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J Sci Food Agric 1998, 76, 505È514
Eþect of Down-Regulation of Cinnamyl Alcohol
Dehydrogenase on Cell Wall Composition and on
Degradability of Tobacco Stems
Marie Andre e Bernard Vailhe ,1 Jean Michel Besle,1* Marie Paule Maillot,1
Agnès Cornu,1 Claire Halpin2 and Mary Knight2
1 SRNH, INRA de Clermont Ferrand-Theix, 63122 St Genès Champanelle, France
2 Zeneca Seeds, Jealotts Hill Research Station, Bracknell, Berkshire, RG12 6EY, UK
(Received 27 June 1997 ; revised version received 15 July 1997 ; accepted 7 August 1997)
Abstract : The e†ect of down-regulation of tobacco cinnamyl alcohol dehydrogenase (CAD) on cell wall composition and degradability has been assessed. CAD
activity was only 20, 16, 14 and 7%, relative to the controls, in four populations
of plants (designated 40-1, 40-2, 48 and 50, respectively) transformed with CAD
antisense mRNA. Cell wall residues of stem samples were analysed for polysaccharide composition, gravimetric and acetyl bromide lignins and lignin nitrobenzene oxidation products. In situ disappearance and cellulase solubility of both
initial dry matter and CWR were determined. The populations of plants with
depressed CAD activity showed no change in lignin content but some consistent
changes in cell wall composition and digestibility were identiÐed. The syringyl
content of lignins decreased and the syringaldehyde to vanillin ratio (S/V) was
consequently reduced. Dry matter degradability, as measured by both methods,
was signiÐcantly improved in all CAD-depressed samples except for population
40-1, which was the least CAD-depressed. Increased in situ disappearance of cell
wall (ISCWD) was found in all plants exhibiting more than 80% CAD downregulation and was maximal (7 percentage units) in population 50 which had the
greatest CAD depression. The rates of ISCWD increased slightly in some populations (40-2 and 50). The relationship between S/V and ISCWD was signiÐcant
(R \ [0É68) only in the samples from a selected population of mature, most
depleted plants. Other modiÐcations may therefore also contribute to the
improvement in degradability. However the changes in lignin composition that
were observed in CAD-depressed tobacco are largely similar to those seen in
some maize and sorghum mutants with altered ligniÐcation and improved digestibility. These data therefore suggest that depressing CAD activity may be an
e†ective method for improving digestibility in forage crops. ( 1998 SCI.
J Sci Food Agric 76, 505È514 (1998)
Key words : down-regulation ; cinnamyl alcohol dehydrogenase ; CAD ; cell wall ;
lignin ; degradability ; transgenic ; genetic engineering ; tobacco
INTRODUCTION
1980). Several studies suggest that lignins play a role
not only through their concentration but also through
their structural characteristics (Jung and Deetz 1993 ;
Besle et al 1994). The concentration and composition of
lignins varies in di†erent plants (Monties 1986) and it
may be possible to modify lignins without interfering
with normal plant development. One way of modifying
Lignins are the most important compositional factor
a†ecting the utilisation of forages by ruminants (Jarrige
* To whom correspondence should be addressed.
Contract/grant sponsor : EUÈECLAIR programme.
Contract/grant number : AGRE 0021 OPLIGE.
505
( 1998 SCI.
J Sci Food Agric 0022È5142/98/$17.50.
Printed in Great Britain
M A Bernard V ailhe et al
506
ligniÐcation is to inhibit enzymes of the lignin biosynthetic pathway. Cinnamyl alcohol dehydrogenase
(CAD), which catalyses the last step of the synthesis of
lignin monomers, has been successfully inhibited by
chemical inhibitors in poplar tissues (Grand et al 1985)
and in wheat (Moerschbacher et al 1990) but the e†ects
on lignin composition and digestibility of the cell walls
were not determined.
Mutants with altered ligniÐcation have been identiÐed in some graminaceous species. A maize mutant with
a reddish brown pigmentation of the leaf midrib was
discovered in 1924 (Jorgenson 1931). It was classiÐed as
bm1 (Kuc and Nelson 1964) and is now considered to
have altered CAD activity (Halpin et al 1995). Several
studies have shown that the digestibility of this mutant
is greater than that of the normal counterpart both in
vitro and in vivo (Barrière and Argillier 1993). Its rate of
digestibility is either the same or slightly greater than
that of normal plants (Cymbaluk et al 1973). Similarly,
it has been shown that the bmr6 mutant of sorghum has
greater in vitro digestibility (Hanna et al 1981) and
lower CAD activity than its normal counterpart
(Bucholtz et al 1980) but, in this case, a slightly lower
level of O-methyl transferase activity was also detected
(Pillonel et al 1991). The improvement in digestibility
was correlated with a lower lignin content and with differences in lignin structure, particularly a lower syringyl
content in lignins for both mutants (Gee et al 1968 ; Pillonel et al 1991).
