INFLUENCE OF SYRINGYL TO GUAIACYL RATIO AND GRAVITY ON GROWTH
RESPONSES AND PHYSICAL PROPERTIES IN GENETICALLY ALTERED POPLARS
(POPULUS TREMULA X P. ALBA)
By
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Jameel Muein Al-Haddad

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ABSTRACT
INFLUENCE OF SYRINGYL TO GUAIACYL RATIO AND GRAVITY ON GROWTH
RESPONSES AND PHYSICAL PROPERTIES IN GENETICALLY ALTERED POPLARS
(Populus tremula x P. Alba)
By
Jameel Muein Al-Haddad
Lignin is a phenolic polymer that is a fundamental part of the structure and function of plants. It
provides mechanical support, acts as a hydrophobic insulation for the water conducting elements
and is a strong shield against phytopathogens. Lignin is made of variable combinations of three
monomers; syringyl (S), guaiacyl (G) and p-hydroxyphenyl (P) units. For the first time, the
combined effects of modified lignin composition and gravity on the growth responses and
physical properties were studied in genetically altered poplar. We hypothesized that enhancing
the syringyl monomer will have an impact on the gravitropic responses and the mechanical and
physiological properties of upright and inclined trees. Hybrid poplar clone 717 (Populus tremula
x P. alba) was transformed to over express the F5H/Cald5H gene at different levels resulting in
varying syringyl to guaiacyl ratios. Wild type poplar and the transgenic lines were either kept
upright or inclined (45°) from vertical to induce gravitropic responses (tension wood formation)
for three months. Over-expression of F5H in the tilted trees caused reduced growth rate than the
upright wild type controls and a reduction in height to diameter ratio. Interestingly, the tilted
stems with the highest syringyl lignin ratio responded faster and recovered back to vertical at
least 8 days before the tilted wild type trees. Quantification of released growth strain showed less
strain in the trees with the highest syringyl lignin percentage potentially due to a decrease in
growth strain earlier than the other genetic lines at the end of the gravitropic period. When all
stems were collected and mechanically tested while still fresh or oven-dried, four-point bending

and compression tests showed similar elastic properties among the different genetic lines when
the trees were kept upright. However, when tested until rupture, the over-expression lines had
lower modulus of rupture (MOR) indicating an increase in brittleness. To test the effects of the
genetic treatment and/or the gravitropic responses on the trees water conductive properties, the
maximum hydraulic conductivity was measured on the stems after high pressure flushing to
eliminate native emboli. Interestingly, the stems with higher syringyl lignin ratio showed
improved water conductive efficiency in the upright stems. Anatomically, it was found that the
lines with higher syringyl lignin abundance had larger vessel diameters and higher percent vessel
lumen area. Percent total lumen area of vessels and fibers also increased whereas percent fiber
area decreased. However, dry wood density was not affected by the lignin modification. Tension
wood had higher fiber wall thickness but less percent total lumen area due to the formation of the
gelatinous layer in the G-fibers of the tension wood. Moreover, wood samples from the different
treatments and lines were analyzed for cell wall biochemical and structural properties. In the
tension wood, increases in cellulose crystallinity and microfibril angle were among the expected
results. Lignin quantitative analysis showed a slight decrease in total lignin with increasing
syringyl fraction. The acid soluble lignin also increased significantly. Carbohydrate analyses
indicated higher percent total sugars in tension wood than in opposite or normal wood.
Interestingly, increasing syringyl lignin percentage resulted in slight decrease in percent total
sugars. Similar results were observed for glucose and galactose percentages. Percent xylose was
lower in the tension wood than in the opposite or normal woods. Before a large scale plantation
of the genetically altered poplar trees can be assumed, further testing in the field will be required
to verify their ability to withstand more complex stresses.
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DEDICATION
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ACKNOWLEDGMENTS

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LIST OF TABLES
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LIST OF FIGURES
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xv

CHAPTER 1
INTRODUCTION
BACKGROUND
Evolution of lignin
With the evolution of terrestrial plants from aquatic ancestors around 475 million years ago
(Boyce et al., 2004; Peter and Neale, 2004; Kendrick and Crane, 1997) (Figure 1.1) came the
necessity to fortify the cell walls of the plants to withstand the different environmental stresses
and to adopt an erect growth form. Studies of modern era plants showed that a complex
aromatic heteropolymer (lignin) makes a vital contribution to the long-term survival of some
perennials (Pedersen et al. 2005). Lignin provides mechanical support through cementing the
secondary cell wall matrix, binding to cellulose microfibrils (Wainwright et al., 1982) and
cross-linking other cell wall components (Martone et al., 2009; Ralph et al., 2004). In addition,
it provides rigidity to vessel walls enabling them to withstand high negative pressures without
collapse (Kitin et al., 2010). Lignin also provides a defense line against plant pathogens
(Bhuiyan et al., 2009). The timeframe of lignin evolution is still unknown especially with the
discovery of lignin in red algae which diverged from vascular plants more than one billion years
ago (Martone et al., 2009).

1

Figure 1.1 Phylogenetic tree showing the distribution of lignin monomer
composition across major lineages (Vanholme et al. 2010).
S = syringyl lignin, G = guaiacyl lignin and H = p-hydroxyphenyl lignin. For
interpretation of the references to color in this and all other figures, the reader is
referred to the electronic version of this dissertation.
Cell wall components and their roles
The first attempt to study the chemical composition of woody plants was made in 1838 by the
French chemist and botanist, Anselme Payne (Schubert, 1965). He found, despite the
complexity and diversity of wood species, they all contain three major chemical components.
They are cellulose, the hemicelluloses and lignin.
Cell wall polysaccharides: (compiled from Liepman et al., 2007; Somerville, 2006; Somerville
et al., 2004; Higuchi, 1997)
Cellulose, the most abundant organic molecule on earth, is a linear polymer that is built
of 1,4-glucosidic linkage of !-D-glucose forming parallel !-1,4-glucan chains. Every 36 glucan
chains are held together by hydrogen bonding to form cellulose microfibril. The length of the

2

microfibril varies depending on species or activity of the CESA enzymes that build the glucan
chains. The crystalline region of the microfibrils is the part made of those continuous chains
built by the 36 CESA enzymes in the plasma membrane-embedded resette subunits. Making
50% of the cell wall, the microfibrils constitute a skeleton for other wall constituents to anchor
to such as hemicelluloses and structural proteins creating a matrix in which lignin is later
deposited.
Hemicelluloses. Branched heteropolymers cross-link the cellulose microfibrils in almost
all plant cell wall matrices. They are divided into four classes; xyloglucans which are heavily
substituted !-1,4-glucan backbone (mostly xylose and glucose), glucomannans are variably
substituted !-1,4-mannan backbone (glucose and mannose), arabinoxylan made of substituted !1,4-linked xylan backbone (mostly arabinose and xylose), and mixed linkage glucans made of
unsubstituted backbone of !-1,4- and !-1,3-linkages of glucosyl residues.
Pectins. Representing one third of plant cell wall polysaccharides, pectins are major
component of primary cell wall. They are a group of complex polysaccharides containing 1,4-"D-linked galacturonic acid. Various sugars bind to the polygalacturonate including rhamnose,
arabinose, fucose, galactose, and xylose.
Lignin
Lignin is a phenolic compound ranking second behind cellulose in abundance on earth
(Dashtban et al., 2010). It acts like a cementing material in xylem tissues of trees providing
support and insulation for water conduction in fibers and vessels, respectively. Lignin polymer
is made of three types of monolignols; syringyl, guaiacyl and p-hydroxyphenyl residues
(Figures 1.1). Lignin in gymnosperms is predominantly made of guaiacyl whereas phydroxyphenyl occurs at higher concentrations in the grass family but is less pronounced in
3

hardwood (Higuchi, 1997). To understand the role played by each of the monolignols, mainly
syringyl and guaiacyl, in trees mechanics and physiology numerous studies have been made (for
literature refer to Table 1.1).
Types of reaction wood
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4

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8

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Modifying lignin
Due to the economic importance of poplar as a source of wood and fiber in plantation forestry,
and due to the increasing interest in its wood as a potential source of renewable cellulose to
serve the demand for sustainable biofuel, there have been an increasing interest in profiling the
transcriptome of woody plants (Prassinos et al., 2005; Ko and Han, 2004) to genetically modify
the composition of wood to suit the purposes of the industry (Huntley et al., 2003). For better
paper and pulp qualities, attempts have been made to genetically alter the lignin synthetic
pathway to make lignin extraction a more feasible process (Huntley et al., 2003).
Due to the detrimental effect of reducing the total lignin content in poplar, lignin
composition has been modified while keeping the lignin content unchanged. In one study on
poplar, Huntley et al. (2003) found that lignin solubility is positively correlated with syringyl
monomer content in the lignin matrix. As a result, bleaching chemicals can treat the matrix
more effectively. Their results indicated a 60% reduction in pulping time of trees with increased
S:G ratio, which potentially would increase pulp mill capacity while decreasing chemical costs.
Based on the demand of the energy sector, poplar trees with down-regulated lignin have been
produced to provide easier access to renewable cellulose.
As a result of 40% reduction in total lignin, Voelker et al. (2011) found a reduction in
wood stiffness and strength. They also reported a reduction in growth and impaired xylem

9

conductivity. An optimum balance between reduced lignin and biomass growth has been
recently proposed (Novaes et al., 2010).
Whether genetically modified trees can have a sustained growth and tolerate
environmental and internal growth stresses is the primary interest of this investigation. Ferulate
5-hydroxylase (F5H) is a gene that encodes for an enzyme that acts downstream in the lignin
synthesis pathway, converting 5-hydroxy-coniferylaldehyde and 5-hydroxyconiferylalcohol
towards the synthesis of sinapyl alcohol, i.e. syringyl (Franke et al., 2000). The F5H mutants
are prime candidates for use in this study, allowing us to investigate the direct influence of
changed lignin monomer composition on growth responses and physical properties in a well
known model system with no other apparent alterations in cell wall structure or tissue
composition to bias the results.
RESEARCH OBJECTIVES
The main objective of this research was to test the ability of the genetically altered poplar trees
to respond to gravity and maintain physical and growth properties that are comparable to those
of the unmodified wild type trees.
The specific objectives were:
•

To investigate the kinematic responses as well as to quantify the growth strains generated in
the tension wood. And to evaluate the overall stem mechanical properties of the poplar lines.

•

To investigate the water relations in the upright stems as well as in those inclined and
formed tension wood in response to gravity. Also to investigate the leaf area changes and
the anatomy of the three wood tissue types.

10

•

To determine the changes in the wood’s carbohydrate composition and other structural
features such as crystallinity and microfibril angle.
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12

REFERENCES
!
!
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16

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17

CHAPTER 2
EFFECT OF SYRINGYL LIGNIN RATIO ON GROWTH RESPONSES AND
MECHANICAL PROPERTIES OF INCLINED GENETICALLY-ALTERED POPLARS
(POPULUS TREMULA X P. ALBA)
!
ABSTRACT
Lignin is an important phenolic polymer of secondary cell walls that is known to influence the
mechanical properties of secondary xylem (wood). The major monomers of angiosperm lignin
are syringyl and guaiacyl. This study evaluates the effect of modifying lignin composition on
the response of trees to gravity following inclination. Changes in kinematics and mechanical
properties were investigated in one-year old hybrid poplar clone 717 (Populus tremula x P.
alba) that was transformed with C4Hprom::F5H resulting in an increased syringyl lignin ratio.
Wild type poplars and the transgenic lines were inclined 45° from vertical for three months to
induce a negative gravitropic response (stem curvature and tension wood formation).
Inclination, and the resulting gravitropic response stimulated tension wood formation in stems
and caused decreases in tree allometry and stem growth. Interestingly, increasing the syringyl
lignin content of stems resulted in a faster adjustment to gravity, by up to eight days, compared
to the unmodified wild-type controls. The stems of the upright and inclined trees had similar
elastic properties. However, the stems of the inclined trees were stiffer when the tension wood
side was under tension during the bending test. Evaluating the gravitropic response of the
transgenic poplar lines to inclination improved our understanding of the mechanical and
physiological role of lignin monomer composition in normal and tension wood.

18

INTRODUCTION
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19

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The mechanical properties of tension wood have not been extensively studied despite
their distinctive physical properties. These properties are evident in the differential shrinkage
characteristics of tension wood, and the difficulty to achieve a smooth surface in wood samples
that contain it (Illston et al., 1979; Hoadley, 2000). In a study comparing tension wood to
normal wood in young poplar, Coutand et al. (2004) reported higher Young’s modulus and
higher level of longitudinal residual strain as proof of the unique mechanical properties of this
specialized tissue.
Similarly, little is known about the effect of modified lignin content and composition on
the mechanical properties of woody plants such as poplar. The ability of the trees to sustain
mechanical support of their own crown weight was reported to been affected by the lignin
content (Gindl and Teischinger, 2002). Due to the relatively longer time to form a useable
volume of wood, it was necessary to utilize young transgenic trees to investigate the effect of
modified lignin content on trees wood properties. To test the mechanical properties of young
genetically modified trees, newly developed testing techniques were tailored to the age and size
of these new stock plants (Kasal et al., 2007). Subsequently, Horvath et al. (2010) studied the
mechanical properties of young genetically modified aspen trees with modified lignin content or
structure. They found a significant reduction in the mechanical properties due to reduced lignin
content, and a slight reduction due to increased S:G ratio. A more recent study showed
comparable losses in wood strength and stiffness and lower growth efficiency due to reduced
lignin content in young poplar (Voelker et al., 2011a and b). Other similar studies have focused
on the effects of either lignin modification or the formation of tension wood in trees. However,

20

no study has investigated the interaction between lignin modification and tension wood
formation. Therefore, this chapter describes, for the first time, a study that combined the effects
of both tension wood formation and lignin structure modification on the mechanical properties
of stems of a perennial model plant. The study was carried out by investigating the gravitropic
response to displacement with respect to gravity leading to tension wood formation in the
transgenic hybrid poplar (Populus tremula x P. alba) trees with modified lignin. Investigating
the impact of lignin composition in transgenic lines of the same species will minimize possible
bias introduced by other anatomical parameters when different plant species or different growth
forms of the same species are compared. It was hypothesized that genetically modified lignin
monomer ratio will have an impact on hybrid poplar growth responses in the form of altered
allometry and kinematics. In addition, significant changes in the mechanical properties of the
wood are also expected. It was predicted that elevation of syringyl lignin ratio and inclination of
the trees, leading to the formation of tension wood in the stems, would cause increases in the
modulus of elasticity (MOE) and modulus of rupture (MOR) in poplar stems.
MATERIAL AND METHODS
Experimental Plan
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Morphology and Allometry
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"LJ_!

"KLJ!

"J_!

"LJ!

"KL!

