a first lignification-associated tissue specific
promoter and at least one open reading frame encoding for a
Medicago sativa caffeic acid 3-O-methyltransferase enzyme or
fragment thereof under expression control of a second
lignification-associated tissue specific promoter, wherein said
first and second lignification-associated tissue specific promoter
can be the same or different, can be used in this method. The open
reading frame can be in either a sense orientation or an antisense
orientation. An exemplary lignification-associated tissue specific
promoter is a bean PAL2 promoter.
[0018] In another aspect, the present invention is a method for
producing a woody plant having altered lignin composition
comprising transforming a woody plant cell with a DNA construct
comprising at least one open reading frame encoding for a Medicago
sativa caffeoyl CoA 3-O-methyltransferase enzyme or a Medicago
sativa caffeic acid 3-O-methyltransferase enzyme or fragment
thereof under expression control of a lignification-associated
tissue specific promoter to form a transgenic cell; and cultivating
the transgenic cell under conditions conducive to regeneration and
plant growth. In another embodiment, the woody plant cell is
co-transformed with one DNA construct comprising at least one open
reading frame encoding for a caffeoyl CoA 3-O-methyltransferase
enzyme or fragment thereof under expression control of a
lignification-associated tissue specific promoter and another DNA
construct comprising at least one open reading frame encoding for a
Medicago sativa caffeic acid 3-O-methyltransferase enzyme or
fragment thereof under expression control of a
lignification-associated tissue specific promoter. In yet another
embodiment, a DNA construct comprising in tandem at least one open
reading frame encoding for a caffeoyl CoA 3-O-methyltransferase
enzyme or fragment thereof under expression control of a first
lignification-associated tissue specific promoter and at least one
open reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or fragment thereof under expression
control of a second lignification-associated tissue specific
promoter, wherein said first and second lignification-associated
tissue specific promoter can be the same or different, can be used
in this method. The open reading frame can be in either a sense
orientation or an antisense orientation. An exemplary
lignification-associated tissue specific promoter is a bean PAL2
promoter.
[0019] In another aspect, the present invention is a method for
modulating the lignin content of a woody plant comprising
transforming a woody plant cell with a DNA construct comprising at
least one open reading frame encoding for a Medicago sativa
caffeoyl CoA 3-O-methyltransferase enzyme or a Medicago sativa
caffeic acid 3-O-methyltransferase enzyme or fragment thereof under
expression control of a lignification-associated tissue specific
promoter to form a transgenic cell; and cultivating the transgenic
cell under conditions conducive to regeneration and plant growth.
In another embodiment, the woody plant cell is co-transformed with
one DNA construct comprising at least one open reading frame
encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or
fragment thereof under expression control of a
lignification-associated tissue specific promoter and another DNA
construct comprising at least one open reading frame encoding for a
Medicago sativa caffeic acid 3-O-methyltransferase enzyme or
fragment thereof under expression control of a
lignification-associated tissue specific promoter. In yet another
embodiment, a DNA construct comprising in tandem at least one open
reading frame encoding for a caffeoyl CoA 3-O-methyltransferase
enzyme or fragment thereof under expression control of a first
lignification-associated tissue specific promoter and at least one
open reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or fragment thereof under expression
control of a second lignification-associated tissue specific
promoter, wherein said first and second lignification-associated
tissue specific promoter can be the same or different, can be used
in this method. The open reading frame can be in either a sense
orientation or an antisense orientation. An exemplary
lignification-associated tissue specific promoter is a bean PAL2
promoter.
[0020] In another aspect, the present invention is a method for
making lignins with altered dimer bonding patterns comprising
transforming a plant cell with a DNA construct comprising at least
one open reading frame encoding for a caffeoyl CoA
3-O-methyltransferase enzyme or a caffeic acid
3-O-methyltransferase enzyme or a fragment thereof under expression
control of a lignification-associated tissue specific promoter to
form a transgenic cell; and cultivating the transgenic cell under
conditions conducive to regeneration and plant growth. In another
embodiment, the plant cell is co-transformed with one DNA construct
comprising at least one open reading frame encoding for a caffeoyl
CoA 3-O-methyltransferase enzyme or fragment thereof under
expression control of a lignification-associated tissue specific
promoter and another DNA construct comprising at least one open
reading frame encoding for a Medicago sativa caffeic acid
3-O-methyltransferase enzyme or fragment thereof under expression
control of a lignification-associated tissue specific promoter. In
yet another embodiment, a DNA construct comprising in tandem at
least one open reading frame encoding for a caffeoyl CoA
3-O-methyltransferase enzyme or fragment thereof under expression
control of a first lignification-associated tissue specific
promoter and at least one open reading frame encoding for a
Medicago sativa caffeic acid 3-O-methyltransferase enzyme or
fragment thereof under expression control of a second
lignification-associated tissue specific promoter, wherein said
first and second lignification-associated tissue specific promoter
can be the same or different, can be used in this method. The open
reading frame can be in either a sense orientation or an antisense
orientation. An exemplary lignification-associated tissue specific
promoter is a bean PAL2 promoter.
[0021] In yet another aspect, the present invention is a plant
transformed by any of the methods disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A and 1B depict proposed biochemical pathways to
lignin monomers. The "metabolic grid" shown in this scheme
incorporates the results of recent studies suggesting previously
unexpected substrate specificities for ferulate 5-hydroxylase (F5H)
and COMT (Humphreys, et al. 1999. Proc Natl Acad Sci USA
96:10045-10050; Osakabe, et al. 1999 Proc Natl Acad Sci USA
96:8955-8960; Li, et al. 2000. J Biol Chem 275:6537-6545).
[0023] FIG. 2 depicts binary constructs used for genetic
modification of COMT and CCOMT expression in transgenic alfalfa.