These Ðndings prompted attempts to carry out downregulation of CAD by genetic engineering. The Ðrst successful attempts to regulate CAD activity were made by
transforming tobacco (Knight et al 1992 ; Halpin et al
1994) and poplar (Baucher et al 1996) with antisense
genes for CAD. In these plants, lignin composition was
modiÐed in ways similar to that found in CADdepressed maize and sorghum mutants. There was a
decrease in syringyl units and an increase in the amount
of lignin that could be extracted with alkali. However,
unlike the mutants, the transgenic plants had normal
levels of lignin.
The aim of the present study was to assess whether
cell wall modiÐcation in the CAD depressed tobacco of
Halpin et al (1994) a†ected degradability.
MATERIALS AND METHODS
Plants
Tobacco plants (Nicotiana tabacum var Samsun), transformed via Agrobacterium with a CAD antisense construct, were produced as described in Halpin et al
(1994). Three independent primary transformants
(plants 40, 48 and 50) that have a single transgenic locus
and reduced levels of CAD activity were selected. These
were backcrossed and selfed to produce F1
(backcrossed) and F1 (selfed) seed.
Progeny (120 plants) of the backcross of plants 40
and 50 were grown from seed in a greenhouse in the
summer of 1992. The genotype of each plant was determined by PCR of the transgene ; segregation of the
transgene was as expected (50% hemizygous antisense
plants and 50% azygous control plants). In lines 40 and
50, CAD activity of antisense plants was only 20 and
7%, respectively, of that of the corresponding controls.
Ten antisense and 10 control plants for each line were
harvested after 16 weeks and stems were fresh-frozen.
These samples were designated 50 (from line 50) or 40-1
(from line 40).
Homozygous line 40 plants were produced by selÐng
F1 (selfed) plants and studying the segregation of kanamycin resistance in F2 seedling progeny. Twenty homozygous antisense and 20 control F2 plants were
identiÐed from F2 seeds. These were randomly positioned in a greenhouse and grown during the spring of
1993. The CAD activity in antisense plants was 16%
that of azygous controls. At 8 and 14 weeks, 5 antisense
and 5 control plants for each stage were harvested and
stems were fresh-frozen. Samples from 8-week-old and
from 14-week-old plants were designated 40-2-8 and
40-2-14, respectively.
Homozygous line 48 antisense plants were produced
as for line 40. F2 plants were grown in a greenhouse
during the winter of 1993. Four antisense plants, with
14% of the CAD activity of the controls, and 4 control
plants were harvested after 16 weeks and the stems were
fresh-frozen. These samples were designated 48.
The control and antisense stems were freeze-dried.
Stems of lines 40-1 and 50 were separated into internodes from the top (5È6 internodes), the middle (3È4
internodes), the base (3È4 internodes) and a remaining
fraction. The whole stems of lines 40-2 and 48 were
studied. All the fractions or whole stems were ground
with a blade mill (Brabender 880803, Duisburg) through
a 2 mm screen and sieved through a 1 mm sieve. The
retained fractions were repeatedly ground to pass
through the sieve. The Ðne particles passing through a
100 lm sieve were discarded to minimise physical losses
from the bags used for the in situ degradability determinations. The proportions of these Ðne particles were
similar for the controls and antisense samples (about
4È7% DM for the di†erent lines). The particles originated mostly from the pith and had a very low acetyl
bromide lignin content. For lines 40-1 and 50, whole
stem samples were reconstituted from all ground fractions.
Chemical analyses
Analyses were performed on the cell wall residues
(CWRs) prepared in duplicate after sequential washing
Cell wall composition and degradability of tobacco stems
507
of the initial sample with water (three times for 15 min
each at 40¡C) and Soxhlet reÑuxing with ethanol 95%
(v/v) and toluene/ethanol (2 : 1, v/v) (Jarrige 1961).
Lignin content was determined in duplicate by the
gravimetric method (JL) of Jarrige (1961) and in triplicate by the acetyl bromide (ABL) spectrophotometric
method of Iiyama and Wallis (1990) with ferulic acid as
standard. Uncondensed units in lignin were determined
in duplicate as aldehydes released by the nitrobenzene
oxidation method of Venverloo (1971), modiÐed according to Monties (pers comm). NaOH (2 M, 5 ml) and
nitrobenzene (0É5 ml) were added to the sample (20 mg) ;
hydrolysis was performed at 160¡C for 3 h in an oil
bath ; the sample was extracted into dichloromethane
and, after vacuum removal of the solvent by evaporation, dissolved in 2 ml methanol. The main products
obtained
(p-OH
benzaldehyde,
vanillin \ V,
syringaldehyde \ S) were designated as NBO. The
methanolic extracts were analysed by high-performance
liquid chromatography (HPLC) with a reverse-phase
column (Lichrosorb RP-18, 250 ] 4É6 mm id) as
described by Mosoni et al (1994).
