THE EFFECTS OF INCREASED SYRINGYL TO GUAIACYL LIGNIN MONOMER
RATIOS ON XYLEM HYDRAULIC PROPERTIES IN HYBRID POPLAR (POPULUS
TREMULA X P. ALBA)
By
Jeffrey Arnold Pierce

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Plant Biology
Ecology, Evolutionary Biology, and Behavior
2012

ABSTRACT
THE EFFECTS OF INCREASED SYRINGYL TO GUAIACYL LIGNIN MONOMER
RATIOS ON XYLEM HYDRAULIC PROPERTIES IN HYBRID POPLAR (POPULUS
TREMULA X P. ALBA)
By
Jeffrey Arnold Pierce

The goal of this research was to examine how changes in the syringyl to guaiacyl
(S:G) lignin monomer ratio will affect the conductive efficiency and resistance to
embolism in hybrid poplar. I used two lines of hybrid poplar clone 717 (Populus tremula
x P. alba) over-expressing the Arabidopsis gene encoding ferulate-5-hydroxylase (F5H)
which resulted in an increased S:G ratio in xylem tissue. The two lines selected for use,
F5H37 and F5H64, expressed 88% and 94% syringyl, respectively, compared to 66%
syringyl expressed in wild type (WT) plants. To quantify the effects of increased S:G on
hydraulic properties, two studies were performed. First, the hydraulic conductivity and
resistance to embolism of F5H and wt lines were tested under well watered and water
stress conditions. The goal of the second experiment was to examine the response of
the F5H and wt lines to winter freeze-thaw stress and spring recovery. Results from this
research show that increasing S:G has no significant effect on the hydraulic conductivity
of xylem, but lines with increased S:G were observed to be more vulnerable to
cavitation. Data for pressure at 75% embolism (P75) showed that F5H64 was
approximately 75% more vulnerable to cavitation and in 2009 F5H37 was twice as
vulnerable to cavitation as the WT. No significant differences were found between lines
in response to drought and winter freeze-thaw stress.

Copyright by
JEFFREY ARNOLD PIERCE
2012

ACKNOWLEDGEMENTS

I would like to thank my family and friends for their continued support and
patience with me throughout this research. I would especially like to thank my parents,
Jack and Deborah Pierce. I would also like to thank Collin Castle and Laura Bennett for
providing a place to stay in East Lansing while I worked to complete this thesis.
I thank my research committee – Drs. Frank W. Telewski, Frank Ewers, and Jim
Flore – for their help in designing, and performing the research described in this thesis,
as well as, their patience with me as I finished writing this thesis.
I have had the privilege of working with many great people during my time at
Michigan State University. All of whom I would like to thank for volunteering their time
and resources to help me through my research. Jason Kilgore for teaching me how to
operate the equipment I used for my physiological studies. Jameel Al’Haddad for
training me in the use of the Sperry apparatus and for all his help with developing my
experiments and statistical analyses. Peter Carrington for all of his advice on the
design and formatting, and aid in data collection. Phillip Kurzeja for keeping me
company late into the night while we performed experiments in the lab and field. Jeff
Wilson and Campus Planning and Administration for help with the design and printing of
posters used at conferences.
Thanks to Dr. Clint Chapple, Purdue University, and David Ellis, USDA, for
providing the transgenic lines used in this study.
The Departmental staff has been invaluable in their assistance. I thank Kasey
Baldwin, Tracey Barner, Jan McGowan, Stacy LaClair, and Adelle Rigotti for their
iv

support and assistance.
Finally, I would like to thank those groups that have provided financial support for
this research. The Department of Plant Biology supported considerable amount of
travel and conference expenses through the Paul Taylor Fund. The college of Natural
Science for supporting me through teaching assistantships. This research and
equipment expenses were supported by the National Research Initiative of the USDA
CSREES grant number 2005-35103-15269 to Dr. Frank W. Telewski.

v

TABLE OF CONTENTS

LIST OF TABLES

vii

LIST OF FIGURES

ix

CHAPTER 1
INTRODUCTION

1

CHAPTER 2
HYDRAULIC CONDUCTIVITY AND VULNERABILITY TO CAVITATION IN HYBRID
POPLAR (POPULUS TREMULA X P. ALBA) WITH INCREASED SYRINGYL TO
11
GUAIACYL LIGNIN MONOMER RATIOS
CHAPTER 3
HYDRAULIC RESPONSE TO FREEZE-THAW STRESS IN GENETICALLY MODIFIED
HYBRID POPLAR WITH INCREASED SYRINGYL:GUAIACYL LIGNIN MONOMER
40
RATIOS
CHAPTER 4
GENERAL CONCLUSIONS

53

LITERATURE CITED

56

vi

LIST OF TABLES

Table 2-1. ANOVA results for yearly initial specific conductivity (ksinitial) for WT,
F5H37 and F5H64 lines exposed to increasing water stress over time (day).
Significant (α<0.05) terms are in bold

20

Table 2-2. ANOVA results for yearly maximum specific conductivity (ksmax) for WT,
F5H37 and F5H64 lines exposed to increasing water stress over time (day).
Significant (α<0.05) terms are in bold

21

Table 2-3. ANOVA results for yearly xylem pressure at 75% loss of conductivity
(P75) for WT, F5H37 and F5H64 lines exposed to increasing water stress over
time (day). Significant (α<0.05) terms are in bold

23

Table 2-4. ANOVA results for yearly dark acclimated chlorophyll fluorescence
(Fv/Fm) and light acclimated chlorophyll fluorescence (Fv'/Fm') for WT, F5H37 and
F5H64 lines exposed to increasing water stress over time (day). Significant
(α<0.05) terms are in bold