PAL2 is the bean phenylalanine ammonia-lyase PAL2 promoter from
-183 to -1226 bp (Liang, et al. 1989. "Developmental and
environmental regulation of a phenylalanine ammonia-lyase
13-glucuronidase gene fusion in transgenic tobacco plants." Proc
Natl Acad Sci USA 86:9284-9288) and NOS, the nopaline synthase
terminator. Directionality of COMT and CCOMT is indicated by the
arrows relative to the direction of the PAL2 promoter. Constructs
containing both COMT and CCOMT in sense or antisense orientations
were made by duplication of the PAL2/COMT/NOS and PAL2/CCOMT/NOS
cassettes, and therefore, each cDNA is under control of a separate
PAL2 promoter. Introduction of both transgenes into a single plant
was also achieved by co-transformation (Irdani, et al. 1998.
"Construction of a new vector conferring methotrexate resistance in
Nicotiana tabacum plants," Plant Mol Biol 37:1079-1084) with single
COMT and CCOMT constructs. All constructs are in the binary vector
pCAMBIA3300.
[0024] FIGS. 3A-3J depict COMT or CCOMT activities in stem tissue
of control lines and COMT and/or CCOMT transgenic lines. FIG. 3A
shows COMT activity in control plants transformed with empty
pCAMBIA3300 vector. FIG. 3B shows COMT activity in plants
transformed with COMT in the sense orientation ("SC"). FIG. 3C
shows COMT activity in plants transformed with a construct
containing both COMT and CCOMT in the sense orientation ("DS"), or
by co-transformation with individual antisense COMT and CCOMT
constructs. FIG. 3D shows COMT activity in plants transformed with
COMT in the antisense orientation ("AC"). FIG. 3E shows COMT
activity in plants transformed with a construct containing both
COMT and CCOMT in the antisense orientation ("DA"), or by
co-transformation with individual antisense COMT and CCOMT
constructs. FIG. 3F shows CCOMT activity in control plants
transformed with empty pCAMBIA3300 vector. FIG. 3G shows CCOMT
activity in plants transformed with CCOMT in the sense orientation
("SCC"). FIG. 3H shows CCOMT activity in plants transformed with a
construct containing both COMT and CCOMT in the sense orientation
("DS"), or by co-transformation with individual antisense COMT and
CCOMT constructs. FIG. 3I shows CCOMT activity in plants
transformed with CCOMT in the antisense orientation ("ACC"). FIG.
3J shows CCOMT activity in plants transformed with a construct
containing both COMT and CCOMT in the antisense orientation ("DA"),
or by co-transformation with individual antisense COMT and CCOMT
constructs. The bars represent the means (solid lines) and standard
deviations (dashed lines) of the respective control populations.
Enzyme activities were determined in the 6.sup.th-9.sup.th
internodes of stems of identical developmental stage.
[0025] FIG. 4 depicts typical gas chromatographs showing
thioacidolysis products from lignin samples of wild-type (WT),
COMT-suppressed (SC5), and CCOMT-suppressed (ACC305) alfalfa
plants. G, S and 5-hydroxyguaiacyl (5OHG) units are marked. The
peaks appear as doublets because of the formation of erythro and
threo isomers of each degradation product.
[0026] FIG. 5 depicts the full nucleotide sequence for the 1097 bp
coding region of alfalfa COMT (nucleotides 31-1128 of GenBank
Accession No. M63853).
[0027] FIG. 6 depicts the full nucleotide sequence for the 743 bp
coding region of alfalfa CCOMT (nucleotides 36-779 of GenBank
Accession No. U20736).
DETAILED DESCRIPTION
[0028] Using the methods of the present invention, the lignin
content and composition of a forage legume such as alfalfa can be
modified. Forage legumes are transformed with genes encoding
O-methyltransferase (OMT) enzymes from the lignin biosynthetic
pathway inserted in the sense or antisense orientations and under a
lignification-associated tissue specific promoter. This
transformation method can result in a variety of outcomes: a
down-regulation of the corresponding homologous OMT genes, gene
silencing, reduced OMT activity levels, reduced lignin content, and
modified lignin composition in transgenic plants, and increased
digestibility of transgenic plant materials in ruminant animals.
Transforming forage legumes with OMT enzymes has now made it
possible to produce plants having modified lignin content and
composition for direct comparison of the effects of lignin content
and/or composition on forage digestibility. A preferred embodiment
of the invention includes genetically engineering forage varieties
with modified lignin to increase forage digestibility in animals.
In another embodiment, plants are modified to alter lignins and
improve pulping characteristics for the paper industry.
[0029] Transformation methods of the present invention utilize
binary constructs comprising DNA sequences encoding
O-methyltransferase (OMT) enzymes from the lignin biosynthetic
pathway, preferably in conjunction with a gene promoter sequence
and a gene termination sequence. In the present invention, full or
partial DNA sequences either isolated from alfalfa or produced by
recombinant means and encoding or partially encoding
O-methyltransferase (OMT) enzymes, are used in the transformation
process. Preferably, a full length alfalfa COMT or CCOMT cDNA
sequence in the sense or antisense orientation is placed in a
binary vector with the cDNA being driven by a
lignification-associated promoter. Alternatively, constructs can
contain tandem COMT and CCOMT cDNAs in sense or antisense
orientations, with each cDNA being driven independently by a
lignification-associated promoter. While full length COMT and CCOMT
cDNA sequences are preferred, a genomic DNA sequence or a cDNA
sequence encoding a portion of COMT or CCOMT can be used in the
present invention, provided that the DNA sequence is of sufficient
length so as to encode a fragment of the enzyme wherein the
fragment is effective for causing antisense inhibition or gene
silencing of OMT expression.
[0030] To drive expression of transgenes in forage legumes, a
lignification-associated promoter is utilized. Any
lignification-associated promoter known in the art can be useful in
the present invention. However, since COMT and CCOMT enzymes are
expressed in the xylem and phloem parenchyma in alfalfa,
lignification-associated promoters selective for vascular tissue
are preferred. The promoter gene sequence can be endogenous to the
target plant, or it can be exogenous provided that the promoter is
functional in the target plant. A lignification-associated tissue
specific promoter can be used to target the production of sense or
antisense RNA in the tissue of interest. An exemplary gene promoter
sequence for use in forage legumes is the bean (Phaseolus vulgaris)
PAL2 promoter.