C.6+;0!JOLO!"O!X.?1P/=>0!781!/;78<608.4!>2>?7;!/;00<!.84?.801!YZ[!@;2-!:0;/.47?O!(;00<!2@!
1.@@0;08/!%\]!;7/.2!;0<>281!/2!6;7:./=!7/!:7;=.86!106;00<O!#.6./7?!.-760<!N0;0!/7M08!7/!
1.@@0;08/!</760<!781!N0;0!+<01!/2!1;7N!/5;00!7,<2?+/0!786?0<!UHW!781!/N2!;0?7/.:0!786?0<!
UBW!+<.86!/5;00!</0-!<06-08/<^!?2N0;I!-.11?0!781!+>>0;O!

24

&

Measuring growth Strains
"!</;7.8!67+60!-0/521!UG2<5.17!781!)M+=7-7I!JKKJW!N7<!+<01!/2!-07<+;0!6;2N/5!</;7.8!
.8!>2>?7;!/;00<!.8!N5.45!</;7.8!67+60<!N0;0!6?+01!/2!@;0<5I!,7;MP@;00!</0-!<+;@740<!+810;!
N5.45!/08<.28!N221!N7<!0b>04/01!/2!@2;-!U+>>0;!<+;@740!2@!/50!.84?.801!>2>?7;!</0-<WO!
(50!67+60<!U&DPKitJZKH]PLJKI!V.<57=!D;04.<.28!];2+>I!37?:0;8I!D088<=?:78.7I!9%"W!
N0;0!,28101!/2!/50!N221!<+;@740<!?286./+1.87??=!7?286!/50!6;7.8!UC.6+;0!JOJW!+<.86!7!
428/74/!7150<.:0!42->7/.,?0!N./5!N0/!<+;@740<!U3PH281!JKKI!,281.86!N7<!47/7?=g01!,=!
B7/7?=</PB!JKKI!V.<57=!D;04.<.28!];2+>I!37?:0;8I!D088<=?:78.7I!9%"WO!"!/5;00PN.;0!
A+7;/0;P,;.160!4.;4+./!N7<!+<01!/2!7??2N!@2;!7+/2-7/.4!42->08<7/.28!@2;!/50!0@@04/<!2@!
/0->0;7/+;0!457860<!.8!/50!?071!N.;0<!28!/50!,;.160!,7?7840I!781!@2;!.84;07<01!
-07<+;0-08/!<08<./.:./=O!(2!;01+40!/08<.28!.81+401!,=!N.;0<I!/0;-.87?!</;.><!UB&]PSZBW!
N0;0!6?+01!/2!/50!b=?0-!<+;@740<!781!428804/01!/2!/50!</;7.8!67+60<!+<.86!/5.8!42>>0;!
N.;0<!UL_YP"XDW!7<!N7<!;042--08101!,=!/50!-78+@74/+;0;!UV.<57=!D;04.<.28!];2+>I!
37?:0;8I!D088<=?:78.7I!9%"WO!(2!7??2N!;042:0;=!2@!N221!<5;.8M760I!4+/<!N0;0!-710!7,2:0I!
,0?2N!781!+810;807/5!0745!67+60!/2!7!10>/5!0A+.:7?08/!/2!7,2+/!57?@!/50!;71.+<!2@!/50!
;0<>04/.:0!</0-<O!B28/.8+2+<!;071.86<!N0;0!/7M08!0:0;=!<04281!1+;.86!/50!4+//.86!
>;240<<!+<.86!7!</;7.8!.81.47/2;!781!;042;10;!UD_!%/;7.8!$81.47/2;!781!'042;10;I!V.<57=!
D;04.<.28!];2+>I!37?:0;8I!D088<=?:78.7I!9%"W!+8/.?!7!</7,.?.g01!;071.86!N7<!745.0:01O!(50!
/0;-.87?!;071.86<!N0;0!428<.10;01!@2;!<+,<0A+08/!787?=<0<!781!42->7;.<28<O!

25

!
!
C.6+;0!JOJO!UF0@/W!"!</;7.8!67+60!N7<!,28101!/2!/50!N221!<+;@740!?286./+1.87??=!7?286!/50!
6;7.8!2@!.84?.801!5=,;.1!>2>?7;!/;00<!/57/!;042:0;01!/50!:0;/.47?!>2<./.28O!U'.65/W!'0?07<01!
</;7.8!N7<!-07<+;01!0:0;=!<04281!1+;.86!/50!4+//.86!>;240<<!+<.86!7!D_!%/;7.8!$81.47/2;!
781!'042;10;O!
Mechanical properties
$8/74/!6;008!781!2:08P1;.01!10,7;M01!</0-<!N0;0!+<01!@2;!-04578.47?!/0</.86O!(50!17/7!
7??2N01!@2;!/50!47?4+?7/.28!2@!/50!-21+?+<!2@!0?7</.4./=!U3)&WI!-21+?+<!2@!;+>/+;0!U3)'W!
781!@?0b+;7?!</.@@80<<!U"$WO!"?<2!M82N8!7<!G2+86s<!321+?+<I!3)&!.<!0A+7?!/2!/50!@?0b+;7?!
</.@@80<<!1.:.101!,=!/50!<04281!-2-08/!2@!b=?0-!4;2<<!<04/.287?!7;07!U*.M?7<I!LeeJWO!(50!
"$!781!3)'!N0;0!47?4+?7/01I!781!3)&!N7<!10;.:01!@;2-!"$!781!/50!<04281!-2-08/!2@!
4;2<<!<04/.287?!7;07!U$WO!X50;07<!3)&!N7<!47?4+?7/01!N./5.8!/50!0?7</.4!;7860!2@!/50!</0-<I!
</0-<!N0;0!?27101!+8/.?!@7.?+;0!.8!2;10;!/2!10/0;-.80!3)'!UX7680;!6-084OI!LeeiWO!!%.bP/;00!
;0>?.47/.28!N7<!+<01!.8!JKK`I!781!JJP/;00!;0>?.47/.28!.8!JKKiO!
Four-point bending'&H081.86!6080;7/0<!7!42-,.87/.28!2@!/08<.287?!@2;40<!7/!/50!?2N0;!<.10!
2@!/50!<7->?0!781!42->;0<<.:0!7/!/50!+>>0;!<.10O!$8!/50!47<0!2@!@2+;P>2.8/!,081.86I!82!
,.7<.86!<507;!@2;40<!N0;0!6080;7/01O!H0@2;0!10,7;M.86!/50!</0-<I!/50=!N0;0!4+/!/2!,0!
-2;0!/578!/08!/.-0<!?2860;!/578!/50.;!1.7-0/0;I!7<!N7<!;0A+.;01!@2;!/50!,081.86!/0</<!
U*.M?7<I!LeeJI!D;+=8!0/!7?OI!JKKKW!781!.8!42->?.7840!N./5!/50!/0</.86!</7817;1!"%(3!#LY_!

26

ULeeYWO!9<.86!78!$8</;28(!98.:0;<7?!3745.80!U3210?!YJKJI!$8</;28!B2;>2;7/.28I!B78/28I!
3"I!9%"W!UC.6+;0!JO_W!0A+.>>01!N./5!7!LK!M*!?271!40??!781!7!4;2<<5071!<>001!2@!JK!
--r-.8I!LYP4-!<06-08/<!2@!6;008!</0-<!N0;0!?27101!N./5.8!/50!0?7</.4!;7860O!(50!</0-<!
@;2-!/50!.84?.801!/;00<!N0;0!/0</01!N./5!/50!/08<.28!N221!<.10!@74.86!7N7=!@;2-!/50!
4;2<<5071I!781!767.8!N5.?0!@74.86!/50!4;2<<5071O!(50!<7-0!</0-<!N0;0!/508!1;.01!/2!7!
428</78/!N0.65/I!0A+.?.,;7/01!/2!;22-!/0->0;7/+;0!781!-2.</+;0!?0:0?I!781!/508!?27101!
767.8!+8/.?!;+>/+;0!7??2N.86!@2;!/50!47?4+?7/.28!2@!3)'O!(2!47?4+?7/0!/50!<04281!-2-08/!2@!
7;07I!78!.107?.g01!2:7?!<57>0!N7<!7<<+-01!@2;!/50!4;2<<!<04/.28<O!(5.<!10>08101!28!/50!
;0<>04/.:0!1.7-0/0;<!2@!/50!7b0<I!N5.45I!.8!/50!.84?.801!</0-<I!.84;07<01!>7;7??0?!/2!6;7:./=!
;7/50;!/578!>0;>081.4+?7;O!

!
Figure 2.3. An Instron® Universal Machine was used for mechanical testing (left) and can be
fitted with a crosshead specific for four-point bending tests (right).
!
Compression testing'0(50!42->;0<<.:0!-21+?+<!N7<!-07<+;01!/5;2+65!/0</.86!/50!</0-<!
+810;!42->;0<<.:0!@2;40O!B=?.81;.47?!</0-!<06-08/<!@;2-!+>;.65/!/;00<I!/N.40!7<!?286!7<!

27

/50.;!1.7-0/0;I!N0;0!0b>2<01!/2!42->;0<<.:0!@2;40!>7;7??0?!/2!/50!6;7.8!+<.86!/50!<7-0!
$8</;28(!98.:0;<7?!3745.80!0A+.>>01!N./5!KOZ!M*!?271!40??!781!7!4;2<<5071!<>001!2@!_!
--r-.8O!!
Replication and Data Analysis
(50!10<.68!.84?+101!7!N.?1P/=>0!781!/5;00!()*!2:0;P0b>;0<<.28!?.80<!@2;!0745!2@!/50!/N2!
/;07/-08/<!U+>;.65/!:<O!.84?.801WI!781!N./5!7!;0>?.47/.28!2@!<.b!/;00<I!/50!/2/7?!N7<!Yi!/;00<!
@2;!/50!0b>0;.-08/O!(50!M.80-7/.4<!0b>0;.-08/!.84?+101!N.?1P/=>0!UmSZ!-2?Oh!<=;.86=?W!
781!/N2!6080/.4!?.80<!Umi_!781!meY!-2?Oh!<=;.86=?WO!(2!/0</!@2;!</0-<f!-04578.47?!
>;2>0;/.0<!.8!JKKiI!JJP+>;.65/!/;00!;0>?.47/.28!@2;!0745!>2>?7;!?.80!N7<!+<01O!].:08!/50!
,7?78401!10<.68I!7!JPN7=!787?=<.<!2@!:7;.7840!U"*)V"W!N7<!+<01O!#7/7!N0;0!787?=g01!
+<.86!/50!<2@/N7;0!'!U'!#0:0?2>-08/!B2;0!(07-I!JKKSWO!"<<+->/.28<!2@!82;-7?./=!781!
52-26080./=!2@!:7;.7840<!N0;0!/0</01O!(50!M.80-7/.4!;0<>28<0<!17/7!N0;0!787?=g01!+<.86!
;0>07/01!-07<+;0<!"*)V"O!
RESULTS
(50!7??2-0/;.4!17/7!@;2-!/50!>2>?7;!</0-<!N0;0!42??04/01!.8!JKK`!7@/0;!/50!</0-<!N0;0!
7??2N01!/2!;0<>281!/2!6;7:./=!781!;0</2;0!/50!:0;/.47?!>2<./.28O!$84;07<.86!<=;.86=?!?.68.8!
>0;408/760!;0<+?/01!.8!7!104;07<0!.8!</0-!50.65/!/2!1.7-0/0;!;7/.2!.8!+>;.65/!781!.84?.801!
/;00<!U'dKOKL`I!42;;0?7/.28!420@@.4.08/d!PKOJeW!UC.6+;0!JOY"WO!(50!<?2>0<!2@!/50!/N2!
;06;0<<.28!?.80<!N0;0!</7/.</.47??=!<.-.?7;O!(50;0@2;0I!/50!1.@@0;0840!.8!/50!.8/0;40>/!
;0>;0<08/01!/50!1.@@0;0840!,0/N008!/50!+>;.65/!781!.84?.801!/;00<O!H7<01!28!/50!0</.-7/01!
.8/0;40>/<I!/50!.84?.801!/;00<!<52N01!7!104;07<0!.8!/50.;!50.65/P/2P1.7-0/0;!;7/.2!,=!7<!
-+45!7<!L`O`e!c!ZOLJ!Uc!!"WI!;0?7/.:0!/2!/50!+>;.65/!/;00<!U'dKOKKJWO!#+;.86!/50!0b>0;.-08/I!

28

,2/5!/50!+>;.65/!781!/50!.84?.801!/;00<!6;0N!7/!7!</7/.</.47??=!<.-.?7;!;7/0!UC.6+;0!JOYHWO!
(50!/N2!;06;0<<.28!?.80<!571!<.-.?7;!<?2>0<!781!.8/0;40>/<O!E2N0:0;I!7!<?2N0;!6;2N/5!;7/0!
N7<!82/.401!N./5!.84;07<.86!<=;.86=?!?.68.8!>0;408/760!.8!7??!2@!/50!/0</01!/;00<!U'dKOKKiI!
42;;0?7/.28!420@@.4.08/d!PKOLSWO!!
150

Height gained during the
experiment (cm)

B

200
175
150
125
100
75
50

130

Height / Diameter

A

110

Upright
60

Inclined

70
80
90
Syringyl (mol.%)

100

90
70

Upright

50
60

Inclined

70
80
90
Syringyl (mol.%)

100

C.6+;0!JOYO!D2>?7;!/;00!7??2-0/;=!;0<+?/<!2@!78!0b>0;.-08/I!781!,.2450-.47?!787?=<0<I!
/57/! N0;0! 4281+4/01! .8! JKK`O! (50! <=;.86=?! -2?0! >0;408/760<! /57/! N0;0! +<01! N./5!
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6;7:./;2>.4!>57<0O&#+;.86!/50!6;7:./;2>.4!;0<>28<0I!/50!/;00<!N./5!/50!5.650</!-2?0!
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?2N0;!<=;.86=?!?.68.8!;7/.2!Ui_!-2?OhWI!2;!/50!+8-21.@.01!N.?1P/=>0!/;00<!USZ!-2?OhWO!

29

!

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781!A+71;7/.4!U'dKOKKLW!/;081<!2:0;!/.-0!N508!42->7;01!/2!/50!/N2!6080/.4!?.80<!N./5!
5.650;!<=;.86=?!?.68.8!>0;408/760O!