30

Table 2-5. ANOVA results for yearly steady-state chlorophyll fluorescence (Fs) for
WT, F5H37 and F5H64 lines exposed to increasing water stress over time (day).
Significant (α<0.05) terms are in bold

31

Table 2-6. ANOVA results for yearly stomatal conductance (gs) and transpiration
(E) for WT, F5H37 and F5H64 lines exposed to increasing water stress over time
(day). Significant (α<0.05) terms are in bold

33

Table 2-7. ANOVA results for yearly predawn (ψpre) and midday (ψmid) pressure
potentials for WT, F5H37 and F5H64 lines exposed to increasing water stress
over time (day). Significant (α<0.05) terms are in bold

36

Table 3-1. ANOVA results for yearly initial specific conductivity (ksinitial) for WT,
F5H37, and F5H64 lines for winter and spring treatments (Trt). 2010 data
includes analysis on effects of transplanted versus non-transplanted plants (tran).
Significant (α<0.05) terms are in bold

46

Table 3-2. ANOVA results for yearly maximum specific conductivity (ksmax) for
WT, F5H37, and F5H64 lines for winter and spring treatments (Trt). 2010 data
includes analysis on effects of transplanted versus non-transplanted plants (tran).
Significant (α<0.05) terms are in bold

46

Table 3-3. ANOVA results for yearly xylem pressure at 75% loss of conductivity
(P75) for WT, F5H37, and F5H64 lines for winter and spring treatments (Trt).
vii

Significant (α<0.05) terms are in bold

48

Table 3-4. ANOVA results for positive pressure manometer measurements of
xylem pressure (Px) for WT, F5H37, and F5H64 lines. Significant (α<0.05) terms
are in bold

51

viii

LIST OF FIGURES

Figure 2-1. Mean specific conductivity (ks) as a function of percent syringyl
content. a) ksmax (solid line) and ksinitial (dashed line) for t=0. b) ksmax against
percent syringyl for watered (solid line), stressed (dashed line) and re-watered
(dashed-dot line) treatments. c) ksmax against percent syringyl for stressed
(dashed line) and re-watered (dashed-dot line) treatments. Mean ± SE

19

Figure 2-2. Maximum specific conductivity (ks) for WT, F5H37, and F5H64
lines over time for 2009 (top) and 2010 (bottom). Rw signifies re-watered
treatments. Mean ± SE

22

Figure 2-3. Pressure at 75 percent loss of conductivity (P75) as a function
of percent syringyl content from watered (solid line), stressed (dashed line),
and re-watered (dashed-dot line) for 2009 (top), 2010 (bottom). Mean ± SE

24

Figure 2-4. Comparison of 2009 (solid line) and 2010 (dashed line) P75 as
function of % syringyl. Mean ± SE

25

Figure 2-5. Vulnerability curves for WT (solid line), F5H37 (dashed-dot line),
and F5H64 (dashed line) lines for 2009 (top) and 2010 (bottom) well watered
treatments (t=1)

26

Figure 2-6. Xylem pressure at 75 percent loss of conductivity (P75) for 2009 (top)
and 2010 (bottom) for wild type (WT), F5H37, and F5H64 lines over time. Rw
signifies re-watered treatment. Mean ± SE

27

Figure 2-7. 2010 dark acclimated chlorophyll fluorescence (Fv/Fm; top) and light
acclimated chlorophyll fluorescence (Fv'/Fm'; bottom) versus time. Mean ± SE

28

Figure 2-8. 2010 steady-state fluorescence (Fs) versus time. Mean ± SE

29

Figure 2-9. 2010 stomatal conductance (gs) for WT (solid line), F5H37
(dashed-dot line), and F5H64 (dashed line) lines over 22 days. Mean ± SE

32

Figure 2-10. 2010 mean leaf pressure potential versus time. Predawn pressure
potential (top) and midday pressure potential (bottom)

34

Figure 3-1. Linear regression of maximum specific conductivity (ksmax) against
syringyl content (%) for winter (solid line) and spring (dashed line) treatments
from 2009 (top) and 2010 (bottom)

47

Figure 3-2. Percent loss of conductivity versus xylem pressure (Px) for winter
ix

(solid) and spring (empty) treatments from 2009 (top) and 2010 (bottom)

49

Figure 3-3. Regression of xylem pressure at 75% loss of conductivity (P75)
against syringyl content (%) for winter (solid line) and spring (dashed line)
treatments from 2009 (top) and 2010 (bottom)

50

x

CHAPTER 1
INTRODUCTION

Lignin is a complex, hydrophobic polymer that functions as a key cell wall
component in vascular plants. One of the most abundant organic compounds in the
world, lignin comprises approximately 1/3 of dry wood biomass. Occurring mainly in the
secondary cell wall of xylem tissue, lignin contributes to the mechanical and hydraulic
properties of the cell wall (Campbell and Sederoff, 1996). Lignin has also been
suggested to provide protection from pathogens (Bruce and West, 1989;
Hammerschmidt, 1984; Hijwegen, 1963).
Lignin can be composed of three monomers: syringyl (S), guaiacyl (G), and phydroxyphenol (H). The composition and deposition of these monomers can be highly
variable across taxa (Campbell and Sederoff, 1996; Towers and Gibbs, 1953), as well
as, within a species (Zobel and van Buijtenen, 1989). Lignin in Gymnosperms primarily
contain guaiacyl and, to a lesser extent, p-hydroxyphenol. Angiosperm lignin is
commonly composed of the guaiacyl and syringyl monomers (Barcelo et al., 2004;
Campbell and Sederoff, 1996; Towers and Gibbs, 1953). This variation in composition
of lignin monomers has also been observed between different cell types and tissues.
For example, fiber cells tend to have a higher ratio of S to G (S:G) lignin, whereas
vessel elements contain more guaiacyl and less syringyl (Campbell and Sederoff, 1996;
Donaldson, 2001; Whetton and Sederoff, 1995; Yoshinaga et al., 1992). Environmental
conditions have also been found to affect the composition of lignin (Donaldson, 2001;
Donaldson, 2002).
1