[0031] Many gene termination sequences known in the art are useful
in the present invention. The gene termination sequence can be from
the same gene as the gene promoter sequence or from a different
gene. An exemplary gene terminator sequence is the 3' end of the
nopaline synthase, or nos, gene.
[0032] The DNA constructs of the present invention can optionally
contain any selection marker effective in plant cells as a means of
detecting successful transformation. Exemplary selection markers
include antibiotic or herbicide resistance genes. Preferred
selectable markers include a neomycin phosphotransferase gene or
phosphinothricin acetyl transferase (bar) gene. For example, a
preferable selection marker is the bar gene encoding
phosphinothricin acetyl transferase which confers resistance to
phosphinothricin-based herbicides.
[0033] Transformation methods of the present invention include any
means known in the art by which forage legumes can be successfully
transformed using the DNA constructs disclosed herein.
Agrobacterium-mediated transformation by leaf disk or biolistic
techniques followed by regeneration through somatic embryogenesis,
direct organogenesis, or vacuum infiltration techniques that
by-pass the need for tissue culture, are preferred.
EXAMPLE 1
Lignin Modification of Alfalfa
[0034] Alfalfa plants were successfully transformed using the
lignin-modifying transformation methods of the present invention.
Alfalfa plants exhibiting changes in both lignin content and
composition were obtained.
[0035] To drive expression of transgenes in forage legumes, we
chose the bean PAL2 promoter, which was previously characterized as
associated with lignification and strongly expressed in the
vascular tissue of transgenic tobacco (Leyva, et al. 1992.
"Cis-element combinations determine phenylalanine ammonia-lyase
gene tissue specific expression patterns," Plant Cell 4:263-271;
Shufflebottom, et al. 1993. "Transcription of two members of a gene
family encoding phenylalanine ammonia-lyase leads to remarkably
different cell specificities and induction patterns," Plant J
3:835-845). A number of gene constructs were made, either to test
the tissue specificity of the bean PAL2 promoter in alfalfa using
the reporter gene GUS, or to drive expression of the alfalfa
O-methyltransferase genes COMT and/or CCOMT in the sense or
antisense orientations. The bean PAL2 promoter was obtained from
the genomic clone gPAL2 (Cramer, et al. 1989. "Phenylalanine
ammonia-lyase gene organization and structure," Plant Mol Biol
12:367-383) and was cloned into the EcoRI/BamHI sites of pUC18.
Site-directed mutagenesis was used to delete the NdeI site in pUC
18 to create the plasmid pUC 18-PAL. The GUS open reading frame was
excised from the plasmid pGN100 (Reimann-Philipp, R. and Beachy, R.
N. 1993. "Coat protein-mediated resistance in transgenic tobacco
expressing the tobacco mosaic virus coat protein from
tissue-specific promoters," Mol Plant Microbe Interact 6:323-330)
by EcoRI/SmaI digestion, and two DNA polylinkers containing
different restriction sites, EcoRI-Bg/II-NdeI-BamHI-SmaI and
EcoRI-BglII-BamHI-NdeI-SmaI, were introduced independently between
the EcoRI and SmaI sites, respectively. A BglII/PstI fragment
containing the nopaline synthase (nos) terminator sequence was
inserted into the BamHI/PstI sites of pUC18-PAL to give the
plasmids pPTN1 and pPTN2, which contain the bean PAL2 promoter and
nos terminator. To create the cassette for gusA gene expression,
the bean PAL2 promoter was released from the plasmid pPTN2 by
digestion with EcoRI, and the ends were filled in with Klenow
fragment and then digested with BamHI. The plasmid ubi3-GUS
(Garbarino J. E. and Belknap W. R. 1994. "Isolation of a
ubiquitin-ribosomal protein gene (ubi3) from potato and expression
of its promoter in transgenic plants," Plant Mol Biol 24:119-127)
was treated with XbaI, Klenow, and BamHI to replace the ubi3
promoter with the isolated bean PAL2 promoter. The gusA expression
cassette was then cloned into HindIII/EcoRI cut pCAMB13 300 to
create the gusA expression construct pCAMGUS.
[0036] Constructs were introduced into Agrobacterium tumefaciens
LBA4404 using the Gibco BRL Lifetechnologies electroporation
procedure (Gibco BRL Lifetechnologies, Rockville, Md.). Leaf disc
transformation of alfalfa (cv Regen SY) was performed based on a
method described previously (Shahin, et al. 1986 "Transformation of
cultivated alfalfa using disarmed Agrobacterium tumefaciens," Crop
Sci 26:1235-1239; Thomas, et al. 1990. "Selection of interspecific
somatic hybrids of Medicago by using Agrobacterium-transformed
tissues," Plant Sci 69:189-198). Phosphinothricin (5 mg/L) was
added to the culture medium for selection of resistant
transformants. Alfalfa plants were grown in the greenhouse under
standard conditions. All transformations were performed with
clonally propagated material of one selected highly regenerable
line named 4D.
[0037] To confirm tissue specificity of the bean PAL2 promoter in
transgenic alfalfa, several independent plants were generated via
Agrobacterium-mediated transformation with the pCAMGUS binary
vector containing the GUS marker gene under control of the full
length (-182 to -1226 bp) bean PAL2 promoter, as illustrated in
FIG. 2. Histochemical GUS assays were then performed to determine
the cellular sites of PAL2 promoter activity. Hand sections of
alfalfa stems, roots, and petioles were incubated on ice for 30
minutes in 2% paraformaldehyde and 100 mM Na-phosphate buffer, pH
7.0. They were then vacuum infiltrated in 2 mM X-gluc in 50 mM
Na-phosphate buffer, pH 7.0, 0.5% Triton X-100 for 10 seconds,
followed by a 2 hour incubation at 37.degree. C. After staining,
green tissues were bleached in 70% ethanol several times to allow
visualization of the blue staining. Transverse sections from these
plants (containing the PAL2-GUS construct pCAMGUS) stained blue
with the chromogenic substrate X-gluc revealed GUS expression in
the vascular tissue of roots, stems, and petioles that was absent
from similarly stained non-transgenic control tissue (containing
empty pCAMBIA3300 vector). Although the majority of the staining in
stem and petiole tissue was localized to vascular parenchyma cells,
there was also some staining of mesophyll cells and epidermal cells
of petioles. These results were reproduced in other independent
transformants. The relatively selective vascular tissue staining
indicated that the bean PAL2 promoter would be suitable for
directing expression of COMT and CCOMT sense and antisense
transgenes, as these enzymes are expressed in xylem and phloem
parenchyma in alfalfa (Kersey, et al. 1999. Protoplasma
209:46-57).