!
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30

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31

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42

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43

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44

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45

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46

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,.2450-.</;=!781!</;+4/+;7?!42->2808/<!U0745!.<!1.<4+<<01!.8!7!<0>7;7/0!457>/0;WO!
CONCLUSION
Poplar stems, after inclination with respect to gravity, adjusted their stems back to vertical
position by means of tension wood formation on the upper side of the tilted stems.
Subsequently, they became shorter with thicker stems where tension wood was formed.
Modifying lignin composition so as to contain higher percentage of syringyl lignin caused the
stems to grow less during the experiment than the unmodified controls. When the tilted trees
began to respond to the gravitropic stimulus they went through three stages: latent phase,
gravitropic phase and autotropic phase. Increasing the syringyl lignin percentage in the stems
led to shorter latent phase and faster overall kinematic response. The stems with the highest
47

syringyl lignin ratio corrected their stems and completed the curving and decurving responses
eight days earlier than the unmodified controls. Contrary to the expectation, the released growth
strain on the tension wood surface was lower in the stems with the highest syringyl lignin
percentage, when measured at the end of the experiment, potentially due to faster recovery to
the vertical position. The mechanical analyses of stems of the upright and inclined trees showed
no change in the elastic properties in response to the lignin modification, suggesting a limited
role for lignin composition in determining stem elastic properties, or the trees ability to cope by
means of other structural and/or compositional changes. Nevertheless, a slight increase in
brittleness was observed due to enhancing the syringyl lignin percentage in the xylem of the
dried poplar stems. Investigating the interaction between tension wood formation and modified
lignin monomer composition in hybrid poplar stems unveils novel information for the scientific
community, and conforms with the increasing interest in utilizing wood as an industrial
feedstock.
ACKNOWLEDGEMENTS
X0!/578M!#7:.1!&??.<!U*7/.287?!B08/0;!@2;!]080/.4!'0<2+;40<!D;0<0;:7/.28W!781!B?.8/!
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</+1=I!H;71?0=!37;M<!781!%/0:0!37;A+.0!U3.45.678!%/7/0!98.:0;<./=W!@2;!>;2:.1.86!7440<<!
/2!/50.;!$8</;28(!98.:0;<7?!3745.80O!(5.<!;0<07;45!N7<!<+>>2;/01!,=!/50!*7/.287?!
'0<07;45!$8./.7/.:0!2@!/50!9%#"!B%'&&%!6;78/!8+-,0;!JKKZP_ZLK_PLZJ`e&
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48

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REFERENCES
!
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).<E%.&FFG&H8$/=+&*I!ULeS_W!304578.4<!2@!/50!42->;0<<.28!N221!;0<>28<0O!$$O!)8!/50!
?247/.28I!74/.28!781!1.</;.,+/.28!2@!42->;0<<.28!N221!@2;-7/.28O!D?78/!D5=<.2?!
J2\SSStSiJO!
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)B!;&02K4!ULeeYW!%/7817;1!-0/521<!2@!/0</.86!<-7??!4?07;!<>04.-08<!2@!/.-,0;O!"%(3!
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I"+,& LMNG& L$"8.& *G& O.8$& PG& C89& BMQ! UJKKiW! ];2N/5! </;0<<0<! 7;0! 5.65?=! 428/;2??01! ,=! /50!
7-2+8/!2@!]P?7=0;!.8!>2>?7;!/08<.28!N221O!$"X"!3RU_W\J_SPJY`!
&
N%./<E#"<E& L& "+-& S=:.8T"& B! UJKKJW! (;78<608.4! /;00<! 7<! /22?<! .8! /;00! 781! >?78/!
>5=<.2?26=O!(;00<!2U\JZKPJ`L!
&
N="-$%1&F*!UJKKKW!9810;</781.86!N221\!7!4;7@/<-78f<!6+.10!/2!N221!/04582?26=I!J81!01O!
(7+8/28!D;0<<I!*0N/2N8I!B28804/.4+/I!9%"O!
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S%.+&S)G&V6%./&IHG&!%$%6/W8&IHG&S=%E$%.&C!UJKKZW!304578.47?!>0;/+;,7/.28!7@@04/<!
4281+4/.:./=I!-04578.47?!>;2>0;/.0<!781!7,2:06;2+81!,.2-7<<!2@!5=,;.1!>2>?7;<O!
(;00!D5=<.2?!3JULKW\!LJY_PLJZLO!
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42->+/.86O!'!C2+817/.28!@2;!%/7/.</.47?!B2->+/.86I!V.0887I!"+</;.7O!$%H*!_PeKKKZLPKSP
KI!9'F!5//>\rrNNNO'P>;2T04/O2;6O!
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51

Voelker SL, Lachenbruch B, Meinzer FC, Strauss SH (2011a) Reduced wood stiffness and
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>2>?7;<!N./5!;01+401!?.68.8!<52N!.->7.;01!b=?0-!4281+4/.:./=I!6;2N/5!0@@.4.084=!
781!<+;:.:7?O!D?78/!B0??!&8:.;28!4K\`ZZP``iO!
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&
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<+;@740!+<.86!</;7.8!781!1.7?!67+60<O!E2?g@2;<45+86!JU\Y`LPY`SO!
&
[=/E8+","& )G& I9]85"& ;G& B"8W8& N& ULeeJW! '0?7/.28<5.><! ,0/N008! 40??! 0:2?+/.28! 781! ?.68.8!
</;+4/+;7?! :7;.0/.0<! .8! 27M! b=?0-! 0:7?+7/01! ,=! -.4;2<42>.4! <04/;2>52/2-0/;=! N./5!
<0>7;7/01!40??PN7??<O!32M+g7.!]7MM7.<5.!4^\`JeP`_SO!

52

CHAPTER 3
INFLUENCE OF SYRINGYL TO GUAIACYL RATIO ON ANATOMY AND WATER
RELATIONS IN GENETICALLY ALTERED POPLARS (POPULUS TREMULA X P. ALBA)

ABSTRACT
Lignin is an important phenolic polymer of secondary cell walls that is known to influence the
physiological properties of secondary xylem (wood). Plants have been shown to alter their
lignin composition to meet internal functional requirements as well as due to external
environmental stimuli. Angiosperm lignin is majorly composed of two monomers, syringyl and
guaiacyl units. The role these monomers play to help the plants maintain their vital hydraulic
functions were investigated. It was hypothesized that increasing the syringyl content in the
xylem will improve water conductivity and resistance to implosion without affecting the
xylem’s anatomical properties. Hybrid poplar clone 717 (Populus tremula x P. alba)
transformed to over express the C4H::F5H gene at different levels resulting in varying syringyl
to guaiacyl ratios was used in this study. Wild type poplar trees and the transgenic lines were
kept upright or inclined (45°) from vertical for three months to induce tension wood formation.
Specific water conductivity was improved in the upright poplar trees with higher syringyl lignin
ratio but not in the inclined trees. On the other hand, changing the lignin monomer ratio did not
affect the upright and the inclined stems vulnerability to cavitation under negative pressure.
Increases in the average leaf area, vessel diameter and percent vessel lumen area in response to
increased syringyl lignin ratio in normal wood were detected, and coincided with the changes in
water conductive properties. The anatomical properties of tension wood were not affected by

53

the lignin modification, nor did xylem density. Evaluating the response of poplar trees to
inclination and altered lignin composition improves our understanding of the role of lignin
monomers in affecting the xylem anatomy and physiological properties in normal and tension
wood.
INTRODUCTION
(50!-04578.47?!781!N7/0;!4281+4/.:0!>;2>0;/.0<!2@!:7<4+?7;!>?78/<!7;0!4;./.47?!/2!/50!
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.8/0;87?!<+45!7<!N.81!UQ0;8!0/!7?OI!JKKZW!2;!6;7:./=!UD.?7/0!0/!7?OI!JKKYWI!/50!<5;.8M760!2@!
/08<.28!N221!UB?7.;!0/!7?OI!JKLLWI!781!/50!8067/.:0!>;0<<+;0!.8<.10!/50!4281+4/.86!b=?0-!
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54

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55

N7<!5=>2/50<.g01!/57/!-21.@=.86!?.68.8!42->2<./.28!.8!/50!/.<<+0!N.??!57:0!82!0@@04/!28!./<!
N7/0;!4281+4/.:0!>;2>0;/.0<O!!
MATERIALS AND METHODS
Plant Material
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>;2>767/01!2:0;!-+?/.>?0!6080;7/.28<!+<.86!;22/!<>;2+/<O!G2+86!/;00<!N0;0!6;2N8!+810;!
6;00852+<0!4281./.28<!28!/50!3.45.678!%/7/0!98.:0;<./=!47->+<O!D2/<!ULiP?./0;W!N./5!<2.?P
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/50!<7->?0O!!
Water transport and Vulnerability
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56

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57

Xylem anatomical measures
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4+/!7/!_K!µ-!/5.4M80<<I!N0/!-2+8/01!781!2,<0;:01!+810;!7!42->2+81!-.4;2<42>0!@.//01!
N./5!7!1.6./7?!47-0;7O!(50!;7812-?=!47>/+;01!1.6./7?!.-760<!N0;0!+<01!/2!-07<+;0!7!
<7->?0!<.g0!2@!JKK!:0<<0?<!781!LKK!@.,0;<!@;2-!0745!</0-!+<.86!%4.28!$-760!U%4.28!
B2;>2;7/.28WO!
Wood density
X221!108<./=!N7<!10/0;-.801!+<.86!/50!-0/521!10<4;.,01!,=!"%(3!%/7817;1!30/521!
#J_eZ!N./5!-21.@.47/.28O!X221!<7->?0<!ULYP4-!?286W!N0;0!2:08!1;.01!7/!`K°B!+8/.?!
428</78/!N0.65/!/508!?0@/!/2!0A+.?.,;7/0!/2!;22-!-2.</+;0!?0:0?O!(50!<7->?0<!N0;0!N0.6501!
,0@2;0!781!7@/0;!/50=!N0;0!1.>P427/01!N./5!?.A+0@.01!>7;7@@.8O!(50!71101!>7;7@@.8!:2?+-0!
N7<!7442+8/.86!@2;!,=!1.:.86!/50!N0.65/!457860!@2;!0745!<7->?0!,=!/50!108<./=!2@!>7;7@@.8O!
9<.86!/50!N7/0;!1.<>?740-08/!-0/521I!/50!10/0;-.801!:2?+-0!2@!/50!N221!<7->?0<!N7<!
+<01!/2!47?4+?7/0!N221!108<./=O!!
Mäule staining
C2;!1.@@0;08/.7?!</7.8.86!2@!40??!/=>0<!781!/.<<+0<!N./5!-21.@.01!?.68.8!7!3l+?0!</7.8.86!
-0/521!N7<!+<01!7<!N7<!10<4;.,01!,=!B57>>?0!0/!7?O!ULeeJWO!(;78<:0;<0!<04/.28<!N0;0!
@.b01!.8!Yh!6?+/7;7?105=10!@2;!`K!-.8+/0<!781!;.8<01!N./5!N7/0;O!(508I!/50=!N0;0!/;07/01!
N./5!LKh!EB?!@2;!Z!-.8+/0<!781!;.8<01!N./5!N7/0;!,0@2;0!/50=!N0;0!-2+8/01!.8!
428408/;7/01!*EY)EO!#.6./7?!.-760<!N0;0!47>/+;01!+<.86!7!?.65/!-.4;2<42>0!/57/!N7<!
@.//01!N./5!7!1.6./7?!47-0;7O!

58

Leaf area
H0@2;0!/50!,06.88.86!2@!/50!YZ°.84?.87/.28!/;07/-08/!2@!/50!/;00<I!7!>0;-7808/!-7;M0;!N7<!
+<01!/2!?7,0?!/50!@.;</!@+??=!0b>78101!?07@!2@!0745!/;00!U?07@!>?7</245;28!.810bI!FD$W!/2!
@74.?./7/0!/;74M.86!2@!/50!80N!6;2N/5I!781!/2!-7;M!/50!,06.88.86!2@!/50!<042817;=!6;2N/5!
U&;.4M<28!781!3.450?.8.I!LeZSWO!(5;00!-28/5<!?7/0;I!/50!?07:0<!7,2:0!/50!-7;M!N0;0!
57;:0</01!781!/50.;!717b.7?!<+;@740!7;07<!N0;0!-07<+;01!+<.86!7!?07@!7;07!-0/0;!F$P_LKK!
UF$PB)'!H.2<4.0840<I!$84OI!F.842?8I!*0,;7<M7I!9%"WO!(2/7?!781!7:0;760!?07@!7;07<!7,2:0!/50!
-7;M!N0;0!10/0;-.801!@2;!/50!+>;.65/!781!.84?.801!/;00<O!
Data analysis
(50!42??04/01!17/7!N0;0!</7/.</.47??=!@.//01!N./5!?.807;!-210?<!/508!/0</01!N./5!"87?=<.<!2@!
V7;.7840!U"*)V"W!@2;!,7?78401!0b>0;.-08/7?!10<.68O!"<<+->/.28<!2@!82;-7?./=!781!
52-26080./=!2@!:7;.7840<!N0;0!:0;.@.01O!#7/7!/;78<@2;-7/.28!N7<!4281+4/01!N508!
8040<<7;=O!%/7/.</.47?!787?=<0<!N0;0!1280!+<.86!/50!<2@/N7;0!'!U'!#0:0?2>-08/!B2;0!(07-I!
JKKSWO!
RESULTS
$8!78!0b>0;.-08/!4281+4/01!.8!JKK`I!/50!<>04.@.4!4281+4/.:./=!2@!5=,;.1!>2>?7;!</0-<!1.1!
82/!457860!N./5!.84;07<.86!-2?0!>0;408/!<=;.86=?!.8!/50!.84?.801!/;00<!UC.6+;0!_OLWO!
E2N0:0;I!.8!+>;.65/!</0-<I!.84;07<.86!<=;.86=?!?.68.8!>0;408/760!@;2-!``!-2?Oh!/2!eY!
-2?Oh!;0<+?/01!.8!807;!?.807;!.84;07<0!.8!<>04.@.4!4281+4/.:./=!U'kKOKLWO!

59

!
C.6+;0&_OLO!%>04.@.4!4281+4/.:./=!U%&W!N7<!-07<+;01!28!@;0<5?=!
57;:0</01!LYP4-!?286!+>;.65/!781!.84?.801!5=,;.1!>2>?7;!</0-<O!3078<!
±!!"!7;0!<52N8I!'!k!KOKLI!#!d!Z!</0-<!>0;!/;07/-08/!?.80O!
!
"@/0;!/50!<7-0!</0-<I!+<01!/2!10/0;-.80!/50!<>04.@.4!4281+4/.:./=I!N0;0!@?+<501!/2!
;0-2:0!87/.:0!0-,2?.I!/50=!N0;0!/0</01!@2;!:+?80;7,.?./=!/2!0-,2?.<-!,=!0b>2<.86!/50-!/2!
.84;07<.86!8067/.:0!>;0<<+;0<!N5.?0!;0>07/01?=!-07<+;.86!/50.;!N7/0;!4281+4/.:./=!
UC.6+;0<!_OJ!/2!_OYWO!(50!8067/.:0!>;0<<+;0!745.0:.86!ZKh!?2<<!2@!N7/0;!4281+4/.:./=!
U:+?80;7,.?./=!/2!0-,2?.<-W!N7<!+<01!N508!42->7;.86!/50!5=,;.1!>2>?7;!?.80<!/57/!N0;0!
6;2N8!.8!/N2!4281./.28<I!+>;.65/!2;!.84?.801!UC.6+;0!_OJWO!9810;!,2/5!4281./.28<I!
.84;07<.86!<=;.86=?!?.68.8!>0;408/760!571!82!0@@04/!28!</0-!:+?80;7,.?./=!/2!0-,2?.<-O!
H2/5I!+>;.65/!/;00<!N./5!82;-7?!N221!781!.84?.801!/;00<!N./5!2>>2<./0!781!/08<.28!N221I!
>0;@2;-01!<.-.?7;?=!.8!/50!:+?80;7,.?./=!/0</O!
!