Lignin's Role in Xylem Evolution
The evolution of the lignin biosynthetic pathway was an important step in the
evolution of vascular land plants (Kenrick and Crane, 1997; Sperry, 2003). The
variation in lignin deposition and content described previously has some possible
implications for the overall evolution of the structure and function of xylem. The wood of
Gymnosperms and primitive Angiosperms is composed of tracheids which fulfill the dual
function of mechanical support and water conduction (Carlquist, 2009). Higher
Angiosperms evolved distinct cell types, fiber cells and vessel elements that separately
carry out the functional roles of support and water movement, respectively. Vessels,
composed of multiple vessel elements, can achieve greater lumen diameter and length
than tracheids. The greater diameter and length of vessels facilitates decreased
resistance to the water flow and increased maximum hydraulic conductivity per wood
area (Sperry, 2003; Sperry et al., 2007).
While the evolution of xylem structure and architecture has been examined
extensively from a functional and ecological perspective (Bhaskar et al., 2007; Carlquist,
1975; Carlquist, 2009; Meinzer et al., 2010; Sperry, 2003; Sperry et al., 2008), there has
been very little work on the role of cell wall chemistry, specifically lignin composition, in
the evolution of xylem traits or how xylem evolution affected the deposition and
composition of lignin (Boyce et al., 2004). Yoshinaga et al. (1992) observed that the
proportion of syringyl present in the cell wall is higher in cells that function in mechanical
strength compared to those that function in water conduction. This suggests that
syringyl may be better suited to providing mechanical support to the cell wall and
2

guaiacyl functions more in conduction of water and protection from cavitation. Definitive
experimental evidence for the interaction between the evolution of lignin biosynthesis
and xylem structure may be difficult to obtain. Differences in anatomy and lignin
composition between and within species may confound results. Yet, advances in the
genetic modification of the lignin biosynthetic pathway have provided new opportunities
to experimentally test the function of lignin monomers and their role in the evolution of
xylem anatomy.

Xylem function and the role of lignin
In vascular plants, water is transported from the roots to the leaves through
xylem tissue. The accepted method by which water is conducted through a plant is
known as the cohesion-tension theory, first proposed by Dixon and Joly (1894).
Transpiration at the leaf surface creates the tensional force that drives water movement
through the stem (Tyree, 1997). Xylem conduits must be able to resist the negative
pressures created. The mechanical strength of xylem tissue has been suggested to
contribute to the hydraulic properties of xylem through regulating the vulnerability of
trachiery elements to collapse (Donaldson, 2001; Jacobsen et al., 2005; Raven, 1977;
Sperry, 2003). Lignin provides structural support to the conduit cell wall providing
resistance to the negative pressures (Donaldson, 2001; Sperry, 2003). Fiber cells have
also been shown to have a role in protecting vessel elements from implosion (Jacobsen
et al., 2005). Failure of the conductive tissue to resist high negative pressures can
result in collapse of the conduit walls, leading to cavitation. Formation of gas bubbles,
or embolism, that cause blockage to the flow of water within conduits can result from
3

cavitation. Air-seeding, where gas is pulled into the conduits from neighboring gas-filled
cells or intracellular spaces has also been hypothesized to cause embolism (Tyree and
Zimmerman, 2002; Sperry and Tyree, 1988). Microfractures in the vessel walls, caused
by high negative pressures or weakened cell walls, may also initiate nucleation of gas
bubbles forming embolism (Jacobsen et al., 2005). The vulnerability of conduits to
cavitation due to water stress has been traced to the permeability of intervessel pit
membranes to gas diffusion (Sperry et al., 1991; Sperry and Tyree, 1988). While
embolism impairs the flow of water through the conduit, embolism can be reversed
(Shen et al., 2007) and may also play a developmental role in the formation of
heartwood (Sperry et al., 1991).
The interplay between hydraulic efficiency and mechanical safety of the xylem
has received consideration (Wagner et al., 1998; Hacke et al., 2001; Woodrum et al.,
2003; Jacobsen et. al., 2005; Kern et al., 2005; Pittermann et al., 2006; Meinzer et al.,
2010). It has been hypothesized that tradeoffs may occur between mechanical strength
and hydraulic conductive efficiency at the tissue level (Jacobsen et. al., 2005; Wagner et
al., 1998; Pittermann et al., 2006; Woodrum et al., 2003; Kern et al., 2005). While
tradeoffs were observed in species of conifer (Pittermann et al., 2006), evidence for the
existence of tradeoffs in angiosperm species is less conclusive (Wagner et al., 1998;
Woodrum et al., 2003; Jacobsen et. al., 2005; Kern et al., 2005). The hydraulic
conductivity of the stem can be influenced by frequency and diameter of conduits.
According to Poiseuille's law, an increase in conduit lumen diameter will result in an
increase in the hydraulic conductivity proportional to the summation of the lumen
diameters to the fourth power (Ewers, 1985; Tyree and Zimmerman, 2002; Wagner et
4

al., 1998). While an increase in vessel diameter will result in an increase in specific
conductivity, ks, this may result in decreased frequency of fiber cells, for example, in
Lianas.