[0038] Full length alfalfa COMT and CCOMT cDNA sequences in the
sense and antisense orientations were placed under control of the
bean PAL2 promoter in the binary vector pCAMBIA3300, as summarized
in FIG. 2. Additional constructs contained tandem COMT and CCOMT
cDNAs, in the sense or antisense orientations, with each cDNA
driven independently by a bean PAL2 promoter, as shown in FIG. 2.
The COMT and CCOMT coding sequences were isolated from separate OMT
cDNA constructs in pET vectors (Inoue, et al. 1998. Plant Physiol
117:761-770), which contained the 1097 bp full length alfalfa COMT
cDNA (Gowri, et al. 1991. Plant Physiol 97:7-14; GenBank Accession
No. M63853) (SEQ ID NO:1 and FIG. 5) or the 743 bp full length
alfalfa CCOMT cDNA (GenBank Accession No. U20736) (SEQ ID NO:2 and
FIG. 6). The COMT and CCOMT inserts were removed as NdeI/BamHI
fragments and ligated into the NdeI/BamHI sites of pPTN1 and pPNT2,
resulting in plasmids pPTNI-COMT and pPTNI-CCOMT for sense
expression of COMT or CCOMT, respectively, and pPTN2-COMT and
pPTN2-CCOMT for antisense expression of COMT or CCOMT,
respectively. The chimeric genes were then cloned as EcoRI/HindIII
fragments into the EcoRI/HindIII sites of the binary vector
pCAMBIA3300, which has a phosphinothricin resistance gene as
selectable marker. Resulting binary constructs were designated
pCAMC1 (single COMT, sense), pCAMC2 (single COMT, antisense),
pCAMCCI (single CCOMT, sense), pCAMCC2 (single CCOMT, antisense),
pCAMCICC1 (tandem COMT sense, CCOMT sense), pCAMC2CC2 (tandem COMT
antisense, CCOMT antisense), and pCAMGUS, as shown in FIG. 2.
[0039] To make constructs for sense or antisense expression of
tandem COMT and CCOMT genes, plasmids pPTN1-COMT and pPTN2-COMT
were first cut with EcoRI, filled in with the Klenow fragment of
DNA polymerase I, and then digested with HindIII. The isolated
fragments were ligated into NarI-treated, Klenow-treated, and
HindIII-treated pPTN1 to create the shuttle vector pPTN1-D. The
tandem COMT and CCOMT region together with the PAL2 promoter and
nos terminator was cut out with AatII, filled in with Klenow,
digested with EcoRI and finally ligated into SmaI/EcoRI cut
pCAMBIA3300 to give binary expression constructs with both OMTs in
the sense or antisense orientation. These were designated pCAMC1CC1
(tandem COMT sense, CCOMT sense) and pCAMC2CC2 (tandem COMT
antisense, CCOMT antisense). Introduction of both COMT and CCOMT
transgenes into the same plant was also achieved by
co-transformation using the above single COMT and CCOMT constructs.
Constructs were introduced into alfalfa by Agrobacterium-mediated
transformation of leaf discs followed by regeneration through
somatic embryogenesis.
[0040] After regeneration (Thomas, et al. 1990. Plant Science
69:189-198) and transfer to the greenhouse, plants were first
analyzed for integration of COMT and CCOMT transgenes by polymerase
chain reaction (PCR). The primers used were
5'-GGGTTCAACAGGTGAAACTC-3' and 5'-CCTTCTTAAGAAACTCCATGATG-3' for
COMT, and 5'-GGCAACCAACGAAGATCAAAAGC-3' and
5'-CTTGATCCTACGGCAGATAGTGATTCC-3' for CCOMT, which yielded
diagnostic 1.1 kb or 0.75 kb amplification products in COMT or
CCOMT transformants respectively. Approximately 80% of the plants
surviving selection were PCR-positive.
[0041] Internode samples (6.sup.th-9.sup.th internodes) from stems
of putative transformants at the same developmental stage were
harvested and assayed for COMT and CCOMT enzymatic activity, as
shown in FIG. 3. Younger internodes (1.sup.st-4.sup.th) were
excluded from the tissue used for enzyme analysis, because these
contain a second form of COMT that is not recognized by the
antiserum raised against the alfalfa COMT targeted by the present
transgenic strategy (Inoue, et al. 2000. Arch Biochem Biophys
375:175-182). Alfalfa stems (internodes 6-9, counting from the
first fully opened leaf at the top) were collected and homogenized
in liquid nitrogen. Powdered tissue was extracted for 1 hour at
4.degree. C. in extraction buffer (100 mM Tris-HCl, pH 7.5, 10%
glycerol, 2 mM DTT, 0.2 MM MgCl.sub.2, 1 mM PMSF), and desalted on
PD-10 columns (Pharmacia, Piscataway, N.J.). Protein concentrations
were determined using Bradford dye-binding reagent (Bio-Rad) with
bovine serum albumin (BSA) as a standard. Enzyme activities were
assayed essentially as described elsewhere (Gowri, et al. 1991.