The CWRs were saponiÐed in duplicate according to
Scalbert et al (1985) with the following modiÐcations. A
sample (1 g) was extracted with 1 M NaOH (30 ml)
under N at 35¡C for 2 h, then centrifuged. The pellet
2
was treated again and then water-washed through a sintered crucible (porosity 2). The supernatants and Ðltrates were pooled, acidiÐed with 6 M HCl to pH 2, left
for 16 h at 4¡C and centrifuged. The pellets were solubilised in NaOH, dialysed (membrane cuto† 6000È
8000 MW) and freeze-dried to yield the alkali soluble
cell wall polymers (ASCWP). EsteriÐed phenolic acids
were extracted from the acid supernatants with ethyl
acetate and further analysed by HPLC as for the products of nitrobenzene oxidation.
Uronic acids were determined (triplicates) by the
method of Blumenkrantz and Asboe-Hansen (1973) in
hydrolysates prepared according to Englyst et al (1982).
Non-cellulose polysaccharides and cellulose were
sequentially hydrolysed (duplicates) according to Jarrige
(1961) and determined gravimetrically (all intermediate
dryings were performed at 60¡C). In addition, for lines
40-1 and 50, the hydrolysates were analysed colorimetrically for reducing sugars using xylose and glucose
respectively as standards (Besle et al 1981). The dry
weight was measured after 24 h at 60¡C in a conventional oven and the dry matter (DM) after a further
24 h at 103¡C.
40-2, and CWRs for line 48. Losses of water-soluble and
Ðne particulate matter were determined by placing
duplicate samples in bags in a vessel containing water
vigorously shaken for 4 h at 40¡C, and then by Ðltering
the extracts on sintered glass (porosity 2) (MichaletDoreau 1990). After incubation (2, 6, 12, 24, 48 and
72 h), the bags were frozen to detach part of the associated microorganisms, thawed and then thoroughly
washed with tap water (at about 30¡C) in an electric
washing machine and dried at 60¡C. The in situ dry
matter disappearance (ISDMD) was calculated on the
basis of dry weight at 60¡C taking into account the Ðne
particle losses. Since all the soluble cell material would
have been extracted by water during the preparation of
CWRs, the kinetics of CWR disappearance (ISCWD)
were calculated on the assumption that cellular material
was totally solubilised during the Ðrst 2 h of rumen
incubation.
Cellulase digestibility determinations were carried out
in duplicate according to the method of Rexen (1977)
modiÐed by Cordesse (1982). A sample (500 mg) was
incubated with 30 ml of acetate bu†er (0É05 M, pH 4É6)
containing 10 g litre~1 Onozuka R10 cell wall degrading complex and 0É3 g litre~1 NaN , and incubated for
3
24 h at 40¡C with mechanical shaking. The residue was
then Ðltered on a sintered crucible (porosity 2) and
dried for 48 h at 60¡C. The enzyme solution was freshly
prepared and previously Ðltered through a sintered crucible (porosity 2).
Evaluation of digestibility
In situ disappearance was measured with nylon bags
(50 lm porosity) placed in triplicate in cannulated sheep
which were fed lucerne hay (Demarquilly and Chenost
1969). Initial DM was used for lines 40-1, 50 and
Experimental design
The kinetics of in situ disappearance were performed on
whole stems, and 48 h-in situ disappearances and cellulase digestibility were determined on all samples (stems
and stem fractions). The in situ measurements were
made on sheep kept under controlled and conÐned conditions, and receiving the same lucerne forage for all the
trials. In each in situ trial, a standard wheat straw
sample was incubated in triplicate for 48 h and no signiÐcant di†erence in the in situ dry matter disappearance occurred between trials (mean 32É3 ^ 1É3%).
Statistical analysis
The kinetics of in situ dry matter and cell wall disappearance were Ðtted (NLIN procedures of SAS (1989))
to an exponential model without (^rskov and McDonald 1979) or with (Dhanoa 1988) lag time.