The Ligno-Cellulose Resource
Wood biomass represents a significant industrial resource. In 2009, global
production of industrial roundwood totaled at approximately 1.424 billion cubic meters
(http://faostat.fao.org/). This number actually falls short of previously projected trends in
the growth of industrial wood production (Whiteman and Brown, 1999) and represents
the lowest yearly production in the 2000s (http://faostat.fao.org/). Utilization of wood
biomass through the harvesting of cellulose for paper products and biofuel production is
impeded by the presence of lignin. Removal of lignin from wood is accomplished
through the chemical (kraft) pulping process and must be performed through the use of
environmentally harmful chemicals and bleaching. Global production of chemical wood
pulp in 2009 was reported at 118,669,440 tons (http://faostat.fao.org/).
Advances in our understanding of the lignin biosynthetic pathway and
breakthroughs in genetic manipulation of the pathway has opened up new opportunities
for addressing the problems of chemical pulping (Li et al., 2008; Nehra et al., 2005;
Simmons et al., 2010). A number of strategies for increasing the efficiency of industrial
pulping through genetic modification of the lignin biosynthetic pathway have been
attempted. These strategies include reducing the total amount of lignin content within
the wood, and manipulation of lignin monomer ratios, whether through increased
production of syringyl lignin or reduction in guaiacyl lignin (Baucher et al., 2003; Huntley
5

et al., 2003; Li et al., 2003; Li et al., 2008; Meyer et al., 1998; Nehra et al., 2005; Pilate
et al., 2002; Stewart et al., 2006).
Increasing the lignin S:G ratio in poplars has been shown to positively impact the
efficiency of chemical pulping (Huntley et al., 2003; Stewart et al., 2006). While
lowering the total lignin content in the wood could make removal of the lignin easier,
reduction to total lignin content also causes significant impacts on the physiological and
morphological properties of the trees (Coleman et al., 2008; Kitin et al., 2010; Pilate et
al., 2002; Voelker et al., 2011). A similar response to reduced lignin content has also
been observed in non-arborescent plant species, such as Arabidopsis (Franke et al.,
2002) and Nicotiana (Hepworth and Vincent, 1999). The use of genetically modified
hybrid poplars has been suggested as a viable alternative for use in paper
manufacturing and biofuel production (Baucher et al., 2003; Li et al., 2008; Pilate et al.,
2002; Simmons et al., 2010). Modification of the lignin biosynthetic pathway to alter the
ratio of lignin monomers present in the xylem tissue makes the harvesting of cellulose
through pulping more efficient (Huntley et al., 2003; Pilate et al., 2002). However, it is
important to examine how changes to lignin biosynthesis within the plant will affect the
plant's physiological and ecological functioning.
Over-expression of the F5H gene results in an increased syringyl to guaiacyl
(S:G) ratio but no changes to the overall amount of lignin in the plants (Humphreys et
al., 1999; Franke et al., 2000). Over-expression does not compromise conduit function
in the form of collapsed vessel elements seen in other plants with reduced overall lignin
content (Franke et al., 2002; Kitin et al., 2010; Voelker et al., 2011; Zhong et al., 1998).
Huntley et al., 2003, reports that there were no significant differences in the ratio of
6

fibers to vessels, average fiber lumen area, vessel lumen area, or cell wall thickness
between wild type and hybrid poplars (Populus tremula x P. alba) over-expressing C4HF5H. However, Horvath et al. (2010) found that up-regulation of CAld5H in Populus
tremuloides Michx. resulted in decreases to stem diameter, vessel lumen diameter, and
fiber length. However, vessel number and vessel lumen area fraction increased in the
CAld5H mutants, though. This may suggest a response towards narrower vessel
lumens to account for a change in mechanical strength of the cell wall due to the
increase in the syringyl to guaiacyl ratio. How the change in vessel and fiber area
affected the overall mechanical strength and hydraulic properties of the wood was not
reported (Horvath et al., 2010).
Recent research has shown that reduction in total lignin content can have a
drastic effect on anatomical and physiological traits, including reduced growth (Coleman
et al., 2008; Voelker, 2009; Voelker et al., 2010), and increased occurrence of vessel
collapse (Coleman et al., 2008; Franke et al., 2002; Kitin et al., 2010; Voelker et al.,
2010; Zhong et al., 1998). Poplars with reduced lignin content were also found to have
decreased hydraulic conductivity and increased vulnerability to embolism (Coleman et
al., 2008; Voelker, 2009).
The genetic manipulation of the lignin biosynthetic pathway also provides an
opportunity for improved experimentation on lignin's role in xylem functioning and the
effects of altering lignin composition on growth and physiology. Our understanding of
the role of lignin biochemistry on functional traits of the xylem is still lacking (Koehler
and Telewski, 2006). There is some evidence that cell wall lignification affects ionmediated changes in sap flow rate (Boyce et al., 2004). Previous research on lignin's
7

role in xylem function has had to rely on comparing variations in mechanical and
hydraulic traits across different genera and species. Results of these experiments can
be confounded by anatomical and developmental differences between species (Koehler
and Telewski, 2006).

The overall goal of this study is to assess the role of S and G lignin monomers
with respect to water transport and tree physiology, and how changes to S:G will affect
the plant’s response to environmental stress, specifically drought and freeze-thaw
stress. This research will also assess the viability of F5H transgenic hybrid poplars for
use in economic forest plantations. Previous studies have examined how increasing the
S:G ratio can influence the efficiency of pulping methods and there is mixed evidence
towards how genetic modification of the C4H-F5H pathway could result in changes to
anatomical morphology of the transgenic wood (Huntley et al., 2003). However,
attempts to experimentally analyze how changes in the S:G ratio affects mechanical
and hydraulic functions within the wood have not been presented (Koehler and
Telewski, 2006). To assess the viability of S:G mutants, this thesis attempts to provide
more experimental evidence for lignin's role in the functioning of xylem tissue and how
the ratio of lignin monomers within the cell wall contributes to this.
The research presented in the proceeding chapters uses the hybrid poplar clone
INRA 717-1B4 (Populus tremula x p. alba) over-expressing the Arabidopsis gene
encoding ferulate-5-hydroxylase (F5H), driven by the cinnamate-4-hydroxylase(C4H)
promoter. Chapter 2 presents results from greenhouse experiments on the response of
F5H transgenic poplar to water stress. This experiment includes quantitative analysis of
8