Plant Physiol 97:7-14; Ni, et al. 1996. "Stress responses in
alfalfa (Medicago sativa L.) XXI. Activation of caffeic acid
3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase genes
does not contribute to changes in metabolite accumulation in
elicitor-treated cell suspension cultures," Plant Physiol
112:117-726) with the following modifications. The assay mixtures
contained 5 .mu.l of [.sup.14CH.sub.3]-S-adenosyl-L-Met (0.6 mM, 13
.mu.Ci/.mu.mol), 5 .mu.l of caffeic acid (1 mM) or caffeoyl CoA (1
mM), 30 .mu.l of assay buffer (100 mM Tris-HCl, pH 7.5, 10%
glycerol, 2 mM DTT, 0.2 mM MgCl.sub.2), and 5 .mu.l protein
extract. They were incubated at 30.degree. C. for 30 minutes,
stopped by adding 50 .mu.l of 0.2 M HCl (for COMT) or 10 .mu.l of 3
M NaOH for CCOMT, incubated at 37.degree. C. for 10 minutes, then
(for CCOMT) acidified by adding 40 .mu.l of 1 M HCl. Labeled
ferulic acid was extracted into 200 .mu.l of hexane:ethyl acetate
(1:1, v/v), and 150 .mu.l of the separated organic phases were
transferred to scintillation vials for determination of
radioactivity.
[0042] There was a wide variation (nearly 4-fold) in COMT activity
in a control population of 20 independent plants transformed with
empty pCAMBIA3300 vector, as seen in FIG. 3A. Of twenty
transformants containing the single COMT sense sequence shown in
FIG. 3B, three lines (SC4, SC5, and SC52) had strongly reduced COMT
activities, whereas the remainder of the population exhibited, on
average, a small increase in COMT activity compared to the average
value for the control population. A similar situation was seen with
respect to COMT activity in the double sense transformants shown in
FIG. 3C, with one plant (DS14) showing strongly down-regulated COMT
activity and the remainder of the population having a slightly
elevated average COMT activity compared to the controls. In the
COMT antisense population shown in FIG. 3D, a single plant (AC310)
had strongly reduced COMT activity, with the remainder of the
overall population showing on average a small reduction when
compared to the average value for the control population. In the
double antisense lines (FIG. 3E), one plant (DA302) showed strongly
reduced COMT activity.
[0043] There was less variation in CCOMT than in COMT activity in
the control population, as seen by a comparison of FIG. 3A and FIG.
3F. Otherwise, the pattern of CCOMT activities in the transformants
harboring sense and antisense CCOMT constructs was very similar to
that observed for COMT. CCOMT activity was strongly down-regulated
in two CCOMT sense lines (SCC 19 and SCC 20) as shown in FIG. 3G,
in one double sense line (DS 14) as shown in FIG. 3H, in two
antisense lines (ACC305 and ACC315) as shown in FIG. 3I) and in one
double COMT/CCOMT antisense line (DA302) as shown in FIG. 3J.
[0044] Transgene insertion was confirmed in selected COMT and CCOMT
down-regulated alfalfa lines by Southern blot analysis. Total DNA
was isolated from leaf tissue of each alfalfa line using a nucleon
phytopure plant DNA extraction kit (Amersham; Arlington Heights,
Ill.). DNA samples (7 .mu.g) were digested with EcoRI,
electrophoretically separated, and transferred to a nylon membrane
(Hybond-N, Amersham) by standard procedures (Sambrook, et al. 1989.
Molecular Cloning. A Laboratory Manual, 2nd Ed., New York, Cold
Spring Harbor Laboratory Press). Blots were probed with
.sup.32P-labeled 1.1 kb alfalfa COMT or 0.75 kb CCOMT coding
sequence probe and washed at high stringency conditions (final wash
0.1.times.SSC, 0.1% SDS, 65.degree. C.). The probe was labeled with
a .sup.32P-dCTP labeling kit (Amersham). A comparison of the
results for selected control and COMT and/or CCOMT down-regulated
transgenic lines showed transgene integration patterns indicative
of multiple transgene insertions of COMT in independent
transformants: SC4 (single COMT sense), SC5 (single COMT sense),
DS14 (double sense), DA302 (double antisense), and AC310 (single
COMT antisense), all showing 3-5 unique bands. Similar transgene
integration patterns were obtained showing multiple transgene
insertions of CCOMT in independent transformants: DA302 (double
antisense), ACC305 and ACC315 (single CCOMT antisense), and DS14
(double sense), all showing 1-5 unique bands.
[0045] RNA gel blot analysis confirmed that the reduced COMT or
CCOMT activity in the various lines resulted from a severe
reduction in COMT or CCOMT transcript levels. RNA was prepared from
alfalfa leaves using TRIREAGENT (Molecular Research Center, Inc.)
according to the manufacturer's suggested protocol. Total RNA
samples (5-10 .mu.g) were fractionated on a formaldehyde denaturing
gel according to standard protocols (Sambrook, et al. 1989.
Molecular Cloning. A Laboratory Manual, 2nd Ed., New York, Cold
Spring Harbor Laboratory Press), transferred to a Hybond-N nylon
membrane, and hybridized with radiolabeled full length alfalfa COMT
or CCOMT cDNA sequences at high stringency as for DNA gel blots.
COMT transcripts were almost undetectable in the total RNA fraction
from sense lines SC4, SC5, antisense line AC310, the double sense
line DS14 and the double antisense line DA302. CCOMT transcripts
were likewise virtually undetectable in antisense lines ACC305 and
ACC315, and in the double antisense line DA302. However, CCOMT
transcripts were relatively unaffected in the double sense line
DS14, in which CCOMT activity is reduced to approximately 23% of
wild type.
[0046] Comparisons of COMT and CCOMT protein levels in the various
transgenic lines were carried out by western blot analysis. Crude
proteins were extracted from the 6.sup.th to 9.sup.th internodes of
selected individual transformants and two wild type plants,
separated on 8-12% gradient SDS-polyacrylamide gels and
electrotransferred onto nitrocellulose membranes. The membranes
were incubated in blocking buffer (PBS containing 0.05% Tween 20
and 5% skim milk) for 2 hours, then incubated in blocking buffer
with monospecific polyclonal antisera raised against recombinant
alfalfa COMT or CCOMT proteins for 2 hours (Kersey, et al. 1999.