The GLM procedure of SAS (1989) was used. The
data of CWR content and composition, kinetic parameters, 48 h-in situ disappearances and cellulase solubilities were analysed in a complete block design with
two or three replications and subjected to one-way
analysis of variance. The data from line 40-2 degradability were subjected to two-way analysis of variance
M A Bernard V ailhe et al
508
with the e†ects of genetic regulation, of stage of growth
and the interaction between these two factors. The
correlations were also calculated with the GLM procedure.
RESULTS
Cell wall composition
Compared to that of controls, the proportion of CWRs
from the transgenic samples was similar in lines 40-1
and 50 but signiÐcantly lower in lines 40-2-8 and 48
(P \ 0É05) (Table 1). The proportion of CWRs in the
stems also decreased from the base upwards and was
lower (P \ 0É01) in the young (40-2-8) whole stem than
in the older (40-2-14) plants. No di†erence was observed
in gravimetric lignin contents (on CWR basis) between
the controls and their transgenic counterparts. A signiÐcant decrease in ABL content was observed only
between transgenic and control whole stems of samples
40-1. The lignin content of the stems was broadly
similar from base upwards between control and transgenic older plants but decreased from about 154 g kg~1
CWR in the bases to 95 g kg~1 CWR in the tops of the
young 40-2 stems (data not shown). When measured
gravimetrically, the cellulose contents in transgenic
samples were broadly similar to those in controls
(except in samples 40-2-14) whereas non-cellulose polysaccharide contents showed irregular variations. When
estimated as reducing sugars (in acid hydrolysates), the
non-cellulose polysaccharide and cellulose contents in
stems of lines 40-1 and 50 were lower than determined
by the gravimetric method (on average by 90 and
68 g kg~1, respectively) and were found to be higher in
the stems of the two transgenic lines than in the controls (on average 340 g kg~1 compared to 310 g kg~1).
Uronic acid contents were unchanged between control
and transgenic samples (on average 55 ^ 4, 59 ^ 9,
21 ^ 3 g kg~1 CWR for lines 40-1, 50 and 48,
respectively).
TABLE 1
Cell wall content and composition of the samples
Samplea
40-2-8 control
40-2-8 AS
40-2-14 control
40-2-14 AS
40-1 control
40-1 AS
50 control
50 AS
48 control
48 AS
RSE
CW Rb
(g kg~1 DM)
WS
WS
WS
WS
WS
T
M
B
WS
T
M
B
WS
T
M
B
WS
T
M
B
WS
WS
423
393*
611
605
767
656
668
746
767
682*
741*
747
723
638
711
788
726
659
704
771
767
712*
9É9
Cell wall constituents (g kg~1 CW R)c
L ignin monomers (g kg~1 JL )d
JL
ABL
HC
C
V
80
85
123
116
148
140
146
145
147
134
147
150
155
156
155
152
154
154
156
151
156
160
4É4
202
165
207
192
265
200
209
232
229*
217
206
233
241
210
215
207
229
223
212
221
161
186
15É9
457
452
427
452*
421
439
433
419
422
421*
418
401*
409
377
403
387
402
394*
418*
404*
371
370
7É4
457
462
447
425*
422
418
414
426
426
441
434
435
429
460
442
445
433
451
420
355
473
470
6É2
227
190*
210
170*
214
223
202
212
204
208
215
225
180
190
133
198
187
183
143
207
138
122
9É7
S
184
141*
168
121**
196
188
187
202
173*
175
178
190
157
168
123
176
149
149*
105*
165
67
45*
9É4
S/V e
NBO
414
337**
380
293*
413
416
393
416
380
386
392
418
340
360
259
376
340
335
250
375
348
349
16É9
0É68
0É62*
0É67
0É59**
0É77
0É70
0É77
0É80
0É71*
0É70
0É69*
0É71*
0É73
0É74
0É77
0É74
0É67*
0É68*
0É61**
0É67*
0É41
0É31**
0É02
a AS, transgenic (antisense) sample ; WS, whole stem ; T, top internodes ; M, middle internodes ; B, basal internodes ; RSE, residual
standard error.
b CWR, cell wall residue ; DM, dry matter.
c JL, Jarrige lignin ; ABL, acetyl bromide lignin ; HC, hemicelluloses (determined by gravimetry) ; C, cellulose (determined by
gravimetry).
d V, vanillin ; S, syringaldehyde ; NBO, nitrobenzene oxidation products \ V ] S ] p-hydroxybenzaldehyde.
e Molar ratio.
*, ** SigniÐcant di†erences between data of transgenic and control counterpart at P \ 0É05, P \ 0É01, respectively.