the hydraulic conductivity, vulnerability to embolism, and gas-exchange properties,
including transpiration, stomatal conductance, leaf water potentials, and chlorophyll
fluorescence. Changes to the structural properties of the vessel elements and fibers
could compromise the plant's ability to efficiently conduct water and resist increasing
tension forces on the cell wall caused by transpiration. This could also lead to changes
in the plant's ability to carry out other important developmental, biochemical, and
physiological functions. The effects of water stress on physiological properties are well
understood (Kramer, 1983; Larcher, 2003). Reduced water transport can result in
decreased cell turgor pressure, stomatal closure, and inhibit photosynthetic rates (Epron
et al., 1992; Hacke and Sauter, 1995; Hubbard et al., 1999; Brodribb and Field, 2008).
Irrigation regimes have been found to be an important contributor towards traits involved
with growth and productivity in poplar (Shock et al., 2002; Tschaplinski et al., 2006).
This could have important implications towards the commercial use of transgenic
poplars. Chapter 3 examines the effects of increased S:G ratios on F5H mutants'
tolerance to winter freeze-thaw stress. Hydraulic conductivity and vulnerability to
embolism were measured in wild type and transgenic lines during mid-winter and early
spring. Possible mechanisms for recovery from freezing induced embolism in poplar
are also considered. Experimental evaluation of mechanisms for freezing induced
embolism in Populus has been addressed in a number of previous experiments (Hacke
and Sauter, 1996; Sperry and Sullivan, 1992; Sperry et al., 1994). Recovery from winter
induced embolism has been associated with positive xylem pressures in diffuse porous
tree species (Hacke and Sauter, 1996; Sperry, 1993). Yet, Populus species do not
demonstrate similar trends toward xylem refilling (Sperry et al., 1994) and vulnerability
9

to freezing induced embolism (Hacke and Sauter, 1996). Anatomical analyses of the
F5H and wild type lines, including quantification of the active sapwood area of the
samples, were examined. However, this data is still being analyzed and will not be
presented at this time.

Summary
Genetic modification of the lignin biosynthetic pathway can provide a novel
avenue for addressing the increased need for more efficient and environmentally
friendly pulping processes for the wood products industry. It also provides a unique
opportunity to study the role of lignin and monomer composition in xylem function.
Xylem functional traits and physiological properties must be tested in order to fully
evaluate the viability of modifying the lignin biosynthetic pathway in plants' for economic
use. This study presents results on experiments examining the water relations of
transgenic hybrid poplars over-expressing the F5H gene in response to environmental
stress. These results provide a better understanding of lignin's role in the functioning
and evolution of the hydraulic properties of xylem.

10

CHAPTER 2
HYDRAULIC CONDUCTIVITY AND VULNERABILITY TO CAVITATION IN HYBRID
POPLAR (POPULUS TREMULA X P. ALBA) WITH INCREASED SYRINGYL TO
GUAIACYL LIGNIN MONOMER RATIOS

Introduction
In woody vascular plants, water is transported from the root system to leaves
through the secondary xylem tissue. Along with providing water transport, the
secondary xylem also provides mechanical support to the plant. At the cellular level,
this dual function of water transport and mechanical support are fulfilled by tracheids in
Gymnosperms, and vessel elements and fiber cells, respectively, in higher
Angiosperms. Water movement through the tracheids or vessels present within the
xylem tissue is driven by transpiration at the leaf surface. Transpiration creates the
tensional force that pulls the water column upward through the stem (Tyree and Ewers,
1991; Tyree, 1997). Cavitation of the water column can occur when negative pressure
within the conduits becomes too high. This can result in collapse of the xylem conduit
walls, and embolism, formation of gas bubbles, which cause blockage to the flow of
water through the conduit. Embolism can occur when gas is pulled into the conduits
from neighboring cells and intracellular spaces, known as air-seeding (Sperry and
Tyree, 1988), or when nucleation of gas bubbles is initiated by microfractures in the
conduit cell walls (Jacobsen et al., 2005).
Lignin is a complex, hydrophobic polymer that is deposited in the secondary cell
wall of xylem cells. Lignin has been suggested to contribute to the mechanical and

11

hydraulic properties of the cell wall (Campbell and Sederoff, 1996).
While lignin has been shown to provide mechanical support to the cell wall
(Donaldson, 2001; Sperry, 2003), lignin's role in xylem hydraulic properties is still not
fully understood. Genetic modification of the lignin biosynthetic pathway provides
opportunities for experiments on lignin's function. By changing lignin content and lignin
monomer ratios within a single species, while maintaining other xylem anatomical
properties which confound results associated with studies using different taxa can be
eliminated. Some recent research has examined the effects of decreasing total lignin
content on xylem function in hybrid poplar (Coleman et al., 2008; Kitin et al., 2010;
Voelker et al., 2011). Results from these experiments showed that reducing the total
lignin content reduces the plant's ability to efficiently transport water and resist
cavitation, i.e. lower specific conductivity and increased xylem pressure at 50%
embolism (Coleman et al., 2008; Voelker et al., 2011), and increases the incidence of
vessel collapse (Franke et al., 2002; Kitin et al., 2010). These experiments highlight the
important role that lignin plays in protecting vessel elements from the high negative
pressures created within the lumen as water is transported through the vessels.
However, these experiments focus on reduction in total lignin content. Yoshinaga et al.
(1992) proposed that the variation in lignin monomer ratios based on cell type suggests
a possible differentiation in the functional role of the lignin monomers. That is, guaiacyl
is more suitable for cells that function in water movement and syringyl is more suited to
cells dedicated to mechanical support.
Experiments on the mechanical properties of the increased S:G lines found a
decrease in the stems modulus of rupture, MOR, but had no significant effect on the
modulus of elasticity, MOE (Al-Haddad, 2012). This suggests that increasing the
12