Protoplasma 209:46-57). The signals were detected with ECL Western
blotting detection reagents (Amersham) according to the
manufacturer's protocol. The results indicated almost complete loss
of COMT protein in the sense lines SC4, SC5 and SC52, in the
antisense line AC310, in the double antisense line DA302, and in
the double sense line DS14. CCOMT protein levels were almost
undetectable in the antisense lines ACC305 and ACC315, and were
strongly reduced in the double antisense line DA302 and the double
sense line DS14. Complete loss of CCOMT protein in the CCOMT
antisense line ACC305 was unexpectedly accompanied by a strong
increase in COMT protein level, and in COMT enzymatic activity
(Table I). The above results indicate that expression of OMT
sequences from the bean PAL2 promoter results in greater
down-regulation of COMT and CCOMT than obtained in previous studies
(Ni, et al. 1994. Transgenic Res 3:120-126; Atanassova, et al.
1995. Plant J 8:465-477; Van Doorsselaere, et al. 1995. Plant J
8:855-864; Zhong, et al. 1998. Plant Cell 10:2033-2045).
[0047] Reduction of enzymatic activity resulting from reduced
transcript levels in plants expressing gene constructs in the sense
orientation is characteristic of epigenetic gene silencing, which
may occur at the transcriptional or post-transcriptional level
(Vaucheret, et al. 1998. "Transgene-induced gene silencing in
plants," Plant J 16:651-659). To determine the basis for the
reduced COMT and CCOMT activities in some of the sense transgenic
lines, nuclear run-on transcription analyses were performed with
transcripts completed in vitro from nuclei isolated from wild type
and COMT-suppressed or CCOMT-suppressed sense lines SC4 and SCC19.
Nuclei were isolated from fresh leaf tissue as described by Cox and
Goldberg (Cox, K. H. and Goldberg, R. B. 1988. "Analysis of plant
gene expression," Plant Molecular Biology. A Practical Approach. C.
H. Shaw, ed, Oxford, IRL Press, pp. 1-35). Run-on transcription
reaction mixtures contained 125 .mu.l nuclei, 30 .mu.l of 1 M
(NH.sub.4).sub.2SO.sub.4, 12 .mu.l of 100 mM MgCl.sub.2, 3 .mu.l of
100 .mu.M phosphocreatine, 12 .mu.l of creatine phosphate kinase
(0.25 mg/ml), 15 .mu.l of RNasin (Promega; Madison, Wis.), 30 .mu.l
of 5 mM CTP, GTP and ATP mixture, 48 .mu.l of water and 25 .mu.l of
.sup.32P-UTP (NEN, 10 .mu.Ci/.mu.l). The reaction mixture was
incubated at 30.degree. C. for 30 minutes, then treated with
RNase-free DNase (30 units, Promega) and proteinase K (500 .mu.g,
GibcoBRL) at 30.degree. C. for 20 minutes. RNA transcripts were
extracted with an equal volume of phenol-chloroform (1:1), and
extracted again with an equal volume of chloroform. Unincorporated
nucleotide was removed by filtration through Sephadex G-50
(Amersham). Radioactivity incorporated into the synthesized RNA was
then measured by slot blot hybridization. Two hundred ng of COMT,
CCOMT or .beta.-ATPase (positive control) cDNAs were denatured and
transferred to a nitrocellulose membrane by UV cross-linking, and
hybridized with radiolabeled RNA probe. Hybridization and washes
were carried out at 65.degree. C. according to Church and Gilbert
(Church, G. H. and Gilbert, W. 1984. "Genomic sequencing," Proc
Natl Acad Sci USA 81:65-71). Autoradiographs were quantified using
a Molecular Dynamics phosphorimager. The results indicated that the
transcription rates of both COMT and CCOMT were essentially the
same in wild type and down-regulated lines. However, the data from
the RNA gel blot analysis mentioned above indicated that the steady
state transcript levels in the sense COMT and CCOMT lines were only
a fraction of the control levels, consistent with
post-transcriptional gene silencing being responsible for reduced
COMT and CCOMT expression in the sense transgene lines.
[0048] Table I summarizes the COMT and CCOMT activity, lignin
content, and lignin composition of selected transgenic alfalfa
lines harboring alfalfa COMT and CCOMT sequences in the sense or
antisense orientations. Levels of acetyl bromine (AcBr) lignin and
Klason lignin are expressed as % of dry matter. Levels of S, G and
5OHG are expressed as mmol/g dry weight. Down-regulation of COMT
had no effect on the activity of CCOMT, and vice-versa, with one
notable exception. The reduction of CCOMT to less than 4% of wild
type activity in line ACC305 was associated with an approximate
doubling of COMT activity as compared to wild-type levels, a
finding consistent with the western blot data noted above.
[0049] Lignin content in the various lines was determined according
to standard procedures for Klason and acetyl bromide soluble lignin
(Lin, S. Y. and Dence, C. W. eds, Methods in Lignin Chemistry,
Springer Series in Wood Science, Springer-Verlag, Berlin,
Heidelberg, 1992). Two hundred milligrams of dried sample was used
for lignin analysis, and Klason lignin content was calculated as
weight percentage of the extract-free sample.
[0050] Klason lignin levels of three independent control lines
averaged 17.6% of dry matter; this value was reduced to between
15.3% and 12.5% in all lines with down-regulated COMT or CCOMT
activity. The largest reductions in Klason lignin (down to 70% of
the wild type value) were in lines with gene silenced COMT.
However, Klason lignin was also reduced in line ACC305, which has
only 3.6% of the wild type CCOMT
1TABLE I COMT and CCOMT Activities of Select Independent Transgenic
Alfalfa Lines COMT CCOMT AcBr Klason S G 5-OH pkat/mg pkat/mg
Lignin % Lignin % Lignin Lignin Lignin S/G 1 5.98 22.35 16.07 17.21
158.8 305.4 0 0.52 2 6.55 23.77 17.79 17.91 152.8 325.2 0 0.47 48
8.19 21.13 17.52 17.64 156.2 279.4 0 0.56 SC4 1.06 20.11 16.48
12.67 10.4 227.9 7.4 0.05 SC5 1.24 22.26 16.35 12.46 8.6 246.1 8.8
0.04 SC52 1.19 22.36 16.83 14.15 17.1 223.7 1.8 0.07 AC310 0.31
22.17 16.97 15.30 0 248.2 0 0 ACC305 14.39 0.78 16.36 14.58 159.1
150.2 0 1.05 ACC315 8.06 10.7 15.31 15.50 164.0 243.6 0 0.69 DS14
0.78 5.59 16.54 14.72 54.0 223.7 0 0.23 DA302 0.81 1.15 16.78 15.06
14.0 303.0 0 0.05
[0051] activity but nearly double the wild type COMT activity, and
in line AC315, with less than 5% wild type CCOMT activity but
normal COMT activity. Thus, reductions in either COMT or CCOMT
activities can independently reduce Klason lignin levels in
alfalfa. In contrast to the effects on Klason lignin,
down-regulation of neither OMT appeared to have a significant
effect on acetyl bromide extractable lignin.