Cell wall composition and degradability of tobacco stems
509
EsteriÐed phenolic acids were not detected in the
CWRs. Except in line 40-1, the ASCWP fraction was
greater in transgenic CWRs than in controls (241, 136,
182, 202, 265 g kg~1 CWR compared to 230, 115, 124,
175, 189 g kg~1 in lines 40-1, 40-2-8, 40-2-14, 48 and 50,
respectively) (Bernard Vailhe et al 1996). The aldehydes,
resulting from nitrobenzene oxidation of the uncondensed monomers from lignins, were mainly vanillin
and syringaldehyde with a small amount of phydroxybenzaldehyde. The total aldehyde content
amounted to between 290 and 410 g kg~1 of JL in the
di†erent samples (Table 1). The proportion of syringaldehyde from lignins was generally lower in the transgenic samples than in the control fractions, the lowest
changes being observed in the sections of stems from
line 40-1. Except in line 40-2, where it decreased, the
proportion of vanillin was unchanged. The S/V molar
ratio decreased in all samples, with the greatest di†erence being observed in line 48. The NBO content
decreased signiÐcantly only for samples from line 40-2,
which indicates an increase in the “condensedÏ carbon to
carbon inter-residue lignin linkages.
Degradability
The extent of ISDMD of control stems was generally
low with little variation (36È39%) between the 16-weekold plants (40-1, 48 and 50) (Table 2), and was slightly
greater than that of the standard wheat straw. It
increased up to 73% (stems of 40-2-8) for younger
plants. A gradient of increasing ISDMD was observed
in the internodes of 40-1 and 50 from the base upwards.
Compared to that of control line 40-1, the extent of
ISDMD of transgenic whole stems was unchanged.
However, some di†erences were observed between the
corresponding fractions in the internodes, probably
because of inhomogeneity in the internode sampling.
Since the plants had a variable number and size of
TABLE 2
In situ disappearance (48 h) and cellulase solubility of initial dry matter and cell wall
residue (CWR) of the tobacco samples
Samplea
40-2-8 control
40-2-8 AS
40-2-14 control
40-2-14 AS
RSE
40-1 control
40-1 AS
RSE
50 control
50 AS
RSE
48 control
48 AS
RSE
In situ disappearance (%)
WS
WS
WS
WS
WS
T
M
B
WS
T
M
B
WS
T
M
B
WS
T
M
B
WS
WS
Cellulase solubility (%)
Dry matter
CW R
Dry matter
CW R
73É0
77É3**
47É3
51É4*
1É16
38É7
50É5
46É2
26É8
37É9
45É7**
39É2**
28É8**
0É50
36É5
46É7
35É8
27É0
41É2**
47É0
38É2**
29É4**
0É52
38É5
45É3**
0É54
36É2
42É2**
13É8
19É7*
2É02
20É1
24É6
19É5
1É9
19É0
20É4**
17É9**
4É7**
0É73
12É1
16É4
9É6
7É3
19É0**
19É6**
12É3**
8É4*
0É73
19É8
23É2**
0É86
72É5
78É9**
48É4
51É4**
0É70
40É2
51É5
46É4
30É5
39É0
49É0
41É4**
34É4**
1É06
36É2
44É7
38É5
28É6
40É4**
49É2**
42É0**
33É7**
0É82
35É4
41É6**
0É30
35É0
46É3**
15É5
19É7**
1É53
22É0
26É1
19É7
6É8
20É5
25É2
20É9
12É2**
1É49
11É8
13É2
13É5
9É3
17É8**
22É8**
17É6**
14É0*
1É14
15É8
18É0*
0É40
a AS, transgenic sample ; WS, whole stem ; T, top internodes ; M, middle internodes ; B,
basal internodes ; RSE, residual standard error.
*, **, *** SigniÐcant di†erences between data of transgenic and control counterpart at
P \ 0É1, P \ 0É05, P \ 0É01, respectively.
M A Bernard V ailhe et al
510
internodes, it was not easy to make a comparable sampling of upper young tissues and middle and basal older
tissues for all the plants. In contrast, the extent of
ISDMD of transgenic batch 40-2 was signiÐcantly
greater than the controls in both the 8-week and
14-week old plants, independent of the stage. Similarly,
for the other transgenic lines, the extent of ISDMD was
signiÐcantly greater than for the controls in all fractions
studied except for the upper part of the stems of line 50.
The greatest di†erences were observed in lines 50 and
48 (increase of 4É8 and 6É8 percentage units in whole
stems).
Except for samples 40-1, the CWR was degraded to a
greatest extent in all fractions of the transgenic stems,
with the highest increase (7 percentage units) observed
in line 50. In transgenic line 48, the increase in ISCWD
was lower than that of ISDMD, owing to the greater
cell soluble content of the transgenic sample. The variations of cellulase digestibility were roughly similar to
those of in situ disappearances (Table 2). Both methods
were in good agreement : for 22 samples,
CDMD \ 0É93]ISDMD ] 4É12, RSE \ 2É3, R \ 0É98).