60

!
C.6+;0&_OJ'!D2>?7;!</0-<f!:+?80;7,.?./=!/2!0-,2?.<-!7<!;0>;0<08/01!,=!
/50!8067/.:0!>;0<<+;0!745.0:.86!ZKh!?2<<!2@!/50.;!N7/0;!4281+4/.:./=O!
3078<!±!!"!7;0!<52N8I!#!d!`!</0-<!>0;!/;07/-08/!?.80O!
!
C.6+;0<!_O_!781!_OY!>;0<08/!/50!;0<>28<0!2@!/50!</0-<!2@!/50!1.@@0;08/!'12343&!?.80<!
/2!/50!.84;07<.86!8067/.:0!>;0<<+;0!>2/08/.7?!UxV2WI!/57/!/50=!57:0!,008!0b>2<01!/2I!781!
./<!.->74/!28!/50.;!5=1;7+?.4!4281+4/.:./=O!(50<0!</0-<!N0;0!57;:0</01!@;2-!+>;.65/!/;00<!
UC.6+;0!_O_W!2;!@;2-!.84?.801!/;00<!N./5!/08<.28!N221!UC.6+;0!_OYWO!(50!4+;:0<!N0;0!+<01!
/2!47?4+?7/0!/50!>;0<<+;0!>2/08/.7?!/57/!745.0:01!ZKh!?2<<!2@!5=1;7+?.4!4281+4/.:./=O!(50!
6;7>5<!<52N!82!4?07;!0@@04/!@2;!.84;07<.86!-2?0!>0;408/!<=;.86=?!28!/50!/;00<!>0;408/!?2<<!
2@!4281+4/.:./=!+810;!/50!4+;;08/!0b>0;.-08/7?!4281./.28<!781!6;71+7?!457860!.8!>;0<<+;0!
>2/08/.7?O!
!

61

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>2/08/.7?!2@!+>;.65/!</0-<!N./5!82;-7?!N221!781!.84;07<.86!-2?0!>0;408/!<=;.86=?O!&745!
17/7!>2.8/!;0>;0<08/<!/50!7:0;760!2@!<.b!</0-<O!(50!6;7>5!N7<!+<01!/2!47?4+?7/0!/50!
>;0<<+;0!>2/08/.7?!/57/!745.0:0<!ZKh!?2<<!2@!4281+4/.:./=O!!
!

!
C.6+;0&4'KO!D0;408/!?2<<!2@!5=1;7+?.4!4281+4/.:./=!UDFBW!7<!7!@+84/.28!2@!b=?0-!>;0<<+;0!
>2/08/.7?!2@!.84?.801!</0-<!N./5!/08<.28!N221!781!.84;07<.86!-2?0!>0;408/!<=;.86=?O!&745!
17/7!>2.8/!;0>;0<08/<!/50!7:0;760!2@!<.b!</0-<O!(50!6;7>5!N7<!+<01!/2!47?4+?7/0!/50!b=?0-!
>;0<<+;0!7/!N5.45!/50;0!N7<!ZKh!?2<<!2@!4281+4/.:./=O!32?0!>0;408/!<=;.86=?!.8!/5.<!6;7>5!

62

N7<!-07<+;01!,0@2;0!</0-!.84?.87/.28!/;07/-08/I!@2;!:7?+0<!7@/0;!.84?.87/.28!>0;.21!;0@0;!
/2!C.6+;0!YOLO!
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(50!</0-<!N0;0!57;:0</01!7/!/50!081!2@!/50!0b>0;.-08/I!781!@2??2N.86!/50.;!
;0<>28<0!/2!6;7:./=!781!;042:0;=!,74M!/2!/50!:0;/.47?!>2<./.28O!(50!?07:0<!/57/!N0;0!
@2;-01!1+;.86!/50!0b>0;.-08/!N0;0!7?<2!42??04/01I!42+8/01I!781!/50.;!717b.7?!<+;@740!
7;07<!N0;0!-07<+;01!UC.6+;0<!_OZ!781!_O`WO!X508!-2?0!>0;408/!<=;.86=?!N7<!08578401!
@;2-!`ih!/2!eYhI!/50!7:0;760!?07@!7;07!2@!/50!+>;.65/!>2>?7;!/;00<!.84;07<01!,=!+>!/2!YLh!
U'dKOKJSW!UC.6+;0!_OZWO!*2!</7/.</.47??=!<.68.@.478/!.84;07<0!.8!7:0;760!?07@!7;07!N7<!
2,<0;:01!.8!/50!.84?.801!/;00<!1+0!/2!5.65!17/7!:7;.7840!N./5.8!/50!;0>?.47/0<O!X508!/50!
7;07<!2@!7??!?07:0<I!/57/!10:0?2>01!1+;.86!/50!0b>0;.-08/I!N0;0!71101!+>!@2;!0745!/;00I!82!
457860!N7<!2,<0;:01!1+0!/2!<=;.86=?!?.68.8!0857840-08/I!2;!1+0!/2!.84?.87/.28!781!
/08<.28!N221!@2;-7/.28!UC.6+;0!_O`"WO!$8!7!<0>7;7/0!0b>0;.-08/I!/50!?07@!42+8/!@2;!0745!
</0-!1.1!82/!457860!N./5!45786.86!>0;408/!<=;.86=?!.8!+>;.65/!2;!.84?.801!</0-<!UC.6+;0!
_O`HWO!)8!7:0;760I!/50;0!N0;0!-2;0!?07:0<!28!/50!</0-<!2@!+>;.65/!/;00<!/578!28!/52<0!2@!
/50!.84?.801!/;00<!U'kKOKLWO!
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!

63

Average leaf area (cm2)

Average area of leaves developed during
the experiment
110

Upright

100

Inclined

90
80
70
60
50
60

70
80
90
Syringyl Lignin (mol.%)

100

!

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64

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66

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68

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787/2-.47?!457860<!;0@+/01!2+;!5=>2/50<.<!2@!82!457860!.8!/50!N221!787/2-=!7<!7!;0<+?/!
2@!/50!?.68.8!-21.@.47/.28O!
(50!N.?1!/=>0!/;00<!781!/52<0!2@!/50!5.650</!-2?0!>0;408/!<=;.86=?!Ue_!-2?OhW!N0;0!
/0</01!@2;!457860!.8!b=?0-!108<./=!1+0!/2!457860<!.8!787/2-.47?!>;2>0;/.0<O!(50!;0<+?/<!
76

.81.47/01!82!<.68.@.478/!457860!.8!b=?0-!108<./=!1+0!/2!/50!?.68.8!-21.@.47/.28!UC.6+;0!
_OLJWO!(5.<!@.81.86I!781!/50!457860!.8!787/2-=I!@7??<!<52;/!2@!0b>?7.8.86!/50!<?.65/!.84;07<01!
.8!1;=!</0-!,;.//?080<<!N508!/50!-21+?+<!2@!;+>/+;0!U3)'W!N7<!0</.-7/01!.8!/50!>;0:.2+<!
457>/0;O!
CONCLUSION
The water conductive efficiency tests indicated a possible role for syringyl in facilitating water
conductivity in the vessels. One hypothesis suggested that a less electric polarity in the vessel
walls, associated with a syringyl-rich lignin, to be behind the enhanced water conductivity. Yet,
that was not evident with the inclined stems that formed tension wood. The anatomical analyses
showed an increase in vessel diameter and percent area vessel lumen in normal wood but not in
tension wood. These results can partially explain the increased water conductivity of the stems.
There was no clear evidence for an effect on trees vulnerability to embolism due to syringyl
enhancement or tension wood formation. It can be hypothesized that higher syringyl lignin ratio
in vessel walls may provide additional mechanical support against vessel wall fatigue, which is
one of the causes of cavitation. To test this hypothesis, mechanical testing of individual vessel
walls will be required. However, this is beyond the capacity of this study. As a result of the
higher syringyl lignin ratio, the change in normal wood anatomy may have led to the slight
decrease in the stems modulus of rupture (MOR, previous chapter), incurring increased stem
brittleness. However, the lignin modification did not affect xylem density in the stem samples
that were tested.
Whether these genetically altered poplar trees are suitable for plantation forestry is a
question that cannot be answered until the trees are tested outdoors in a natural environment,
rather than under the modified conditions of the greenhouse. Based on the findings of this study,

77

it would appear that under field conditions, trees with a higher syringyl lignin can potentially
perform similarly, or better, than the unmodified wild type trees.
ACKNOWLEDGEMENTS
X0!/578M!#7:.1!&??.<!U*7/.287?!B08/0;!@2;!]080/.4!'0<2+;40<!D;0<0;:7/.28W!781!B?.8/!
B57>>?0!UD+;1+0!98.:0;<./=W!@2;!>;2:.1.86!+<!N./5!/50!/;78<608.4!>2>?7;!?.80<!+<01!.8!/5.<!
</+1=I!"-=!X.?<28!781!&-.?=!C?=88!U3.45.678!%/7/0!98.:0;<./=W!@2;!/50.;!50?>!N./5!/50!
787/2-.47?!-07<+;0-08/<O!(5.<!;0<07;45!N7<!<+>>2;/01!,=!/50!*7/.287?!'0<07;45!
$8./.7/.:0!2@!/50!9%#"!B%'&&%!6;78/!8+-,0;!JKKZP_ZLK_PLZJ`e&
!

78

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79

REFERENCES
!
!
)$-%.&``G&Z=<W7"+&H!G&B:%..1&PBG&`98/7%.&B!ULeeSW!9<0!2@!408/;.@+67?!@2;40!.8!/50!
</+1=!2@!b=?0-!47:./7/.28O!R!&b>!H2/!K^I!``ZP`SYO!
&
)7.E%8+&`G&I."+W&OG&C%77&OG&C9E7"++&N*&ULei_W!$85.,./.28!2@!?.68.8!@2;-7/.28!,=!FP
7?>57P7-.822b=P,0/7P>508=?>;2>.28.4!74.1I!78!.85.,./2;!2@!>508=?7?78.80!
7--28.7P?=7<0O!&+;!R!B0??!H.2?!3RUJW\L_ePLYYO!
&
*=%.]"+&HG&F"$:E&PG&*"9<E%.&;!UJKK_W!F.68.8!,.2<=8/50<.<O!"88!'0:!D?78/!H.2?!JK\ZLeP
ZY`O!
&
*=1<%&LSG&a68%+8%<W8&;)G&L=-1&O0G&P"<=#/%+&LG&H8.8<W&BG&S+=$$&)NG&N=$#.==W&`;!
UJKKYW!&:2?+/.28!2@!b=?0-!?.68.@.47/.28!781!5=1;260?!/;78<>2;/!;06+?7/.28O!D*"%!
2b2UZKW\LSZZZPLSZZiO!
&
*=1-&P0!ULeSSW!H7<.4!47+<0!2@!1.@@0;08/.7/.28!2@!/08<.28!N221!781!42->;0<<.28!N221O!
"+</!C2;0</!'0<!_\LJLPLY_O!
&
L"::$%&LBG&X=,5&!G&V$$8/&*VG&B=7%.T8$$%&LF!ULeeJW!"8!G58/DE12&D&!-+/78/!10@04/.:0!.8!/50!
6080;7?!>508=?>;2>782.1!>7/5N7=O!D?78/!B0??!K\LYL_PLYJYO!
Clair B, Almeras T, Pilate G, Jullien D, Sugiyama J, Riekel C (2011) Maturation stress
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Plant Physiol 155:562-570.
&
V.8<W/=+&F\G&;8<E%$8+8&IP!ULeZSW!(50!>?7</245;28!.810bO!"-!R!H2/!KK\JeSt_KZO!
&
I."+W%& FG& N%77& ;FG& 0%+"9$5& PHG& F9%,,%.& ;\G& N97:E.%1/& P;G& LE"::$%& L! UJKKJW!
B57860<! .8! <042817;=! -0/7,2?.<-! 781! 10>2<./.28! 2@! 78! +8+<+7?! ?.68.8! .8! /50! ;0@i!
-+/78/!2@!G58/DE12&D&O!D?78/!R!4bULWI!YSPZeO!
&
I."+W%& FG& ;<;8<E"%$& L;G& ;%1%.& SG& BE8.$%1& );G& L9/97"+=& PLG& LE"::$%& L! UJKKKW!
321.@.01!?.68.8!.8!/2,7442!781!>2>?7;!>?78/<!2:0;P0b>;0<<.86!/50!";7,.12><.<!6080!
08421.86!@0;+?7/0!ZP5=1;2b=?7<0O!D?78/!R!33\JJ_PJ_YO!
&
O".5+%.&*CG&F=1&PG&N9<&F!UJKK_W!&@@04/<!2@!/08<.28!N221!28!<>04.@.4!4281+4/.:./=!781!
:+?80;7,.?./=!/2!0-,2?.<-!2@!W365@3&0D46V!<001?.86<!6;2N8!7/!/N2!7/-2<>50;.4!B)J!
428408/;7/.28<O!(;00!D5=<.2?!34U`W\_iSP_eZO!
Hacke UG, Sperry JS, and Pockman WT (2001) Trends in wood density and structure are
linked to prevention of xylem implosion by negative pressure. Oecologia 126:457-461.

80

!"#$%&'()*(+,-./,0(1*(+,#%/&%(+*(2%-%/()*(34(3!"#$%$&!'(()*+!,(!-./0.0!/)0)+.*!1,2.(.*3+.,0!
,0!4,,2!303+,15!,(!367)0!+8))69!:;<;!56"%&=#>?@A9!
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36!.02.*3+)2!E5!L-+83F.,-)+!1.*8,6*,759!<,,2!Y*.!S)*I0,-!D=JB?BA9!
(
+4/%&,(Q*(BTU%#@40(:*(3%E#%0-(=*(3,V/T(7M!"#$$J&!S)06.,0!4,,2!36!3!1,2)-!(,8!(L0*+.,03-!
/)0,1.*6!,(!4,,2!(,813+.,09!Z)4!HI5+,-!6WO=K@?P#9!
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?( B,$,/"VN,0&( M"#,( I,%N! "#$$P&! W=! ;! -30/L3/)! 302! )0F.8,01)0+! (,8! 6+3+.6+.*3-!
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$R!^WV!I++7=__4449W?78,`)*+9,8/9!
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a7.0!H-30+!T.,-!H=%JB?%B#9!
Schubert WJ (1965) Lignin Biochemistry. Academic Press Inc., New York.
(
I4N,//(I;!"%>AK&!\,178)66.,0!4,,2!.0!/510,67)8169!],-9!:R!Y78.0/)8?])8-3/R!T)8-.09!
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81

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RbUZW\!`e_P`eeO!!

