Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase Page: 55
This article is part of the collection entitled: UNT Scholarly Works and was provided to UNT Digital Library by the UNT College of Arts and Sciences.
Extracted Text
The following text was automatically extracted from the image on this page using optical character recognition software:
.1. ii. Maria ct a.. PhirochemisitrY 62 (2003) 53 65
reveal whether it was a guaiacyl or syringyl unit
attached to the 5-hydroxyguaiacyl unit within the lignin
and therefore whether coniferyl or sinapyl alcohol had
added to the new 5-hydroxyguaiacyl end of the growing
polymer. Recently, NMR studies have provided further
evidence of the incorporation of 5-hydroxyconiferyl
alcohol into lignins and revealed the occurrence of ben-
zodioxane structures in the lignins of COMT-deficient
poplar at significant levels (Marita et al., 2001; Ralph et
al., 2001b, 2001c).
The present study examines alterations to lignin
structure and composition resulting from independent
down-regulation of COMT and CCoAOMT in alfalfa
plants. Previous work has shown genetically modified
alfalfa to have altered lignin composition (Guo et al.,
2001a) and improved in situ digestibility (Guo et al.,
2001b). The results of this study reveal new details of
the incorporation of novel units in the lignin of COMT-
deficient alfalfa including units not previously identified
and an increase in the cellulose:lignin ratio in
CCoAOMT-deficient alfalfa.
2. Results and discussion
NMR and DFRC methods were used to determine
structural and compositional changes that could be
attributed to the enzyme deficiencies in lignins from
COMT- and CCoAOMT-down-regulated alfalfa. A
previously examined COMT down-regulated hardwood
incorporated novel benzodioxane units (Marita et al.,
2001; Ralph et al., 2001b) into the lignin, but it was not
clear whether these novel units would analogously occur
in an herbaceous legume. A detailed structural analysis
of the lignins and improved compositional data from
both transgenics was therefore required.
2.1. COMT-deficient alfalfa
2.1.1. HMQC NMR spectra
2D NMR experiments (gradient-enhanced HMQC;
Kay et al., 1992) allowed structural analysis of the
major units in isolated lignin from alfalfa. In Fig. 3, theHO1
HO OAr
5H I
H.,Hs OMe
MeO 0
G/S
O OH
OMeOAc
DFRC 5H I
O OMe
MeO 0
AcO OAc
OMe2 3c,3s
Fig. 2. Benzodioxanes flanked by normal n-ethers (structures of gen-
eral type 2) release diagnostic benzodioxane dieters. 3(; and 3s. fol-
lowing DFRC degradation.HMQC data for the wild-type alfalfa lignin were similar
to those for other non-transgenic dicot lignins with
structural features being well resolved and readily
recognized features (Marita et al., 1999; Ralph et al.,
1999). As in previously examined COMT-deficient
poplars (Marita et al., 2001; Ralph et al., 2001b), the
COMT-deficiency in the alfalfa resulted in a lignin with
a striking new feature, the appearance of benzodioxane
units H which can, therefore, now be considered a sig-
nature for COMT-deficiency. Benzodioxanes H result
from the incorporation of 5-hydroxyconiferyl alcohol
into the lignin by typical endwise coupling reactions
(Ralph et al., 1999). The relative H-unit levels in this
spectrum, Fig. 3b, were far higher than in other lignins
we have studied. As will be discussed later, the high level
is due in part to a preferential partitioning of this
structure into the soluble fraction analyzed by NMR.
Other features of the spectrum, compared with the con-
trol, are a relative decrease in normal p-aryl ether units
(3-0-4, A) and resinol units (3-R, C), and an increase in
dibenzodioxocins (5-5/P-0-4, a-O-4, D).
Quantification of interunit type structures based on
measuring volume integrals in the 2D HMQC spectra
(Zhang and Gellerstedt, 2000; Marita et al., 2001; Ralph
et al., 2001b) of wild-type and COMT-deficient alfalfa
are presented in Table 1. Again it is striking that ben-
zodioxane units H (38%) in the COMT-deficient alfalfa
approach the level of the normal 1-ether units A (44%).
They are the second most abundant interunit type, far
exceeding the levels of traditional units such as 3-5 (B)
and 3-3(C). In the wild-type alfalfa, no benzodioxane
units H were detected; the lignin was comprised of
mainly 3-ether units A (81%). The percentage of phe-
nylcoumaran units B did not change as a result of
COMT down-regulation. There was a decrease in resi-
nol units C in the COMT-deficient alfalfa (3%) com-
pared to wild-type (6%) presumably as a result of the
depletion in syringyl units. Cinnamyl end-group X levels
were similar (3-4%). Dibenzodioxocins D, which arise
from coupling reactions of a monolignol with 5-5-cou-
pled G-units, appeared to be elevated (from 1 to 4%). A
higher D and a lower C unit content is consistent with a
lower incorporation of sinapyl alcohol. It suggests that
the lignin may be forming something of a block-copo-
lymer with G-rich regions and 5-hydroxyguaiacyl-rich
regions as will be discussed later. An elevated B level
would also normally be expected in G-rich regions
(paralleling the D content) but this is not seen.
2.1.2. HMBC NMR spectra
Long-range correlation spectra provide valuable
insight into which types of units are connected to each
other. HMBC data reveal the extent of S/G composi-
tional changes in p-aryl ether units A in the extractable
isolated lignins by showing a-proton correlations with
diagnostic carbons in their aromatic rings. Equivalent55
Upcoming Pages
Here’s what’s next.
Search Inside
This article can be searched. Note: Results may vary based on the legibility of text within the document.
Tools / Downloads
Get a copy of this page or view the extracted text.
Citing and Sharing
Basic information for referencing this web page. We also provide extended guidance on usage rights, references, copying or embedding.
Reference the current page of this Article.
Marita, Jane M.; Ralph, John; Hatfield, Ronald D.; Guo, Dianjing; Chen, Fang & Dixon, R. A. Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase, article, August 28, 2002; [New York, New York]. (https://digital.library.unt.edu/ark:/67531/metadc488183/m1/3/: accessed April 23, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT College of Arts and Sciences.