In contrast to the in situ Ðndings, even the tops of
transgenic line 50 were more degradable than those
of the control. In addition, the enhanced degradability due to genetic modiÐcation was signiÐcantly
(P \ 0É05) lower in the older stems of 40-2 than in the
younger stems.
Non-cellulose polysaccharides were more degradable
than cellulose (Table 3). Except for line 40-1, improvements in ISCWR degradability in transgenic stems were
due to a greater degradation of all polysaccharides. Surprisingly, disappearances of Jarrige lignin in the rumen
gave negative values but, in lines 50 and 40-2-8, the
transgenic lignin content of fermented residues was signiÐcantly lower than in controls. In addition, the NBO
content decreased from 34 to 27% JL after in situ incubation of transgenic line 50 but remained unchanged in
control line 50 and in both transgenic and control line
40-1. This indicates a di†erence in the solubilisation of
lignins and an increase in the degree of condensation of
the transgenic lignin after rumen incubation : the latter
was mainly due to preferential losses of syringyl units.
The kinetics of in situ disappearance clearly show the
respective e†ects of soluble cell material and wall degradation on the pattern of the degradation curves of dry
matter and of CWR. Improvement of ISCWD was particularly great in line 50 (Fig 1). The values obtained for
the kinetic parameters (Table 4) showed a good correspondence between calculated and experimental curves.
The indigestible fraction of the transgenic stems was signiÐcantly lower in all samples except line 40-1. The
inÑuence of the soluble fraction was mainly seen in line
48 since less than half of the potential ISDMD (a ] b)
increase in transgenic stem was due to enhanced cell
wall degradation (b). Although for ISCWD the slowly
degradable fraction (b) was not enhanced for the transgenic counterpart of line 40-1, a clear increase was measured in line 40-2, which had a lower residual CAD
activity, without signiÐcant interaction between the
transgenic modiÐcation and stage. The greatest
increases in “bÏ were observed for transgenic lines 50 and
40-2-14.
The rates of degradation (c) increased for transgenic
lines 50 and 40-2-8 but not for 40-1, 40-2-14 and 48.
The lag phases calculated from ISCWD Ðts were e†ectively zero or similar (about 1É6% h~1 in line 40-2-8) in
control and transgenic samples, except in line 50, for
which it was lower for the transgenic than for the
control (0É9 and 3É2% h~1 respectively, P \ 0É01).
Relationships between lignin modiÐcations and
degradability
The stems of transgenic line 48 had the greatest
decrease in S/V and the greatest increase in ISDMD,
TABLE 3
In situ disappearance (48 h) of the cell wall constituents in
whole stems (% initial content)
Samplea
Hemicelluloses
Cellulose
L igninsb
40-2-8 control
40-2-8 AS
40-2-14 control
40-2-14 AS
40-1 control
40-1 AS
50 control
50 AS
47É2
49É6
25É3
30É0
29É3
32É9
19É5
31É2*
38É7
49É0
14É4
18É8
13É5
15É9
4É8
24É8*
[45É1
[40É5
[30É4
[36É7*
[3É4
[12É3
[10É4
[4É7
a AS, transgenic sample.
b Jarrige lignins.
*, ** SigniÐcant di†erences between data of transgenic and
control counterpart at P \ 0É05 and P \ 0É01, respectively.
Fig 1. In situ disappearances of dry matter (squares) and of
cell wall residue (circles) in control (open symbols) and transgenic (closed symbols) whole stems of line 50. Each bar represents ^ 1 SD of the mean.
Cell wall composition and degradability of tobacco stems
511
TABLE 4
Parameters of the kinetics of dry matter and CWR disappearances in whole stems
Samplea
40-2-8 control
40-2-8 AS
40-2-14 control
40-2-14 AS
RSE
40-1 control
40-1 AS
RSE
50 control
50 AS
RSE
48 control
48 AS
RSE
DM disappearanceb
CW R disappearanceb
a
b
c
Ind
43É8
44É9***
25É9
25É5
0É20
22É4
22É4
0É41
18É8
19É3
0É40
23É3
29É1**
0É79
30É0
32É2***
21É7
25É0**
0É48
16É6
15É4
0É85
17É1
20É9**
1É08
14É6
16É9
1É36
0É15
0É17**
0É33
0É30
0É08
0É22
0É17
0É04
0É15
0É20
0É03
0É10
0É06
0É02
26É2
22É8***
52É5
49É5**
0É44
61É0
62É2
0É55
64É1
59É7**
0É78
62É1
54É0***
0É88
b
39É7
43É0*
14É9
21É7**
1É61
20É6
19É0
0É74
14É1
19É2**
1É07
19É0
23É8**
1É40
c
0É08
0É12
0É13
0É07
0É03
0É20
0É16
0É03
0É05
0É11**
0É01
0É10
0É07
0É02
a AS, transgenic sample ; RSE, residual standard error.
b a, rapidly degradable material ; b, slowly degradable material ; c, rate of degradation (%,
h~1) ; Ind, indigestible material.