syringyl content in the secondary cell wall of vessels will result in increased brittleness
of the adjoining cell walls which may result in decreased ability to resist high negative
pressures. This may result in microfractures in the cell wall leading to air seeding and
cavitation. Jacobsen et al. (2005) found a correlation between MOR and resistance to
cavitation. However, these results may result from anatomical differences within the
xylem tissue between several related plant species rather than differences in the lignin
content within the xylem.
In this experiment I examined the effects of increasing S:G on the hydraulic
conductivity and resistance to embolism of hybrid Poplar (Populus tremula x P. alba)
under well watered and water stressed conditions. Two genetic lines with increased
S:G, along with the wild type, were used to examine their efficiency of xylem hydraulic
conductivity (specific conductivity, ks), resistance to cavitation (xylem pressure at 75%
loss of hydraulic conductivity, P75). To test the response to water stress, the lines were
watered to field capacity and then water was withheld throughout the course of the
experiment. Stem segments were sampled periodically throughout the experiment and
tested for ks and P75. To observe how water stress proceeded in the living specimens,
a number of physiological properties were measured throughout the experiment. These
physiological processes include stomatal conductance (gs), transpiration (E), leaf
pressure potential (predawn, ψpre, and mid-day, ψmid), dark acclimated chlorophyll
fluorescence (Fv/Fm), light acclimated chlorophyll fluorescence (Fv'/Fm'), and steadystate fluorescence yield (Fs). Fv/Fm, Fv'/Fm', and Fs have been shown to be useful in
measuring the efficiency of Photosystem II in plants and can be useful in tracking the
effects of stress on photosynthetic efficiency (Maxwell and Johnson, 2000).
It was hypothesized that an increase in the S:G would result in an increased
13

conductive efficiency, but would also increase the plants vulnerability to cavitation. As
syringyl increases we expect to see an increase in ks and a decrease in the resistance
to embolism and P75 values.
It can be expected that water stress will have a greater effect on plants with
decreased resistance to embolism. Thus, I hypothesize that the increased S:G lines will
have a lower ks as water stress increases. Similar effects will also be expected in
physiological processes. Lines with increased ks will show higher values for leaf
physiological properties and lower values in plant lines with increased vulnerability to
embolism as water stress occurs.

Methods
Transgenic lines of hybrid poplar clone INRA 717-1B4 (Populus tremula x P. alba)
over-expressing the Arabidopsis gene encoding ferulate-5-hydroxylase (F5H), driven by
the cinnamate-4-hydroxylase (C4H) promoter were obtained from David Ellis (formerly
with CELLFOR, Richmond, BC, Canada) with permission from Clint Chapple, Purdue
University (Franke et al., 2000; Material use agreement between Michigan State
University and Purdue University executed 12 April 2002). Two transgenic lines
expressing different percentages of syringyl were chosen for use in this experiment:
F5H-37 and F5H-64, expressing 88 percent and 93 percent syringyl, respectively. The
transgenic lines and wild type (WT) trees were propagated from root sprouts in early
spring (March-May) and allowed to grow in a greenhouse for four months. In August, 60
trees were selected for use (20 trees each of the transgenic and WT lines). Two days
before the start of the experiment all trees were watered to field capacity each day. At
t=0, three trees of each line were selected randomly for pre-dawn and mid-day gas
14

exchange, water potential and fluorescence measurements. Stem samples were then
harvested from the selected trees for measurements on hydraulic conductivity and
vulnerability to cavitation. Remaining trees were then subjected to drought stress by
withholding water. Every two days, the pre-dawn and mid-day gas exchange, water
potential, and chlorophyll fluorescence were measured, as described below, on
randomly selected trees (n=3 for each line). Stem samples for hydraulic conductivity
measurements were collected every 12 days.

Hydraulic Conductivity and Vulnerability to Cavitation
At t=0, and every 12 days after, 9 trees (3 of each line) were randomly selected
and stem samples were collected for measurement of hydraulic conductivity (kh; kg m s
1

-1

Mpa ), specific conductivity (ks; kg m

-1 -1

s

-

-1

MPa ), and vulnerability to cavitation.

Segments, approximately 20 cm in length, were excised from the main stem underwater
to avoid air infiltration into vessels and transported to the laboratory. The samples were
then re-cut underwater, using a razor blade, to 14 cm long, and 1 cm of bark was
removed from each end. Stem samples were then connected to a Sperry apparatus to
measure the initial kh (Sperry et al., 1988). After the initial kh (khinitial) was measured,
samples were then connected to a tygon tubing system and flushed with water to
remove any gas emboli present in the vessel elements. To reduce the incidence of
microbial growth, water used for flushing was adjusted to a pH of 2 using HCl, degassed
in a glass carboy connected to a vacuum for approximately 24 hours, and passed
through a 0.1-µm filter. Stems were flushed at a pressure of 14 MPa for 1 hour, and
then replaced in the Sperry apparatus to obtain the maximum kh (khmax; Sperry et al.,
1988). The ks values were determined by dividing the kh by the sapwood cross15

2

sectional area (As; m ) of the sample. The As was calculated as the wood crosssectional area subtracted by the pith cross-sectional area. Wood cross-sectional area
was calculated by taking the mean of two perpendicular diameter measurements of the
wood without bark from each end of the stem sample with digital calipers. Pith crosssectional area was calculated by averaging the measurements of the largest diameter of
the pith at each end of the sample.
To determine vulnerability to cavitation, stem samples were spun in a centrifuge
(RC-5B, Sorvall, Dupont Instruments) with a modified rotor built to hold the stem
samples. Negative pressure, measured in MPa, was generated through spinning the
rotor at specified rpms (Alder et al., 1997). The kh was then measured at sequential
negative pressures. The percent loss of conductivity (PLC) was calculated for each kh
value using the equation: 100 – {(kh / khmax) * 100%}, and used to construct
vulnerability curves. The 75 PLC (P75) values were also obtained for each sample by
fitting a second order polynomial model to each of the PLC values (Jacobsen et al.,
2005).