[0052] A qualitative and semi-quantitative analysis of the lignin
in the transgenic alfalfa lines was made using histochemical
staining methods. Histochemical analysis of lignin in transverse
stem sections (5.sup.th internode) of control (wild type),
antisense COMT line AC310, and antisense CCOMT line ACC305 alfalfa
was performed as follows. For Maule reagent staining, hand sections
of alfalfa stems were immersed in 1% (w/v) potassium permanganate
solution for 5 minutes at room temperature, then washed twice with
3% hydrochloric acid until the color turned from black or dark
brown to light brown. Phloroglucinol-HCl reagent was prepared by
mixing two volumes of 2% (w/v) phloroglucinol in 95% ethanol with
one volume of concentrated HCl. Photographs were taken within 30
minutes of staining. Staining of transverse stem sections with
phloroglucinol-HCl indicated little or no reduction in staining
intensity in COMT or CCOMT antisense as compared to control lines.
Reduction in phloroglucinol staining is often taken as being
indicative of a reduction in lignin content, although the reagent
appears most specific for coniferaldehyde end groups in lignin
(Lewis, N. G. and Yamamoto, E. 1990. "Lignin: occurrence,
biogenesis and biodegradation," Annu Rev Plant Physiol Plant Mol
Biol 41:455-496). In contrast, staining with Maule reagent gave a
red coloration in wild type plants which was lost in COMT
down-regulated lines. Such a color shift is reported to be
diagnostic for reduction of S lignin (Lewis, N. G. and Yamamoto, E.
1990. Plant Physiol Plant Mol Biol 41:455-496).
[0053] Analysis of lignin degradation products by gas
chromatography/mass spectrometry (GC/MS) following thioacidolysis
is a widely used method for analysis of lignin monomer composition
(Lapierre, et al. 1985. "Thioacidolysis of lignin: Comparison with
acidolysis," J Wood Chem Technol 5:277-292), and can be extended to
analyze dimer linkage patterns. Thioacidolysis and the Raney nickel
desulfurization method of Lapierre et al. (Lapierre, et al. 1995.
"New insight into the molecular architecture of hardwood lignins by
chemical degradative method," Res Chem Intermed 21:397-412) were
therefore used to determine lignin composition and resistant
inter-unit bonds in the selected transgenic alfalfa lines. The data
from such analyses shown in FIG. 4 and Table I indicate that
reduction in lignin levels in plants with down-regulated COMT
activity is associated with a much greater decrease in S units than
in G units, resulting in a large decrease in S/G ratio, consistent
with the results of histochemical staining with Maule reagent. In
fact, thioacidolysis products of S lignin were not detected at all
in the COMT antisense line AC310. In contrast, there was no
reduction in S lignin in lines with reduced CCOMT activity, unless
there was a corresponding decrease in COMT activity, as in the
double sense and antisense lines. However, levels of G lignin were
most strongly reduced in transgenic line ACC305, the line with the
greatest decrease in CCOMT activity. Overall, the data clearly
indicate that COMT down-regulation impacts both S and G lignin,
with greatest effects on S lignin, whereas CCOMT down-regulation
only affects G lignin in alfalfa. Reduction of CCOMT to less than
5% of wild-type activity leads to reductions in G lignin with no
apparent effect on S lignin in alfalfa. This contrasts with
reported reductions in both G and S lignin in transgenic tobacco
down-regulated in CCOMT expression (Zhong, et al. 1998. Plant Cell
10:2033-2045). CCOMT would, therefore, appear to function in the
biosynthesis of G lignin in alfalfa, as has been previously
proposed in tobacco (Ye, et al. 1994. Plant Cell 6:1427-1439;
Zhong, et al. 1998. Plant Cell 10:2033-2045) but not in S lignin
biosynthesis, contrary to the model of Li, et al. based on in vitro
studies of enzyme specificity (Li, et al. 2000. J Biol Chem
275:6537-6545).
[0054] Analysis of gas chromatogram traces from the thioacidolysis
reactions revealed new peaks in the reaction products from lignin
extracted from COMT downregulated plants, as shown in FIG. 4. These
peaks were identified as originating from 5-hydroxyguaiacyl
moieties that might be expected to be present if S lignin
biosynthesis were being blocked primarily at the second methylation
stage in COMT down-regulated plants. However, the levels of
5-hydroxyguaiacyl units were always much less than the
corresponding reduction in S units, as shown in Table I.
[0055] In the intact lignin polymer, the various monomeric units
are linked to each other through covalent bonding at a number of
different positions. This gives rise to more than five major types
of lignin dimers that can be analyzed by GC/MS after thioacidolysis
and Raney nickel desulfurization, as illustrated by the five
structures in Table II. 5-5 and 4-O-5 linkages only occur between G
units, whereas .beta.-.beta. linkages only occur between S units.