*, **, *** SigniÐcant di†erences between data of transgenic and control counterpart at
P \ 0É1, P \ 0É05, P \ 0É01, respectively.
but a relatively low increase in “bÏ. Line 50 had relatively lower changes in composition or lignin structure
but a great increase in “bÏ. Line 40-2-14 was the only
material which showed signiÐcant changes in cell wall
composition and lignin structure as well as moderate
increases in ISCWD. Indeed, poor correlations were
observed for pooled samples between V, S or S/V and
the parameters from ISDMD or ISCWD kinetics (“bÏ or
“a ] bÏ) ; the best observed (R \ 0É78) was between the
decrease in S/V and the increase in “a ] bÏ for whole
stems, but it was not signiÐcant. However, for the
mature plants in which CAD activity was the most
altered (ie excluding 40-1 samples), a signiÐcant negative
correlation was observed between S or S/V and ISCWD
(R \ [0É59 and R \ [0É68, respectively, P \ 0É05,
n \ 12).
In addition, although only four paired samples were
studied, for the population of mature stems, the
increases in ASCWP paralleled the improvements in
ISCWD (Fig 2).
DISCUSSION
Cell wall content and composition
Fig 2. Variation across mature plants in (=) improvements of
in situ cell wall disappearance (ISCWD) and (4) increases in
yield of alkali-soluble cell wall polymers (ASCWP) due to the
transgenic modiÐcation.
Our results on lignin content and composition in transgenic plants with reduced CAD activity are consistent
with those of a previous study (Halpin et al 1994), in
which only lines 40-1 and 50 were studied. Reduction of
CAD activity was expected to decrease the formation of
hydroxy-cinnamyl alcohols and therefore reduce the
lignin content. Lignin contents were not changed, but
decreases in the S/V molar ratio and in NBO suggest
changes in lignin composition and structure. The
syringyl content of the lignin was reduced but guaiacyl
content was a†ected to a lesser extent. This suggests a
mechanism exists that di†erentially regulates the deposition of syringyl and guaiacyl units in lignin
(Terashima and Fukushima 1989). Alternatively,
enzymes, distinct from the CAD down-regulated here,
may be able to catalyse the production of coniferyl
512
alcohol, as suggested by Boudet and Grima Pettenati
(1996) for Eucalyptus sp. However, several factors
support the probability that lignin in the transgenic
plants incorporates compositional changes in addition
to those identiÐed here. An increase in the alkaliextractibility of the transgenic lignins was found (Halpin
et al 1994 ; Bernard Vailhe et al 1996). Greater amounts
of vanillin and syringaldehyde could be extracted by
mild alkali from cell wall samples of transgenic plants
than could be extracted from control plants (Bernard
Vailhe et al 1996 ; Chabbert and Monties, pers comm).
Halpin et al (1994) reported that transgenic stem
samples showed a deeper staining with phloroglucinol
(reactive with cinnamaldehydes), compared with control
samples, and larger amounts of coniferyl and sinapyl
aldehydes when analysed by pyrolysis mass spectrometry. It has therefore been suggested that a by-pass of
CAD occurred and that cinnamaldehydes were incorporated into lignins. Since their presence could not be
detected by thioacidolysis (Halpin et al 1994), FTIR or
13C NMR (Bernard Vailhe et al 1996), it is possible that
they are located in condensed moieties, such as pinoresinols, and thereby produce an altered lignin (Halpin et
al 1994).
In contrast to the data on bm1 maize (Muller et al
1971) and bmr6 sorghum (Cherney et al 1986), lignin
content in our transgenic samples did not decrease, but
other similarities with these plants were observed. A
lowering of the CWR (Muller et al 1971), of the S/V
molar ratio and an increase in the degree of condensation have also been noted in bm1 maize (Kuc and
Nelson 1964 ; Kuc et al 1968 ; Gordon and Griffith
1973) and similar variations in lignin composition were
observed in bmr6 sorghum (Chabbert et al 1993). It has
already been suggested that cinnamaldehyde incorporation alters lignins in bmr6 mutant sorghum (Pillonel et
al 1991), and perhaps in bm1 maize, which also had a
brownish xylem (Gee et al 1968).