82

CHAPTER 4
CHEMICAL RESPONSES TO MODIFIED LIGNIN COMPOSITION IN TENSION WOOD
OF HYBRID POPLAR (POPULUS TREMULA X P. ALBA)
!
ABSTRACT
(50!0@@04/!2@!7?/0;.86!/50!<=;.86=?!?.68.8!-282-0;!;7/.2!N7<!.8:0</.67/01!.8!/5;00!N221!
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U'12343&0-567348!b!'9084/8W!/;78<@2;-01!N./5!/50!>,*2517??()*!6080O!X.?1P/=>0!781!/5;00!
/;78<608.4!>2>?7;!?.80<!N0;0!6;2N8!0./50;!+>;.65/!2;!.84?.801!YZ[!@;2-!:0;/.47?!@2;!/5;00!
-28/5<!/2!.81+40!/08<.28!N221!@2;-7/.28O!*2;-7?!N221!.8!/50!</0-<!2@!+>;.65/!/;00<I!781!
/50!/08<.28!781!2>>2<./0!N221!.8!/50!</0-<!2@!.84?.801!/;00<I!N0;0!787?=g01!<0>7;7/0?=!@2;!
47;,25=1;7/0<I!?.68.8I!40??+?2<0!4;=</7??.8./=I!781!-.4;2@.,;.?!786?0O!$8!N.?1P/=>0!>2>?7;!
</0-<I!/08<.28!N221!571I!28!7:0;760I!`OYh!5.650;!-2?0!>0;408/!<=;.86=?!?.68.8!/578!
82;-7?!N221I!781!SO`h!5.650;!/578!2>>2<./0!N221O!(50<0!1.@@0;0840<!1.-.8.<501!.8!/50!
/;78<608.4!/;00<!N./5!.84;07<01!04/2>.4!<=;.86=?!-282-0;!42->2<./.28O!(50!?.68.8!
-21.@.47/.28!;0<+?/01!.8!7!<?.65/!104;07<0!.8!/50!/2/7?!?.68.8!.8!82;-7?!N221O!$8!/08<.28!
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83

82;-7?!N221!N./5.8!/50!;0<>04/.:0!?.80<O!!E2N0:0;I!,2/5!<+67;!781!6?+42<0!>0;408/760<!
N0;0!?2N0;!.8!/50!/08<.28!N221!2@!/;78<608.4!?.80<!N508!42->7;01!/2!/50!/08<.28!N221!2@!
N.?1/=>0!/;00<O!&:7?+7/.86!/50!;0<>28<0!2@!/;00<!N./5!7?/0;01!?.68.8!-282-0;!42->2<./.28!
/2!6;7:./=!N.??!.->;2:0!2+;!+810;</781.86!2@!/50!457860<!.8!40??!N7??!450-.</;=!781!
+?/;7</;+4/+;7?!>;2>0;/.0<!2@!82;-7?I!2>>2<./0!781!/08<.28!N221!.8!>?78/<O!
INTRODUCTION
Gravity induces the formation of a special type of wood called tension wood in the branches and
displaced stems of porous wood in angiosperms. In inclined trunks, it is formed only on the
upper side of the stem, whereas wood formed on the lower side is termed opposite wood.
Generally, tension wood has more cellulose, and less lignin and hemicelluloses than normal
wood (Timell 1969). Tension wood is also characterized anatomically by wood with more fibers
and fewer vessels of smaller diameter. In most woody angiosperms, tension wood fibers have
secondary cell walls with up to two S-layers and an additional layer of gelatinous appearance
(G-layer). In the G-layer, highly crystalline microfibrils are oriented almost parallel to the
longitudinal axis of fiber, or what is referred to as “approaching zero degrees” (Pilate et al.
2004). The microfibrils are of high tensile strength and the angle they form correlates with
shrinkage events in the tension wood at maturation (Boyd 1977). The major components of the
G-layer in the tension wood of woody dicots are, by dry weight of the acid insoluble residue
(AIR), ~70% cellulose, ~15% xyloglucan, ~2% mannan, #8% pectin, and traces of lignin. In
normal wood, the secondary cell walls have three S-layers, S1, S2 and S3. In these layers,
cellulose makes 50% of the dry weight of the AIR. Other major components include 20-30%
xylan, ~5% glucomannan and 10-20% lignin. Composition summaries of wood tissue and cell
wall layers can be found in Nishikubo et al. (2007) and Mellerowicz and Gorshkova (2012).
84

The hemicelluloses help maintain the structure of the primary and secondary cell walls.
Several models have been proposed on how this is achieved, including cross-linking the
cellulose microfibrils (Atalla et al. 1993, Cosgrove 2000), covalently linking to pectins
(Keegstra et al. 1973), and/or interacting with lignin (Sugino et al. 2004). The amount of
hemicellulose varies among species, cell types and cell wall layers. Their backbones share a
similar structure that is made up of glucose, mannose, or xylose. In dicots, the backbones may
be linked to branches consisting of associated sugars to make xyloglucans, xylans, mannans,
and glucomannans. Xyloglucans are the most abundant hemicelluloses in the primary cell walls
of most plants, and play an important role in the G-layer of secondary cell walls (Nishikubo et
al. 2007). Their glucan backbones are substituted with xylose sidechains that often terminate
with arabinose and galactose residues (reviewed in Fry 1989). Xylans are the dominating noncellulosic polysaccharides in the secondary walls of woody angiosperms (Ebringerová and
Heinze 2000, McCartney et al. 2006, Bowling and Vaughn 2008, Kim and Daniel 2012). They
make up 25-35% of the dry biomass of woody dicots, and are comprised of mostly of xylose
and a lower level of arabinose. Xylans are also implicated in strengthening the secondary walls
against xylem collapse and failed water transport (Bernal et al. 2007, Persson et al. 2007).
Mannans are composed of a mannose backbone whereas glucomannans also contain glucose.
These two hemicellulose moieties are usually less abundant in gymnosperms and angiosperms.
In poplar secondary cell walls, glucomannan was mostly found in the S-layers while mannan
was found to a lesser extent in the G-layer (Kim and Daniel 2012, Mellerowicz and Gorshkova
2012). Hemicelluloses are of economic significance as they influence biomass saccharification
and thus increase the level of biofuel produced. To improve production efficiency in plant

85

tissues, several researchers have attempted to modify the composition of hemicelluloses
(reviewed in Scheller and Ulvskov 2010).
Lignins are important phenolic polymers that are abundant in the middle lamellae and
secondary cell walls of terrestrial plants (Boerjan et al. 2003). Generally, they have been
perceived to provide rigidity and a hydrophobic surface to conductive cells in the xylem (Hacke et al. 2001), contributing to the mechanical and physiological importance in woody
tissues. The mechanical strengths of lignin in the opposite wood and cellulose in the gelatinous
fibers of tension wood are necessary for angiosperm trees to establish the gravitropic response
(Fratzl et al. 2008). Lignins are derived from the random polymerization of three monolignols;
coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol, and are commonly referred to as
guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units once incorporated into the lignin
polymer. Across the plant kingdom, where lignin is synthesized, the composition and monomer
ratios vary substantially (reviewed in Vanholme et al. 2010). These ratios have been found to
change in response to external stimuli such as wind (Koehler and Telewski 2006), and gravity
(Baillères et al. 1995). To understand the contribution of the monomers to tree form and
function, several studies have looked at the differing ratios of monomers in different tissues or
plant species (Yoshinaga et al. 1992, Chabbert et al. 1997, Hoffmann et al. 2003). However, the
combined effects of other variables such as anatomy and the chemical composition of these
tissues and species complicates the interpretation of the role of lignin monomer composition on
structure and function. Investigating the affect of lignin composition in transgenic lines of the
same species minimizes the possible bias of other structural and anatomical parameters
introduced when different plant species or different growth forms of the same species are
compared.

86

Ferulate 5-hydroxylase (F5H) is a key enzyme in regulating lignification and,
specifically, in controlling lignin monomer composition in woody plants (Franke et al. 2000).
Using coniferaldehyde and coniferyl alcohol as substrates, F5H diverts phenylpropanoid flux
towards the synthesis of syringyl lignin monomers, and away from guaiacyl synthesis (Chen et
al. 2000). Over-expression of F5H, therefore, drives the lignin biosynthetic pathway and alters
the inherent syringyl to guaiacyl ratio (S:G) without changing the overall lignin content
(Huntley et al. 2003, Stewart et al. 2009). The objective of this research was to investigate the
effect of altering the syringyl lignin percentage in poplar on cell wall components in normal
wood, as well as in opposite and tension wood. In this study, transgenic hybrid poplar was used
as a model for a woody perennial angiosperm containing different levels of syringyl lignin.
MATERIALS AND METHODS
Plant material and sampling
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,=!C;78M0!0/!7?O!UJKKKW!2:0;P0b>;0<<.86!/50!";7,.12><.<!()*!6080O!(50!<=;.86=?!-282-0;!
7,+817840!.8!/50!82;-7?!N221!;78601!@;2-!`iOZ!-2?Oh!.8!/50!N.?1P/=>0!/2!7!5.65!2@!e_OY!
-2?Oh!.8!()*+,0-+/78/O!G2+86!/;00<!2@!:0;.@.01!?.68.8!42->2<./.28!N0;0!6;2N8!+810;!
6;00852+<0!4281./.28<!.8!/50!<+--0;!7/!3.45.678!%/7/0!98.:0;<./=O!D2/<!ULiP?./0;W!N./5!7!
<2.?P?0<<!-01.+-!UH744/2I!3.45.678!D07/!B2->78=I!E2+</28I!(oW!N0;0!+<01!N./5!82!
711./.287?!@0;/.?.g7/.28!2;!<+>>?0-08/7?!?.65/.86O!(;00<!N./5!LKKPLJK!4-!?286!</0-<!N0;0!
.84?.801!YZ[!@;2-!:0;/.47?!/2!.81+40!/08<.28!N221!@2;-7/.28I!781!N0;0!6;2N8!@2;!/5;00!
-28/5<!7?286!N./5!>7.;01!+>;.65/!428/;2?<O!C2;!450-.47?!787?=<0<I!</0-!<7->?0<!N0;0!

87

/7M08!LZ!4-!7,2:0!/50!;22/!42??7;O!(50=!N0;0!10,7;M01!781!7.;P1;.01!7/!`K[B!/2!745.0:0!7!
428</78/!N0.65/O!%/0-!<06-08/<I!/7M08!@;2-!/50!.84?.801!/;00<I!N0;0!4+/!?286./+1.87??=!/2!
<0>7;7/0!/08<.28!N221!@;2-!2>>2<./0!N221!@2;!.810>08108/!450-.47?!787?=<0<O!!
Crystallinity and microfibril angle
3.4;2@.,;.?!786?0!781!40??!N7??!4;=</7??.8./=!N0;0!10/0;-.801!,=!oP;7=!1.@@;74/.28!7442;1.86!
/2!B2?0-78!0/!7?O!UJKKeW!+<.86!7!H;+M0;!#i!#.<42:0;!oP;7=!1.@@;74/.28!+8./!0A+.>>01!N./5!
78!7;07!7;;7=!10/04/2;!U]"##%WO!!H;.0@?=I!1.@@;74/26;7-<!N0;0!2,/7.801!@;2-!<7->?0<!2@!
;71.7?!@740!N221!<04/.28<!>;04.<.28!4+/!ULO`e!--W!@;2-!/50!6;2N.86!</0-!.<2?7/01!LZ!4-!
7,2:0!/50!;22/!42??7;O!X.10P786?0!1.@@;74/.28!N7<!+<01!.8!/;78<-.<<.28!-210I!781!/50!
-07<+;0-08/<!N0;0!>0;@2;-01!N./5!B+Q|L!;71.7/.28!U}!d!LOZY!~WI!/50!oP;7=!<2+;40!N7<!@./!
N./5!7!KOZ!--!42??.-7/2;!781!/50!<47//0;01!>52/28!42??04/01!,=!7!]"##%!10/04/2;O!H2/5!
/50!oP;7=!<2+;40!781!/50!10/04/2;!N0;0!<0/!/2!/50/7!d!K[!@2;!-.4;2@.,;.?!786?0!
10/0;-.87/.28I!N50;07<!/50!J!/50/7!U<2+;40W!N7<!<0/!/2!LS[!@2;!N221!4;=</7??.8./=!
10/0;-.87/.28O!(50!7:0;760!(P:7?+0!2@!/50!/N2!KKJ!1.@@;74/.28!7;4!>07M<!N7<!+<01!@2;!
-.4;2@.,;.?!786?0!47?4+?7/.28<I!7<!>0;!/50!-0/521!2@!9M;7.80/g!0/!7?O!JKKSI!N50;07<!
4;=</7??.8./=!N7<!10/0;-.801!,=!-7/50-7/.47??=!@.//.86!/50!17/7!+<.86!/50!-0/521!2@!V28M!
ULeS_WO!(N2!;71..!N0;0!/7M08!@;2-!0745!/;00!781!/50<0!:7?+0<!N0;0!7:0;7601!@2;!0745!/;00O!
Lignin and carbohydrate analysis
&b/;74/.:0P@;00!6;2+81!N221!N7<!>;0>7;01!,=!6;.81.86!;0>;0<08/7/.:0!<7->?0<!2@!N221!.8!
7!X.?0=!-.??!/2!>7<<!/5;2+65!7!YK!-0<5!<.0:0I!781!%2b5?0/!0b/;74/01!2:0;8.65/!N./5!
740/280!7/!SK[BO!(50!0b/;74/!@;00!N221!N7<!/508!1;.01!2:0;!DJ)Z!781!;0/7.801!@2;!
42->?0/0!450-.47?!787?=<.<O!"!-21.@.01!Q?7<28!-0/521!N7<!0->?2=01!/2!10/0;-.80!/2/7?!