Chlorophyll Fluorescence and Gas Exchange
Measurements of chlorophyll (Chl) fluorescence were obtained using a Li-6400
portable photosynthesis system with a 6400-40 Leaf Chamber Fluorometer (LI-COR
Biosciences Inc.).
Every two days, 9 trees (3 each for transgenic and WT lines) were randomly
selected for measurement. Measurements were taken at pre-dawn, between 4am and
6am, to ensure dark adaptation, and at mid-day, between noon and 2pm, during peak
photosynthetic activity. One leaf from each tree was measured. Chl fluorescence
16

parameters were measured as described (Maxwell and Johnson 2000). Parameters
analyzed were minimum fluorescence yield (dark adapted, Fo; light, Fo'), maximum
fluorescence yield (dark adapted, Fm; light, Fm'), quantum efficiency of open
photosystem II centers (dark adapted, Fv/Fm; light, Fv'/Fm'), and steady-state
fluorescence yield (Fs).
Gas exchange measurements were taken directly following measurement of Chl
fluorescence using the same leaves at mid-day. Transpiration, E, and stomatal
conductance, gs, were measured using a Li-1600 Steady State Porometer (LI-COR
Biosciences Inc.). Before measurements, the Li-1600 porometer was activated and
allowed to reach a stable relative humidity. The sensor head cuvette was attached to
each leaf and held to maintain a normal orientation. The gs was monitored until
reaching equilibrium. Once the rate of conductance stabilized, measurements of E and
gs were recorded.

Leaf Pressure Potential
Leaf water potentials (predawn; mid-day) were measured using a Scholander
pressure-chamber. Directly following pre-dawn and mid-day Chl fluorescence and gas
exchange measurements, one leaf from each plant was harvested, by removing the
base of the petiole from the stem, bagged in a Ziploc sandwich bag, and placed in a
cooler containing ice packs. Leaf samples were then transported to the laboratory. The
petiole of the leaf was cut using a razor blade, and then placed into the Scholander
chamber.

Data Analysis
17

®

Statistical analyses were performed using SAS software (SAS Institute Inc.). A
two-way ANOVA was performed on each data set to analyze differences caused by time
(day), line, and their interaction effects. A comparison of means was performed on each
factor level using Tukey’s HSD.

Results
Xylem conductive efficiency
Mean specific conductivity from all four years of experiments shows a trend
toward increased ks as percent syringyl content at t = 0 (Figure 2-1a). WT and F5H64
were not significantly different from each other, but F5H37 was significantly different
from the other two lines (α < 0.05). Specific conductivity, both maximum and initial, was
severely reduced by water stress (Figure 2-1b). Under the stressed treatment (t=36),
F5H37 shows increased ksmax over the WT, and F5H64 shows a lower ksmax (Figure
2-1c). Statistical analysis shows no significant difference between lines in the stressed
treatment, though (α = 0.3219). Re-watered treatment plants show an increase in ksmax
compared to stressed treatments but they do not return to ksmax values observed in well
watered treatments (Figure 2-1b; Figure 2-1c).
When examining ks separately for each year, results are inconsistent. Figure 2-2
shows how mean ksmax changed over time for 2009 and 2010 experiments. In 2009,
both F5H overexpression lines had a lower mean ksmax then the WT for t=1 and t=8.
ANOVA results show that for 2009 day, line and the interaction effect were significant for
both ksinitial and ksmax (Table 2-1; Table 2-2). For 2010 data, there is no difference in
ksmax between lines but by t=14 F5H64 has a higher ksmax then the WT and F5H37
lines. A Tukey's HSD shows that at t=14 F5H64 is significantly different from F5H37 but
18

not different from the WT (α< 0.05). The interaction effect between day and line was
significant for 2010 but line effect was not (Table 2-1; Table 2-2).

ks (kg m- s- MPa-)

Figure 2-1. Mean specific conductivity (ks) as a function of percent syringyl content. a)
ksmax (solid line) and ksinitial (dashed line) for t=0. b) ksmax against percent syringyl for
watered (solid line), stressed (dashed line), and re-watered (dashed-dot line)
treatments. c) ksmax against percent syringyl for stressed (dashed line) and re-watered
(dashed-dot line) treatments. WT and F5H64 n=12; F5H37 n=6.
a)

10.00
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00

ks max
ksinitial

60

65

70

75
80
% Syringyl

85

90

95

100

ks (kg m- s- MPa-)

b)
10.00
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00

watered
Stressed
re-watered

60

65

70

75
80
85
% Syringyl
19

90

95

100

Figure 2-1 continued.
c)
1.2
Stressed
re-watered

ks (kg m- s- MPa-)

1
0.8
0.6
0.4
0.2
0
60

65

70

75
80
% Syringyl

85

90

95

100

Table 2-1. ANOVA results for yearly initial specific conductivity (ksinitial) for WT, F5H37,
and F5H64 lines exposed to increasing water stress over time (day). Significant
(α<0.05) terms are in bold.
Response