Degradability
No improvement in degradability was observed in
transgenic population 40-1, which had the highest
residual CAD activity. Population 50, with the lowest
CAD activity, and 40-2-14, which was only slightly
more depressed than 40-1, had the greatest improvement in CWR degradability. It is likely, therefore, that
the variations were due both to the extent of CAD
down-regulation and to the heterogeneity of the population (Halpin et al 1994). A threshold of over 80%
reduction in CAD activity is probably necessary to
achieve signiÐcant improvements in digestibility. Wide
variations have also been reported for whole bm1 maize
plants ; the increases in IVDMD were either insigniÐcant (Lechtenberg et al 1972 ; Gordon and Neudoer†er)
or similar to those observed in this study (Barnes et al
M A Bernard V ailhe et al
1971). The cellulase solubilities measured for the
tobacco samples were in good agreement with the
extent of in situ disappearances, and the improvements
in susceptibility to cellulase in severely CAD downregulated transgenic samples were consistent with those
observed for bmr6 sorghum (Pillonel et al 1991). A comparison between dicots and grasses should be made
with caution, since there are structural di†erences
between the two, such as the presence of ferulic acid
bridges between lignins and polysaccharides in grasses.
The main e†ect of genetic modiÐcation was to
enhance CWR degradability but the impact on DM
degradability depended on the concomitant variation in
CWR content, which decreased in line 48. Consistent
with this are the changes previously reported in the
bmr6 sorghum mutant (Porter et al 1978). Negative
lignin disappearances have been observed in other
digestibility trials (Besle et al 1995). Greater lignin disappearances and higher syringyl solubilisation observed
in some tobacco transgenic populations have also been
reported for bm1 maize (Gordon and Neudoer†er
1973).
Wide variations were also observed in the rate of degradation. The slight increase in rate observed for two
lines and the absence of any signiÐcant di†erence in the
other line are in agreement with similar variations in
rates observed for bm1 maize (Cymbaluk et al 1973 ;
Gordon and Neudoer†er 1973). A lower lag phase of
the transgenic stems in line 50 than in the control,
together with a higher rate of degradability, is predictive
of a higher intake of the down-regulated plants. A slight
increase in intake of bm1 maize was found in experiments on young bulls (Barrière et al 1994).
The interaction between transgenic modiÐcation and
stage of growth (line 40-2) was only signiÐcant for cellulase digestibility. The absence of e†ect of downregulation in population 40-1 was probably due to a
lower decrease in CAD activity and to the variability
between plants rather than to greater maturity (2 weeks
older) than batch 40-2-14. Whatever the case, any e†ect
of stage of growth needs to be conÐrmed. In bm1 and
normal maizes, improvements in degradability due to
mutation slightly decreased with maturity (Barnes et al
1971).
Relationships between cell wall modiÐcations and
degradability
In this study, the improvements in degradability were
related to S or S/V. For a similar lignin content, the
decrease in the proportion of syringyl unit (or S/V) in
the secondary wall may increase the accessibility of the
glycanases to the substrate and therefore improve
digestibility (Reeves 1987 ; Jung and Deetz 1993). If this
did occur in CAD down-regulated plants, it would only
constitute part of the explanation since the correlation
Cell wall composition and degradability of tobacco stems
513
between syringyl units or S/V and degradability were
only signiÐcant for a restricted population. Otherwise,
the changes in proportion of ASCWP may derive from
structural changes related to cinnamaldehyde incorporation, similar to those occurring in the bm1 maize
mutation (Gordon and Neudoer†er 1973). Covalent
intermolecular bonds may be a†ected, particularly those
between lignin and carbohydrates, and hence degradability. Further studies are needed to understand which
structures in addition to syringyl units in transgenic
lignins have a causal e†ect on improvements in degradability.
Except in rare cases (Sommerfeldt et al 1979), it is not
possible to di†erentiate the e†ects of lignin content from
those of lignin structure on digestibility in natural
mutants. The data from plants with a severely depleted
CAD activity provide Ðrm additional evidence of the
e†ect of lignin structure on the extent, and possibly the
rate, of cell wall degradability. Tobacco is not used in
ruminant nutrition so the tobacco model plant has a
limited relevance to feeding applications. It is not possible with such a model to predict the e†ects of genetic
manipulations on forage plants. Nevertheless, genetic
engineering may be a means of obtaining forages with
improved digestibility but without reduction of lignin
content.
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ACKNOWLEDGEMENTS
The authors thank Michel Fabre for his contribution in
carrying out the in situ disappearance measures. This
work was supported by the European Union under the
ECLAIR
programme
AGRE
0021
OPLIGE
(Optimisation of lignin in crop and industrial plants
through genetic engineering).
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