.beta.-1, .beta.-5 and .beta.-6 linkages can occur between two G
units or between a G and an S unit. Thus, the five basic linkage
types can result in nine different lignin dimers. The levels of
these various dimers were analyzed by GC/MS, from the series of
control and COMT or CCOMT down-regulated alfalfa plants previously
analyzed for lignin content and monomer composition. Table II
depicts the dimer bonding patterns of lignin samples from wild
type, COMT-suppressed, and CCOMT-suppressed alfalfa plants
following determination of dimer composition by thioacidolysis
followed by Raney nickel desufurization. Units are mmol/g dry
weight. The Klason lignin levels and S/G ratios of the various
lines are given in Table I. The chemical structures of a selection
of the dimer linkages recovered from lignin after thioacidolysis
and Raney nickel desulfurization are shown. The results in Table II
indicate that reduction of COMT activity resulted in at most a
small increase in the recovery of dimers consisting of two G units
(5-5, 4-O-5, .beta.-1 (G), .beta.-5 (G), .beta.-6 (G)). However,
there was a total loss of recovered dimers with .beta.-.beta. or
mixed .beta.-1 or .beta.-6 linkages, which all involve S units, in
plants with reduced COMT activity. In contrast, reduction of CCOMT
activity did not lead to a reduction in dimers containing S units.
Rather, CCOMT down-regulation appeared to lead to increased
recovery of .beta.-5 (G) dimers but a reduction in .beta.-6 (G)
dimers. Lignin from line ACC305 had the highest proportion of
.beta.-.beta. linked S units.
[0056] Taken together, the above data indicate that the reduction
in S/G ratio caused by down-regulation of COMT results in a
decreased proportion of linkages involving S units. This indicates
that lignin linkage pattern is determined by monomer availability.
However, there were also qualitative changes in lignin dimers
resulting from OMT down-regulation. Thus, gas chromatograms of
thioacidolysis/Raney nickel desulfurization products of lignin from
five independent COMT down-regulated plants exhibited a major peak
at 52.9 minutes retention time that was absent from corresponding
traces from wild type or CCOMT down-regulated plants. The compound
was analyzed by MS and shown to have a molecular ion with a
mass/charge ratio (m/z) of 504, identical to that of the
.gamma.-p-coumarate ester of an S unit, a dimer previously
identified in maize lignin (Grabber, et al. 1996. "p-Coumaroylated
syringyl units in maize lignin: implications for .beta.-ether
cleavage by thioacidolysis," Phytochemistry 43:1189-1194). However,
the retention time of the new
2TABLE II Dimer Bonding Patterns of Lignin Samples from Wild Type,
COMT-suppressed and CCOMT-suppressed Alfalfa Line 5-5,G 4-0-5,G
b-1,G b-1,M b-5,G b-5,M b-b,S b-6,G b-6,S H-S,ester 2 13 3.6 17.9
3.2 19.4 16.9 9.6 11 5 0 48 13.3 3.6 17.5 2.4 20.4 14 9.5 12.4 6.3
0 SC4 16.6 4.1 21.9 0 24.6 19 0 14.2 0 9.4 SC5 17.3 3.6 20.3 0 24.7
14 0 19.2 0 7.1 AC310 15.5 3.4 19.1 0 23.7 15 0 10.6 0 12.4 DA302
16.9 4 21.9 0 30.7 6.4 0 9 0 10.5 DS14 15 3.3 20.7 0 21.5 18.9 0
12.8 0 7.5 ACC305 11.2 2.9 18.6 5.2 22.2 19.5 14.8 10.1 6.6 0
ACC315 14.8 3.6 23.5 5.7 30.1 10.3 4.5 6.2 3.1 0 1 2 3 4 5
[0057] dimer and its MS fragmentation pattern were similar but not
identical to those of an authentic sample of the S unit coumarate
ester. The appearance of the new dimer correlated with the loss of
S-linked dimers from the lignin in COMT down-regulated plants
(Table II).
[0058] On the basis of the above analyses, line SC5 was chosen as a
severely COMT down-regulated line in which recoverable S lignin was
virtually absent, and line ACC305 chosen as a severely CCOMT
down-regulated line in which G lignin was reduced and S/G ratio
increased. Line CK48 was chosen as a control. The lines were
vegetatively propagated, and greenhouse grown plants were harvested
at the late bud stage, dried at 120.degree. F., ground into 1 mm
powder and put into preweighed nylon bags (approximately 5 g/bag).
These bags were put into the rumens of fistulated steers for 12,
24, 36, or 72 hours of digestion. At each time point, duplicate
samples for each line were analyzed in three different steers.
After digestion, bags were taken out from the rumen, washed in a
commercial washing machine and vacuum-dried in a freeze drier.
Digestibility was calculated based on sample weight before and
after digestion. The results in Table III show that total digestion
of forage from all three lines reached a value of approximately 80%
by 12 hours within the rumen. However, there was no further
digestion of forage from the control and COMT down-regulated lines
beyond 24 hours in the rumen. In contrast, the forage from the
CCOMT down-regulated line continued to be digested up to at least
76 hours within the rumen, attaining a value of approximately 89%
digestibility.
[0059] Down-regulation of CCOMT by antisense or gene-silencing
approaches was shown to be a valid method for improving forage
digestibility in alfalfa, and presumably other forage legumes such
as clovers and trefoils. The lack of effectiveness of strong
down-regulation of COMT in significantly improving forage
digestibility indicates that, contrary to current opinion, reducing
S lignin is not a valid strategy for improving digestibility.
Rather, it is the reduction in G lignin, which may result in a
reduced level of lignin condensation, that has the major impact on
digestibility of alfalfa.
3TABLE III In vivo Digestibility of Alfalfa in Fistulated Steers
Digesitibility (%) Plant Line Time in Rumen Steer 1 Steer 2 Steer 3
Average CK48 12 h 80.00 78.19 80.83 79.68 24 h 83.04 82.95 83.87
83.29 36 h 83.98 83.20 83.91 83.70 72 h 84.15 82.34 82.02 82.83 SC5
12 h 78.89 77.21 81.52 79.21 24 h 84.39 84.82 85.75 84.99 36 h
84.27 84.92 85.00 84.73 72 h 85.12 85.00 84.35 84.82 ACC305 12 h
80.91 77.97 81.91 80.26 24 h 84.26 85.21 87.21 85.56 36 h 86.22
86.86 87.21 86.76 72 h 88.55 90.92 87.74 89.07
[0060]
Sequence CWU 0
0
* * * * *