88

47;,25=1;7/0!781!?.68.8!;7/.2<!UE+8/?0=!0/!7?O!JKK_WO!(50!74.1P.8<2?+,?0!;0<.1+7?!U"$'W!N7<!
A+78/.@.01!6;7:.-0/;.47??=!7@/0;!74.1!5=1;2?=<.<I!N50;07<!/50!74.1P<2?+,?0!?.68.8!N7<!
A+78/.@.01!+<.86!7,<2;>/.28!<>04/;2<42>=!7/!JKZ!8-!U("DD$!9<0@+?!30/521!93PJZKI!LeeLWO!
B7;,25=1;7/0!428408/;7/.28!N7<!10/0;-.801!,=!5.65P>0;@2;-7840!?.A+.1!45;2-7/26;7>5=!
U#.280b!H.2FBI!#.280bI!B"W!0A+.>>01!N./5!78!.28!0b457860!D"L!42?+-8I!7!>+?<01!
7->0;2-0/;.4!10/04/2;!N./5!7!62?1!0?04/;210I!781!7!"%ZK!7+/2P.8T04/2;O!D;.2;!/2!.8T04/.28I!
<7->?0<!N0;0!@.?/0;01!/5;2+65!KOYZ!--!@.?/0;<!U3.??.>2;0I!H01@2;1I!3"WO!"!JK!-F!:2?+-0!
N7<!?27101!28!/50!42?+-8!0A+.?.,;7/01!N./5!JZK!-3!*7)E!781!0?+/01!N./5!10P.28.g01!
N7/0;!7/!7!@?2N!;7/0!2@!LOK!-F!-.8PLI!@2??2N01!,=!7!>2</!42?+-8!711./.28!2@!JKK!-3!*7)E!
7/!7!@?2N!;7/0!2@!KOZ!-F!-.8PLO!&745!<7->?0!N7<!10/0;-.801!.8!/;.>?.47/0O!!
%=;.86=?!/2!6+7.74=?!/2!2P5=1;2b=>508=?!;7/.2<!N0;0!10/0;-.801!,=!/5.274.12?=<.<!
U'2,.8<28!0/!7?O!JKKeW!+<.86!LK!-6!<7->?0<!2@!0b/;74/.:0P@;00!N221O!]7<!45;2-7/26;7>5=!
787?=<0<!N0;0!>0;@2;-01!28!7!ED!ZieK!%0;.0<!$$!@./!N./5!7!LZ!-!b!KOJZ!--!#HPZ!42?+-8!
URX!%4.08/.@.4W!781!C$#!10/04/2;O!(50!]B!-0/521!+<01!7!JOK!yF!.8T04/.28!:2?+-0I!78!.8./.7?!
.8T04/2;!/0->0;7/+;0!2@!JZK[BI!781!7!10/04/2;!/0->0;7/+;0!2@!JSK[BO!(50!.8./.7?!2:08!
/0->0;7/+;0!N7<!<0/!/2!L_K[B!U50?1!@2;!_!-.8OW!781!/50;07@/0;!;7->01!7/!7!;7/0!2@!_[B!-.8P
L!/2!J`K[B!781!50?1!@2;!Z!-.8O!

Data analysis
(5;00!.81.:.1+7?!/;00<!@2;!0745!6080/.4!?.80!781!/;07/-08/!N0;0!<7->?01!@2;!</7/.</.47?!
787?=<0<O!#7/7!N0;0!787?=g01!+<.86!/50!<2@/N7;0!'!U'!#0:0?2>-08/!B2;0!(07-!JKKSWO!
X221!/=>0!781!?.80!0@@04/<!N0;0!0:7?+7/01!,=!7!/N2PN7=!787?=<.<!2@!:7;.7840!U"*)V"WO!
"<<+->/.28<!2@!82;-7?./=!781!52-26080./=!2@!:7;.7840<!N0;0!/0</01O!

89

RESULTS
Effects of genetic and environmental modifications on total lignin and lignin components
$84?.801!5=,;.1!>2>?7;!/;00<!2@!/50!N.?1P/=>0!2;.6.8!781!/50!/5;00!CZE!2:0;P0b>;0<<.28!
?.80<!@2;-01!/08<.28!N221!28!/50!+>>0;!<.10!2@!/50!?078.86!</0-<O!X508!42->7;01!/2!
82;-7?!N221I!/50!/08<.28!N221!2@!N.?1P/=>0!U828P/;78<@2;-01!428/;2?<W!>2>?7;!</0-<!571!
7!`OY!-2?Oh!.84;07<0!U'dKOKYW!.8!<=;.86=?!-282-0;!>0;408/760!7/!/50!0b>08<0!2@!6+7.74=?!
-282-0;!UC.6+;0!YOLI!"WO!(;78<608.4!?.80!()*+,!571!7!</7/.</.47??=!<.-.?7;!>0;408/760!2@!
me_!-2?Oh!.8!7??!/5;00!N221!/.<<+0<O!$8!N.?1P/=>0!5=,;.1!>2>?7;I!>0;408/!/2/7?!?.68.8!.8!
/08<.28!N221!N7<!<.68.@.478/?=!?2N0;!U'dKOKLLW!/578!.8!,2/5!82;-7?!2;!2>>2<./0!N221!
UC.6+;0!YOLI!HWO!"-286!82;-7?!N221I!>0;408/!/2/7?!?.68.8!N7<!JO`h!5.650;!.8!N.?1P/=>0!
/;00<!/578!.8!/;78<608.4!/;00<!U'dKOKKZI!42;;0?7/.28!420@@.4.08/!d!PKOZ`WO!H2/5!N221!/=>0!
781!<=;.86=?!-282-0;!7,+817840!7@@04/01!/50!>0;408/!74.1!<2?+,?0!781!.8<2?+,?0!?.68.8<!
UC.6+;0!YOLI!B!781!#WO!)8!7!N221!<7->?0!N0.65/!,7<.<I!.8!711./.28!/2!?2N0;!/2/7?!?.68.8I!
/08<.28!N221!428<.</08/?=!571!7!?2N0;!>0;408/760!2@!<2?+,?0!781!.8<2?+,?0!?.68.8<!
42->7;01!/2!82;-7?!2;!2>>2<./0!N221!U,2/5!'kKOKKLWO!$8!/50!82;-7?!N221!2@!/;00<!
0b>;0<<.86!78!.84;07<.86!;7/.2!2@!<=;.86=?!-282-0;I!?.68.8!74.1!<2?+,.?./=!.84;07<01!,=!JO_!
@2?1!U'kKOKKLI!42;;0?7/.28!420@@.4.08/!d!KOeKWI!N50;07<!74.1!.8<2?+,?0!?.68.8!UQ?7<28W!
104;07<01!,=!7<!-+45!7<!ZOZh!U'kKOKKLI!42;;0?7/.28!420@@.4.08/!d!PKOiiWO!

90

!
Figure 4.1. Lignin ratio and composition of hybrid poplar clone 717 (Populus tremula x P. alba)
wild-type and transgenic lines F5H37, F5H64, and F5H82. Trees were kept upright or inclined
with respect to gravity to form tension wood. Wood samples of normal, opposite and tension
wood were dried and analyzed separately. (A) Poplar lines showing different levels of mole
percent syringyl. (B) Effect of modified mole percent syringyl on percent total lignin. (C)
Percent lignin acid solubility and (D) acid insolubility, as affected by syringyl enhancement.
Error bars represent means±SE (n=3 trees).
Effect on cellulose microfibril angle (MFA) and cellulose crystallinity
H2/5!N.?1P/=>0!781!/;78<608.4!>2>?7;!/;00<!N5.45!N0;0!1.<>?7401!N./5!;0<>04/!/2!/50!
6;7:7/.287?!:04/2;I!>;21+401!!7!/=>.47?!6;7:./;2>.4!;0<>28<0O!!(5.<!.84?+101!/50!>;21+4.86!
2@!/08<.28!N221!28!280!<.10!2@!/50!</0-!;0<+?/.86!.8!/50!;02;.08/7/.28!2@!/50!1.<>?7401!
</0-!/2!/50!:0;/.47?!>2<./.28O!UC.6+;0!YOJWO!X./5.8!78=!N221!/.<<+0I!.84;07<01!<=;.86=?!
-282-0;!7,+817840!1.1!82/!7@@04/!/50!40??+?2<0!3C"!UC.6+;0!YO_I!"WI!N5.?0!/08<.28!N221!
571!7!<.68.@.478/?=!?2N0;!3C"!U'kKOKKLWO!B0??!N7??!4;=</7??.8./=I!N5.45!120<!82/!7>>07;!/2!

91

,0!7@@04/01!,=!/50!457860!.8!?.68.8!42->2<./.28I!N7<!<.68.@.478/?=!5.650;!.8!/08<.28!N221!
U'kKOKKLW!42->7;01!/2!2>>2<./0!2;!82;-7?!N221!UC.6+;0!YO_I!HWO!

!
Figure 4.2. Poplar trees, of varying mole percent syringyl, responding to gravity (left).
Transverse section of wild-type poplar stem after three months of inclination treatment (right).
Tension wood (TW) and opposite wood (OW) were labeled, and methylene blue staining was
used.

Figure 4.3. Effect of varying mole percent syringyl on (A) microfibril angle and (B) cell wall
crystallinity in hybrid poplar clone 717 (Populus tremula x P. alba) expressing C4Hprom::F5H
at different levels. Trees were kept upright or inclined with respect to gravity to form tension
wood. Wood samples of normal, opposite and tension wood were dried and analyzed separately.
Error bars represent means±SE (n=3 trees).

92

Effect on wood carbohydrate content
$8!N.?1P/=>0!781!/;78<608.4!5=,;.1!>2>?7;!?.80<I!82;-7?!N221!571!7!<.68.@.478/?=!?2N0;!
>0;408/760!2@!/2/7?!<+67;<!U'kKOKKLW!/578!/08<.28!2;!2>>2<./0!N221!UC.6+;0!YOYI!"WO!
$8/0;0</.86?=I!/50;0!N7<!7!<?.65/I!=0/!<.68.@.478/I!.8/0;74/.28!,0/N008!>0;408/!<=;.86=?!
-282-0;!.8!/50!?.68.8!@;74/.28!781!>0;408/!/2/7?!<+67;!U/2!1;=!-7<<W!.8!/08<.28!N221!
U'dKOK_iI!42;;0?7/.28!420@@.4.08/!d!PKO`_W^!7!LeOL!-2?Oh!.84;07<0!.8!<=;.86=?!-282-0;!
7,+817840!.8!/08<.28!N221!N7<!7442->78.01!,=!7!_O`h!104;07<0!.8!/2/7?!<+67;!;0?7/.:0!
7,+817840O!"!<.-.?7;!/;081!N7<!2,<0;:01!N508!/50!?.68.8!-21.@.47/.28!N7<!7442->78.01!
,=!7!YOLh!104;07<0!.8!6?+42<0!;0?7/.:0!7,+817840!UC.6+;0!YOYI!HWO!)8!7:0;760I!>0;408/!
67?74/2<0!N7<!5.650;!.8!/08<.28!N221!/578!.8!82;-7?!N221!U'dKOK_iW!2;!2>>2<./0!N221!
U'dKOKKYWUC.6+;0!YOYI!BWO!E2N0:0;I!>0;408/!67?74/2<0!.8!/08<.28!N221!104;07<01!N./5!
.84;07<.86!7,+817840!2@!<=;.86=?!?.68.8!781!0A+7??01!./<!?0:0?!.8!82;-7?!N221O!(50!2:0;7??!
7:0;760!>0;408/!b=?2<0!.8!/08<.28!N221!N7<!JOJh!?2N0;!/578!.8!82;-7?!N221!U'kKOKKLWO!
"?/0;.86!/50!<=;.86=?!-282-0;!>0;408/760!1.1!82/!<.68.@.478/?=!7@@04/!b=?2<0!;0?7/.:0!
7,+817840!.8!/50!/5;00!N221!/.<<+0<!UC.6+;0!YOYI!#WI!N50;07<!7!<.68.@.478/!.8/0;74/.28!N7<!
<52N8!,0/N008!>0;408/!b=?2<0!781!>0;408/!/2/7?!?.68.8!.8!78=!2@!/50!N221!/.<<+0<!
U2ÄKOKK`W!UC.6+;0!YOZWO!(50!7:0;760!;57-82<0!>0;408/760!N7<!5.650;!.8!82;-7?!N221!
U'kKOKKLW!/578!.8!0./50;!/08<.28!2;!2>>2<./0!N221O!E2N0:0;I!.8!<>./0!2@!/50!1.@@0;.86!
-282-0;!;7/.2<!.8!/50.;!?.68.8I!/50!;0?7/.:0!7,+817840!2@!;57-82<0!N./5.8!0745!N221!/=>0!
;0-7.801!428</78/!UC.6+;0!YOYI!&WO!378.>+?7/.86!/50!<=;.86=?!-282-0;!;7/.2!.8!>2>?7;!
/;00<!1.1!82/!7@@04/!/50!;0?7/.:0!7,+817840!2@!-7882<0!UC.6+;0!YOYI!CW!2;!7;7,.82<0!U17/7!
82/!<52N8W!.8!/50!/5;00!N221!/=>0<O!

93

!
Figure 4.4. Effect of varying mole percent syringyl on the percentages, to dry mass, of (A) total
sugars, (B) total glucose, (C) galactose, (D) xylose, (E) rhamnose, and (F) mannose in hybrid
poplar clone 717 (Populus tremula x P. alba) expressing C4Hprom::F5H at different levels.
Trees were kept upright or inclined with respect to gravity to form tension wood. Wood samples
of normal, opposite and tension wood were dried and analyzed separately. Error bars represent
means±SE (n=3 trees).

&

94

Figure 4.5. Regression plot showing significant correlation between percent total lignin (soluble
and insoluble) and percent xylose in three wood types: normal (Norm), opposite (Oppos) and
tension (Tens) in hybrid poplar clone 717 (Populus tremula x P. alba) expressing
C4Hprom::F5H at different levels. The trees were inclined with respect to gravity to induce
tension wood formation whereas upright trees were used as controls. In addition to normal
wood in upright trees, opposite and tension wood from each inclined tree were separated, dried
and analyzed separately.
DISCUSSION
&8:.;28-08/7?!</.-+?.I!<+45!7<!6;7:./=I!7?/0;!/50!<57>0!781!@2;-!2@!786.2<>0;-!/;00<!2@/08!
;0<+?/.86!.8!</0-!;02;.08/7/.28!/2!7!80N!+>;.65/!>2<./.28!1;.:08!,=!/50!/08<.?0!@2;40!2@!
<>04.7?.g01!b=?0-!/.<<+0!47??01!/08<.28!N221!UD.?7/0!0/!7?O!JKKYWO!#.@@0;08/!7>>;27450<!
57:0!,008!712>/01!/2!.81+40!/08<.28!N221!@2;-7/.28O!"810;<<28P]+880;Å<!0/!7?O!UJKK`W!
781!C2</28!0?!7?O!UJKLLW!.81+401!/50!@2;-7/.28!2@!/08<.28!N221!/5;2+65!,081.86!/50!</0-<!
U5.650;!-04578.47?!</;0<<W!42->7;01!/2!1.<>?74.86!/50!/;00<!N./5!;0<>04/!/2!6;7:./=!U?2N0;!
-04578.47?!</;0<<WI!7!-0/521!0->?2=01!.8!/5.<!</+1=O!$84?.87/.28!2@!+8<+>>2;/01!
786.2<>0;-!</0-<!.<!+<+7??=!@2??2N01!,=!/08<.28!N221!@2;-7/.28!28!/50!+>>0;!<.10I!,+/!

95

28?=!.8!/50!?2N0;!>7;/!2@!/50!</0-I!?071.86!/2!</0-!;0P2;.08/7/.28!/2!/50!:0;/.47?!>2<./.28O!
H081.86!/50!</0-<!?071<!/2!/50!@2;-7/.28!2@!7!N.10;!<>;071!2@!52-26082+<!/08<.28!N221!
28!/50!<.10!+810;!/08<.28O!(50!0@@04/!2@!</0-!,081.86!.<!787?262+<!/2!/57/!2@!<82N!,+.?1!+>!
28!/50!</0-!2;!,;78450<I!781!/50!0@@04/!2@!/;00!.84?.87/.28!.<!<.-.?7;!/2!/57/!2@!<+1108!
457860<!/2!?781!<+;@740!/2>26;7>5=!@2??2N.86!?781<?.10<O!"!-7T2;!787/2-.47?!
457;74/0;.</.4!2@!/08<.28!N221!.8!>2>?7;<!.<!7!@.,0;!N./5!78!711./.287?!828P?.68.@.01!40??!
N7??!?7=0;!2@!60?7/.82+<!7>>07;7840!U]P?7=0;W!/57/!.<!-2</?=!42->2<01!2@!5.65?=!4;=</7??.80!
40??+?2<0!-.4;2@.,;.?<O!D;0:.2+<!787?=<0<!+<.86!'12343&0-567348!b!84/8!;0>2;/01!JYh!?.68.8!
.8!/08<.28!N221!42->7;01!/2!JSh!.8!2>>2<./0!2;!82;-7?!N221!UC2</28!0/!7?O!JKLLWO!
#0<>./0!/50!@74/!/57/!/08<.28!N221!>;0<08/<!7!>;2,?0-!/2!/50!?+-,0;!.81+</;=I!./<!?2N!
?.68.8!781!5.65!40??+?2<0!457;74/0;.</.4<!7;0!2@!.8/0;0</!!7<!7!@001</24M!@2;!7?/0;87/.:0!
080;6=!>;21+4/.28!U'767+<M7<!0/!7?O!JKK`WO!
Effect on lignin
%7;M7808!781!E0;60;/!ULeSLW!;0>2;/01!78!.84;07<0!.8!<=;.86=?>;2>780!+8./<!.8!/08<.28!
N221!42->7;01!/2!82;-7?!N221!.8!,.;45!UX6-348!<>OW!781!-71;287!UG5/3-3&!<>OWO!C2</28!0/!
7?O!UJKLLW!;0>2;/01!78!.84;07<0!.8!<=;.86=?!/2!6+7.74=?!;7/.2!.8!/08<.28!N221!42->7;01!/2!
82;-7?!N221!.8!5=,;.1!>2>?7;!U'12343&0-567348!b!84/8WI!7!457860!/57/!.<!0A+.:7?08/!/2!
LJOZh!,7<01!28!*3'!17/7O!)/50;!</+1.0<!2,<0;:01!7!<.-.?7;!/;081!UH?781!781!%4+;@.0?1!
Le`YI!H7.??q;0<!0/!7?O!LeeZI!G2<5.17!0/!7?O!JKKJI!"6+7=2!0/!7?O!JKLKWO!(50!>;0<08/01!;0<+?/<!
.8!5=,;.1!>2>?7;!4284+;!N./5!/50<0!;0>2;/<I!781!<52N!7!/=>.47?!;0<>28<0!/2!/50!6;7:./;2>.4!
</.-+?+<O!X.?1P/=>0!>2>?7;!571!78!7:0;760!2@!`OYh!.84;07<0!.8!<=;.86=?!-282-0;!;7/.2!.8!
/08<.28!N221!N508!42->7;01!/2!82;-7?!N221I!781!78!7:0;760!2@!SO`h!.84;07<0!42->7;01!
/2!/50!>7.;01!2>>2<./0!N221!<04/.28O!"!<.-.?7;!.84;07<0!.8!/50!-282-0;!;7/.2!N7<!7?<2!
96

;0>2;/01!@2;!/50!<7-0!/=>0!2@!5=,;.1!>2>?7;!/;00<!7<!7!;0<+?/!2@!-04578.47??=!>0;/+;,.86!
/50-!/2!<.-+?7/0!N.81!<N7=!UQ205?0;!781!(0?0N<M.I!JKK`WO!];7:./=P.81+401!.84;07<0!.8!
<=;.86=?!?.68.8!7,+817840!.8!/08<.28!N221!1.-.8.<501!.8!/50!/;78<608.47??=!-21.@.01!/;00<!
.8!/50!>;0<08/01!</+1=O!&b26082+<!>,*2517??()*!2:0;P0b>;0<<.28!.8!/50<0!/;00<!?01!/2!7!
807;P<7/+;7/.28!2@!?.68.8!N./5!/50!<=;.86=?!-282-0;O!
"!8067/.:0!42;;0?7/.28!0b.</01!,0/N008!>0;408/!<=;.86=?!-282-0;!781!>0;408/!
/2/7?!?.68.8!.8!82;-7?!N221!.8!>2>?7;!</0-<O!$8!7!</+1=!+<.86!L_!1.@@0;08/!>2>?7;!4?280<!U'90
E64-1DE6&I!'90E64-1DE6&!b!'9078VD71KD@YDD!CL!5=,;.1<I!'90E64-1DE6&!b!'90#DF58!CL!5=,;.1<!781!
+81.<4?2<01!'12343&!<>04.0<WI!./!N7<!@2+81!/57/!7!</;286!8067/.:0!42;;0?7/.28!0b.</01!
,0/N008!%\]!;7/.2!781!?.68.8!428/08/!UH2<0!0/!7?O!JKKeWO!$8!/50!>;0<08/01!</+1=I!>0;408/!
/2/7?!?.68.8!U<2?+,?0!781!.8<2?+,?0W!N7<!<.68.@.478/?=!5.650;!.8!82;-7?!781!2>>2<./0!N221I!
42->7;01!/2!/50!>7.;01!/08<.28!N221!<7->?0<O!(08<.28!N221!.842;>2;7/0<!7!-2</?=!
40??+?2<.4!?7=0;!/57/!711<!/2!/50!/2/7?!-7<<!2@!/50!40??!N7??O!"!</+1=!0->?2=.86!0+47?=>/+<!
<52N01!<.-.?7;!;0<+?/<!N50;0!/08<.28!N221!@2;-7/.28!571!.->7.;01!?.68.@.47/.28!
42->7;01!/2!82;-7?!N221!U"6+7=2!0/!7?O!JKLKWO!$8!<>./0!2@!/50!<?.65/!104;07<0!.8!>0;408/!
/2/7?!?.68.8!.8!82;-7?!N221!2@!>2>?7;!2:0;P0b>;0<<.28!?.80<!.8!/50!4+;;08/!</+1=I!74.1!
<2?+,?0!?.68.8!>0;408/760!.84;07<01!,=!+>!/2!JO_!@2?1!.8!/50!<7-0!/.<<+0O!(50!.84;07<0!.8!
?.68.8!74.1!<2?+,.?./=!N7<!78!0b>04/01!2+/42-0!781!.<!.8!76;00-08/!N./5!>;0:.2+<!</+1.0<!
7<!<=;.86=?!-2.0/.0<!7;0!-2;0!74.1!?7,.?0!/578!6+7.74=?!U%/0N7;/!0/!7?O!JKK`WO!"<!7!;0<+?/!2@!
/50!>2<./.:0!42;;0?7/.28!,0/N008!?.68.8!74.1!<2?+,.?./=!781!<=;.86=?!-282-0;!7,+817840I!
.84;07<.86!/50!>;2>2;/.28!2@!<=;.86=?!?.68.8!;0<+?/01!.8!-2;0!/578!`Kh!;01+4/.28!.8!
>+?>.86!/.-0!781r2;!7!4284+;;08/!;01+4/.28!.8!/50!>;240<<.86!450-.47?<!UE+8/?0=!0/!7?O!
JKK_WO!

97

!
Effect on cellulose microfibril angle (MFA) and cellulose crystallinity
B0??+?2<0!-.4;2@.,;.?!786?0!781!4;=</7??.8./=!N0;0!@2+81!/2!42;;0?7/0!N./5!/2/7?!?.68.8I!
6;2N/5!</;0<<0<I!781!<5;.8M760!0:08/<!.8!/50!</0-<!UH2=1!LeSSI!H7.??q;0<!0/!7?O!LeeZI!
G2<5.17!0/!7?O!JKKJWO!$8!/50!5=,;.1!>2>?7;!/;00<!/57/!N0;0!0:7?+7/01!.8!/5.<!</+1=I!/50!3C"!
104;07<01!U4?2<0;!/2!/50!>7;7??0?!N./5!/50!@.,0;!7b.<W!.8!/08<.28!N221!781!/50!40??+?2<0!
4;=</7??.8./=!.84;07<01O!!(50<0!/N2!+?/;7</;+4/+;7?!457860<!478!,0!42;;0?7/01!N./5!/50!
@2;-7/.28!2@!/50!40??+?2<0P;.45!60?7/.82+<!?7=0;O!(50<0!@.81.86<!7;0!428<.</08/!N./5!
>;0:.2+<?=!;0>2;/01!457;74/0;.</.4<!2@!/08<.28!N221!UH2=1!LeSSI!D.?7/0!0/!7?O!JKKYWO!
"?/0;.86!/50!<=;.86=?!-282-0;!>0;408/760!.8!/50!/;78<608.4!?.80<!1.1!82/!.8@?+0840!
<042817;=!40??!N7??!3C"!2;!4;=</7??.8./=!.8!/50!1.@@0;08/!N221!/.<<+0<O!(50!;0<+?/<!;0>2;/01!
50;0!<+660</!/57/!?.68.8!-282-0;!42->2<./.28I!.8!786.2<>0;-!N221I!120<!82/!7@@04/!/50<0!
+?/;7</;+4/+;7?!/;7./<O!!
Effect on wood monosaccharides
$8!'12343&084/80FJ!b=?2<0!.<!7!42->2808/!2@!b=?26?+478I!7!50-.40??+?2<0!/57/!0b.</<!.8!/50!]P
?7=0;!2@!/08<.28!N221!@.,0;<O!o=?26?+478!-7M0<!+>!LKPLZh!2@!/50!?7=0;!1;=!-7<<I!-7M.86!./!
/50!-2</!7,+8178/!828P40??+?2<0!47;,25=1;7/0!2@!/50!?7=0;!U*.<5.M+,2!0/!7?O!JKKSI!
30??0;2N.4g!0/!7?O!JKKiI!Q7M+!0/!7?O!JKKeI!30??0;2N.4g!781!]2;<5M2:7!JKLJWO!(50!17/7!
>;0<08/01!50;0!<52N!<.68.@.478/?=!?2N0;!b=?2<0!>0;408/760!.8!/08<.28!N221!UL`O`h!2@!1;=!
-7<<W!42->7;01!/2!2>>2<./0!ULiOZhW!2;!82;-7?!N221!ULeOihWO!(50<0!;0<+?/<!7;0!<.-.?7;!
/2!>;0:.2+<!;0>2;/<!UC+T.!0/!7?O!LeiJI!C2</28!0/!7?O!JKLLWO!E2N0:0;I!2/50;!</+1.0<!<52N01!
5.650;!b=?2<0!.8!10:0?2>.86!/08<.28!N221!2@!>2>?7;!U"810;<<28P]+880;Å<!0/!7?O!JKK`WI!781!

98

.8!iP=07;!2?1!0+47?=>/+<!U"6+7=2!0/!7?O!JKLKWO!(50!?7//0;!</+1=!7?<2!@2+81!78!.84;07<0!.8!
/50!428/08/!2@!50-.40??+?2<0<!.8!/08<.28!N221!428/;7;=!/2!78!07;?.0;!;0>2;/!,=!(.-0??!
ULe`eWO!$8!<2-0!.8</7840<!/50!<7-0!>?78/!=.0?1<!1.@@0;08/!;0<+?/<!N508!787?=g01!7/!
1.@@0;08/!10:0?2>-08/7?!</760<O!37/+;0I!?.65/P6;2N8!>07!U'D&370&8-DZ37!FO!4:O!"?7<M7W!
</0-<!=.0?101!?0<<!b=?26?+478!/578!0?2867/.86I!17;MP6;2N8!</0-<!.81.47/.86!0./50;!?0<<!
b=?26?+478!?7/0;!.8!/50!/.<<+0!2;!?0<<!7440<<.,.?./=!/2!0b/;74/.28!08g=-0<!1+0!/2!7?/0;01!
</;+4/+;0!UD7+?=!0/!7?O!JKKLWO!%.-.?7;?=I!/08<.28!N221!<7->?0<!-7=!;0/+;8!1.@@0;.86!;0<+?/<!
N508!787?=g01!7/!:7;=.86!10:0?2>-08/7?!</760<O!"810;<<28P]+880;Å<!0/!7?O!UJKK`W!
;0>2;/01!5.650;!b=?2<0!.8!10:0?2>.86!/08<.28!N221!2@!@.0?1P6;2N8!5=,;.1!7<>08!U'12343&0
-567348!FOW!LL!17=<!7@/0;!,081.86I!N50;07<!<7->?0<!2@!@+??=!10:0?2>01!/08<.28!N221!2@!
5=,;.1!>2>?7;!U'12343&0-567348!FO0b0'9084/8!FOW!,08/!@2;!`K!17=<!<52N01!<.68.@.478/?=!?2N0;!
b=?2<0!/578!/52<0!2@!82;-7?!N221!UC2</28!0/!7?O!JKLLWO!(5.<!N2+?1!.81.47/0!/57/!
</7817;1.g7/.28!@2;!/.<<+0!760!-7=!,0!;0A+.;01!@2;!42->7;7/.:0!</+1.0<!28!40??!N7??!
42->2<./.28O!*2/7,?0!1.@@0;0840<!,0/N008!/08<.28!N221!781!82;-7?!N221!0b.</!N508!/50!
1.@@0;08/!40??!N7??!?7=0;<!7;0!787?=g01!<0>7;7/0?=O!$8!-2</!N221=!786.2<>0;-<I!/50!]P?7=0;!
2@!/08<.28!N221!@.,0;I!N5.45!;0>?740<!/50!%_!?7=0;!.8!82;-7?!N221!@.,0;I!57<!1.</.84/!
40??+?2<0!+?/;7</;+4/+;0I!828P40??+?2<0!47;,25=1;7/0<I!781!?.68.8!428/08/!U;0:.0N01!.8!
30??0;2N.4g!781!]2;<5M2:7!JKLJWO!(50<0!@07/+;0<!6.:0!/08<.28!N221!./<!1.</.84/!450-.47?!
781!</;+4/+;7?!>;2>0;/.0<O!!
"!104;07<0!.8!-7882<0!781!LIYP"P-78878!.8!/08<.28!N221!42->7;01!/2!82;-7?!
N221!2@!7<>08!N7<!>;0:.2+<?=!;0>2;/01!U"810;<<28P]+880;Å<!0/!7?O!JKK`I!E0108</;Ç-!0/!
7?O!JKKeWI!N50;07<!82!<.68.@.478/!457860!.8!>0;408/!-7882<0!N7<!2,<0;:01!,0/N008!/50!
/N2!/.<<+0<!.8!iP=07;!2?1!0+47?=>/+<!U"3@84A2-3&0F41/343&0F7,.??OW!<7>N221!U"6+7=2!0/!7?O!
99

JKLKWO!(50!-7882<0!A+78/.@.47/.28!;0<+?/<!.8!/50!>;0<08/!</+1=!N0;0!.84284?+<.:0!1+0!/2!
5.65!:7;.7840!7-286!/50!;0>?.47/0<O!]7?74/2<0!.<!7!-7T2;!42->2808/!2@!<0:0;7?!>2?=-0;<!.8!
>?78/!40??!N7??<O!$8!/50!]P?7=0;I!67?74/2<0!781!7;7,.82<0!7;0!42->2808/<!2@!,2/5!
7;7,.8267?74/78!$$!781!b=?26?+478!<.10457.8<O!(50!?7//0;!N0;0!;0>2;/01!/2!4;2<<P?.8M!/50!]P
?7=0;!/2!/50!71T7408/!%J!F7=0;!U*.<5.M+,2!0/!7?O!JKKSWO!%.-.?7;!/2!/50!;0<+?/<!>;0<08/01!50;0!
@2;!5=,;.1!>2>?7;I!67?74/2<0!N7<!@2+81!/2!,0!5.650;!.8!/08<.28!N221!/578!.8!82;-7?!N221!
2@!,0045!<>04.0<I!781!/50;0@2;0!57<!,008!>;2>2<01!/2!,0!78!.81.47/2;!2@!/50!>;0<0840!2@!
/08<.28!N221!U'+0?!781!H7;82+1!LeSiI!"g+-7!0/!7?O!Lei_WO!E2N0:0;I!7!<.68.@.478/!
104;07<0!.8!>0;408/!67?74/2<0!N7<!2,<0;:01!.8!/08<.28!N221!2@!iP=07;!2?1!0+47?=>/+<!
<7>N221!U"6+7=2!0/!7?O!JKLKW!.81.47/.86!428/;7</.86!;0<+?/<!@;2-!1.@@0;08/!<>04.0<!2;!
/.<<+0<O!(50!.84;07<0!.8!<=;.86=?!?.68.8!7,+817840!.8!>2>?7;!.8!/5.<!</+1=!N7<!42;;0?7/01!
N./5!7!<?.65/!104;07<0!.8!67?74/2<0!7,+817840!.8!/08<.28!N221O!(5.<!104;07<0!-7=!,0!7!
;0<+?/!2@!;01+401!67?74/2<0!,.81.86!/2!b=?26?+478!781r2;!7;7,.8267?74/78!$$!.8!/50!]P?7=0;!
1+0!/2!?2N0;!67?74/2<=?/;78<@0;7<0<!74/.:./=I!2;!1+0!/2!;01+401!0>.-0;.g7/.28!2@!6?+42<0!
.8/2!67?74/2<0O!!]?+42<0!7,+817840!N7<!7?<2!@2+81!/2!<?.65/?=!104;07<0!N./5!.84;07<.86!
<=;.86=?!-282-0;!7,+817840!.8!/50!<7-0!<7->?0<!2@!/08<.28!N221O!%.-.?7;!/2!67?74/2<0I!
1.@@0;08/!</+1.0<!;0>2;/01!428/;7</.86!;0<+?/<!@2;!;57-82<0^!280!@;74/.28!2@!7?M7?.80!40??P
N7??!0b/;74/<!N7<!@2+81!/2!428/7.8!5.650;!?0:0?!2@!;57-82<0!.8!/08<.28!N221!/578!82;-7?!
N221!2@!R7>780<0!,0045I!U(8F3&0@56#8-8!H?+-0W!U"g+-7!0/!7?O!Lei_WI!N50;07<!?2N0;!?0:0?<!
N0;0!@2+81!.8!/08<.28!N221!.8!0+47?=>/+<!<7>N221!U"6+7=2!0/!7?O!JKLKWO!'57-82<0!781!
67?74/+;28.4!74.1!-7M0!+>!/50!,74M,280!2@!;57-8267?74/+;2878!$!U']!$W!>04/.8<O!$8!>2>?7;!
U'12343&084/80FWI!;57-82<0!N7<!;0>2;/01!/2!0b.</!.8!.<2?7/01!]P?7=0;!2@!/08<.28!N221!
U*.<5.M+,2!0/!7?O!JKKSWO!$8!"-0;.478!,0045!U(8F3&0F58#EDU14D8!&5;5OWI!;57-82<0!N7<!>7;/!
100

2@!67?74/78!.8!/50!]P@.,0;!2@!/08<.28!N221!<+660</.86!/50!>;0<0840!2@!']!$!.8!/50<0!@.,0;<!
UQ+2!781!(.-0??!Le`eWO!E2N0:0;I!7442;1.86!/2!30??0;2N.4<!781!]2;<5M2:7!UJKLJWI!7!
,.2450-.47?!>;22@!2@!/50!>;0<0840!2@!']!$!.8!/08<.28!N221!.<!</.??!?74M.86O!
(50!0</.-7/01!6?+42<0!>0;408/760<!.8!/5.<!</+1=!/7M0!.8/2!7442+8/!/50!7,+817840!
>;0<08/!.8!,2/5!40??+?2<0!781!50-.40??+?2<0<O!$8!/08<.28!N221I!7!<?.65/!104;07<0!.8!
0b/;74/7,?0!6?+42<0!N2+?1!,0!0b>04/01!1+0!/2!/50!>;0:.2+<?=!;0>2;/01!104;07<0!.8!
50-.40??+?2<0<O!(5.<!104;07<0!.<!+<+7??=!2@@<0/!,=!/50!.84;07<0!.8!40??+?2<0!10>2<./.28!.8!/50!
]P?7=0;^!N5.45!0b>?7.8<!/50!5.650;!>0;408/!6?+42<0!2@!/08<.28!N221!42->7;01!/2!82;-7?!
N221O!E2N0:0;I!/50!<?.65/I!=0/!</7/.</.47??=!<.68.@.478/I!104;07<0!.8!/08<.28!N221!6?+42<0!
781!2:0;7??!<+67;!>0;408/760<!.8!;0<>28<0!/2!<=;.86=?!-282-0;!0857840-08/!;7.<0<!
@+;/50;!A+0</.28<!7<!/2!N50/50;!/50!@0;+?7/0!ZP5=1;2b=?7<0!UCZEW!08g=-0I!2;!78=!2@!./<!
>;21+4/<I!.<!.8:2?:01!.8!/50!<.687?.86!>;240<<!@2;!/08<.28!N221!@2;-7/.28I!2;!/57/!/50!
0?0:7/01!0b>;0<<.28!2@!CZE!>;2/0.8!.8/0;@0;0<!N./5!/50!428:0;<.28!2@!<+4;2<0!.8/2!6?+42<0O!
#+;.86!/08<.28!N221!@2;-7/.28I!47;,28!-7=!,0!+/.?.g01!/2N7;1<!,+.?1.86!/50!1.@@0;08/!
42->2808/<!2@!/50!60?7/.82+<!?7=0;!U%45+,0;/!Le`ZWO!(5.<!7??247/.28!2@!47;,28!-7=!0b>?7.8!
/50!;01+4/.28!.8!/2/7?!<+67;!781!6?+42<0!>0;408/760<!2,<0;:01!.8!/50!()*!2:0;P0b>;0<<.86!
>2>?7;!?.80<!+<01!.8!/5.<!</+1=O!].:08!/57/!/50<0!>0;408/760<!7;0!;0?7/.:0!7,+817840<I!7!
A+78/./7/.:0!457860!.8!78=!</;+4/+;7?!42->2808/!;0<+?/<!.8!7!47<4710!<5.@/!.8!/50!47?4+?7/01!
>0;408/760<!2@!/50!;0-7.8.86!42->2808/<O!
CONCLUSION
(5.<!</+1=!428@.;-<!/50!;0<+?/<!2@!>;0:.2+<!</+1.0<!28!/08<.28!N221!781!>;2:.10<!7!80N!
.8<.65/!.8/2!/50!;2?0!/57/!7?/0;.86!-282?.682?!;7/.2!.8!?.68.8!57<!28!/50!40??!N7??!450-.</;=!
2@!5=,;.1!>2>?7;!.8!82;-7?I!/08<.28!781!2>>2<./0!N221O!H2/5!N.?1P/=>0!781!/;78<608.4!
101

/;00<!;0<>28101!/2!6;7:./=!,=!>;21+4.86!/08<.28!N221!28!/50!+>>0;!<.10!2@!/50!</0-!781!
42;;04/.:0!6;2N/5!;0/+;8.86!1.<>?7401!</0-<!/2!/50!:0;/.47?!>2<./.28O!B28@.;-.86!07;?.0;!
</+1.0<!7;0!/50!2,<0;:7/.28<!/57/!/08<.28!N221!.8!/50!N.?1P/=>0!>2>?7;!.<!457;74/0;.g01!,=!
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ACKNOWLEDGMENTS
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REFERENCES
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#0/0;-.87/.28!2@!1.@@0;0840<!.8!787/2-.47?!781!450-.47?!457;74/0;.</.4<!2@!/08<.28!781!
2>>2<./0!N221!2@!iP=07;!2?1!"3@84A2-3&0F41/343&O!3710;7<O!B.084.7!=!/0482?26É7!
23U_W\JYLPJZLO!
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)+-%.//=+MO9++%.i/&BG&;%$$%.=68<Y&VPG&C=T%&PG&B%,%.7"+&*G&\E781"&[G&L=958+E=&Z;G&
`8$//=+&ZG&N%+.8//"5&*G&;=.85Y&!G&B9+-#%.,&*&UJKK`W!H.2<=8/50<.<!2@!40??+?2<0P
08;.4501!/08<.28!N221!.8!'12343&\!6?2,7?!787?=<.<!2@!/;78<4;.>/<!781!-0/7,2?./0<!
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_3\LS_PLiLO!
Ukrainetz NK, Ritland K, Mansfield SD (2007) Identification of quantitative trait loci for
wood quality and growth across eight full-sib coastal Douglas-fir families. Tree Genet
Genomes 4:159-170.
Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W (2010) Lignin biosynthesis and
structure. Plant Physiol 153:895–905.
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CHAPTER 5
SUMMARY AND CONCLUSIONS
The growing awareness of the limited availability of energy sources like oil and coal has
brought simultaneously a significant energy price hikes and increased interest in finding
alternative sources. In the industry, the production costs also increased but so did the need to
reduce the chemical wastes that are partially blamed for changing the global climate. The paper
and pulp industries started looking for ways to increase the efficiency of the operations and to
reduce the chemicals used along the process. Lignin extraction is costly in terms of energy and
use of hazardous chemicals. A research team had modified the lignin composition in poplar
trees allowing more than 60% increase in lignin extraction efficiency while using less
disposable chemicals. The trees were genetically modified to over-express ferulate-5hydrovylase (F5H) gene which encodes an enzyme that diverts the conversion of lignin
intermediates into producing more syringyl than guaiacyl. In angiosperms, lignin in the
secondary cell walls is majorly made of these two monomers. The ratio of which was found to
change across different plant species and in response to environmental stimuli such as gravity.
The influence of changing the syringyl to guaiacyl ratio and gravity on poplar trees growth
responses and physical properties was investigated in this study.
Increasing syringyl to guaiacyl ratio in hybrid poplar trees (Populus tremula x P. alba)
was found to result in a lower plant height to stem diameter ratio. The trees with the higher
syringyl content gained less height than the control trees throughout the experiment. However,
when the syringyl-enriched trees were tilted they spent eight days less than the wild type
controls to adjust to gravity and recover back to vertical. Whether that was due to higher tensile
force in the tension wood this research was not able to confirm. Growth strain measurements

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showed less strain released from the tension wood of the faster responding trees. That could be
due to the measurements being done at the end of the gravitropic phase when the tensile force is
at its minimum. Four-point bending and compression tests showed no differences in the elastic
properties due to the lignin modification when the stems were tested while fresh or after drying.
Accordingly, two assumptions were made; one assumption was that the elastic properties of the
stems are more influenced by the cellulose microfibrils than by the lignin structure. The other
assumption was that the trees demonstrated another example of their ability to adapt or
compensate to changes in the lignin structure. When the dry stems were loaded to rupture a
decrease in the modulus of rupture was found. This increase in brittleness raised further
questions about the possible changes in the tissue anatomy and the chemical and structural
properties of the cell walls.
Dry Wood density estimation using the water displacement method showed a decrease
in the density in the poplar line with the highest syringyl content when compared to the wild
type. Wood density is considered a good predictor of the woods mechanical properties. The
brittle nature of the trees with increased syringyl content may be explained by the decrease in
the density. Transverse sections were created and stained before they were visualized under a
light microscope. Percent area tension wood was similar among all tested poplar lines. Digital
images were captured for further measurements to be done on the cellular level. Major findings
were a decrease in percent fiber wall in the upright trees with higher syringyl. Moreover, it was
found that those trees have higher percent vessel diameter and percent vessel lumen, which
raises a question whether these anatomical changes, as a result of the syringyl enrichment, will
cause changes in the water conductive properties in poplar. Among the expected results were
lower total lumen in the tension wood and an increase in percent fiber wall, which can be

112

explained by the fact that fibers in the tension wood have an additional cell wall layer of
gelatinous nature hence the name G-fibers.
To investigate the influence of syringyl over-expression and gravity on the water
conductive properties of the poplar trees maximum hydraulic conductivity (kmax) was measured
in 14-cm long stem segments allowing for calculation of water specific conductivity (ks). It was
found that the upright trees with the higher syringyl conducted water more efficiently that the
wild type. This answered the question whether the larger vessel lumen, in poplar lines with
higher syringyl, increases water conductivity. To determine if the vessel walls had been affected
by the lignin modification vulnerability curves were constructed but failed to lead to a
conclusion due to the high variance of the data.
Further analyses of the wood were carried out through analyzing the lignin and
carbohydrate composition of the cell wall. Increasing syringyl content was found to lead to a
slight drop in total lignin. It cannot be rules out that this slight change in total lignin might have
some effect on the results of the mechanical analyses. Increasing syringyl content did increase
the percent acid soluble lignin but had no impact on cell wall crystallinity or microfibril angle.
An interesting biochemical result was a slight decrease in percent sugars with increasing
syringyl in the tension wood. Similar result was observed for percent glucose and galactose.
Percent xylose came out lower in the tension wood than in the normal wood which was contrary
to what was reported by a different group. No effect of syringyl enrichment was found on
percent xylose in any of the tissue types.
An attempt had been made during the study to create new genetic lines with RNAisuppressed F5H gene. If successful a broader spectrum of syringyl to guaiacyl would allow for
higher resolution data. In addition, there have been very few studies on the influence of guaiacyl

113

enrichment on the plants characteristics and behavior. Even though the transformation attempt
had been successful, no significant change occurred on the syringyl to guaiacyl ratio in the
modified poplar stems. A change in the design of the transformation construct might have been
necessary. Another possible explanation was that the introduced siRNA was successful at
hybridizing with the RNA of only one out of two copies of the F5H gene in poplar.
Overall, no major altercations due to syringyl enrichment were detected in the tested
poplar trees under the current greenhouse setting. Before a large scale plantation of the
genetically altered poplar trees can be assumed, further testing in the field will be required to
verify their ability to withstand more complex stresses.

114