Year

Adj R2

F (p-value) Term

Df

Type III SS

F-value

P-value

ksinitial

2007

0.5432

<.0001

Day
Line
Line*Day
Residuals

3
1
4
40

94.6248
5.7678
3.4491
87.3359

14.45
2.64
0.53

<.0001
0.1119
0.6666

2008

0.8492

<.0001

Day
Line
Line*Day
Residuals

3
1
3
46

112.8121
0.0057
4.3346
21.2048

81.58
0.01
3.13

<.0001
0.9118
0.0344

2009

0.8697

<.0001

Day
Line
Line*Day
Residuals

3
2
6
24

45.7281
8.6529
7.2485
13.7036

39.61
11.24
3.14

<.0001
0.0004
0.0204

2010

0.9574

<.0001

Day
Line
Line*Day
Residuals

3
2
6
24

387.3637
0.4907
12.8769
17.8431

173.68
0.33
2.89

<.0001
0.7221
0.0291

20

Table 2-2. ANOVA results for yearly initial specific conductivity (ksmax) for WT, F5H37,
and F5H64 lines exposed to increasing water stress over time (day). Significant
(α<0.05) terms are in bold.
Response

Year

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

ksmax

2007

0.4760

0.0003

Day
Line
Line*Day
Residuals

3
1
3
40

74.1750
0.2819
2.9619

11.60
0.13
0.46

<.0001
0.7180
0.7094

2008

0.8463

<.0001

Day
Line
Line*Day
Residuals

3
1
3
46

124.0421
0.0058
4.9796
23.7403

80.12
0.01
3.22

<.0001
0.9163
0.0313

2009

0.8807

<.0001

Day
Line
Line*Day
Residuals

3
2
6
24

79.7400
11.1174
10.2737
13.7036

46.55
9.74
3.00

<.0001
0.0008
0.0249

2010

0.9581

<.0001

Day
Line
Line*Day
Residuals

3
2
6
24

598.3527
3.4418
19.4217
27.1466

176.33
1.52
2.86

<.0001
0.2387
0.0301

21

Figure 2-2. Maximum specific conductivity (ksmax) for WT, F5H37, and F5H64 lines over
time for 2009 (top) and 2010 (bottom). Rw signifies re-watered treatments. Mean ± SE.
n=3.
2009
6.00
WT

5.00
ks (kg m- s- MPa-)

F5H37
4.00

F5H64

3.00
2.00
1.00
0.00
1

8

22
Time (days)

37

rw

2010
14.00
WT

12.00

F5H37

Ks (kg m-1 s-1 MPa-1)

10.00

F5H64

8.00
6.00
4.00
2.00
0.00
1

14

23
Time (days)
22

36

rw

Resistance to cavitation
Vulnerability curves for the watered treatments from 2009 and 2010 show some
variation between the two years (Figure 2-5). Both F5H37 and F5H64 are more
vulnerable to cavitation than WT in 2009. In 2010 there is no difference in vulnerability
between WT and F5H37, but F5H64 shows a slight increase in vulnerability. No clear
trend was observed between P75 values and percent syringyl content for watered,
stressed and re-watered treatments (Figure 2-3). ANOVA results for 2009 showed
significance for day and line effects, but the day effect was the only significant variable
for 2010 (α < 0.05; Table 2-3). When comparing P75 values for watered treatment from
2009 and 2010, WT and F5H64 lines were similar, but F5H37 showed higher
vulnerability to cavitation in 2009 then 2010 (Figure 2-4; Figure 2-6).

Table 2-3. ANOVA results for yearly xylem pressure at 75% loss of conductivity (P75) for
WT, F5H37 and F5H64 lines exposed to increasing water stress over time (day).
Significant (α<0.05) terms are in bold.
Response

Year

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

P75

2009

0.5277

0.0330

Day
Line
Line*Day
Residuals

3
2
6
24

1.4639
1.4944
0.9464
3.4953

3.35
5.13
1.08

0.0357
0.0140
0.4003

2010

0.6782

0.0009

Day
Line
Line*Day
Residuals

3
2
6
24

1.8853
0.3685
0.7365
1.4191

10.63
3.12
2.08

0.0001
0.0627
0.0941

23

Figure 2-3. Pressure at 75 percent loss of conductivity (P75) as a function of percent
syringyl content from watered (solid line), stressed (dashed line), and re-watered
(dashed-dot line) for 2009 (top), 2010 (bottom). Mean ± SE. n=3.
2009
60

65

70

% Syringyl
75
80
85

90

95

100

0
-0.2
P75 (MPa)

-0.4
-0.6
-0.8
-1
-1.2
-1.4
watered
stressed
Re-watered

-1.6
-1.8
-2

2010
60

65

70

% Syringyl
75
80
85

90

95

100

0

P75 (MPa)

-0.2
-0.4
-0.6
-0.8
-1
-1.2
-1.4
-1.6

watered
stressed
Re-watered

-1.8
-2

24

Figure 2-4. Comparison of 2009 (solid line) and 2010 (dashed line) P75 as function of
% syringyl. Mean ± SE. n=3.

P75 (MPa)

60
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2

65

70

75

% Syringyl
80
85

2009
2010

-1.4
-1.6
-1.8
-2

25

90

95

100

Figure 2-5. Vulnerability curves for WT (solid line), F5H37 (dashed-dot line), and F5H64
(dashed line) lines for 2009 (top) and 2010 (bottom) well watered treatments (t=1). n=3.
2009
100

80
70
60
50
WT – t=1

40

F5H37 – t=1

30

F5H64 – t=1

20
10

Percent Loss Conductivity (%)

90

0
-3

-2.5

-2

-1.5
MPa

-1

-0.5

0

2010

90
80
70
60
50
WT – t=1

40

F5H37 – t=1

30

F5H64 – t=1

20
10
0

-3

-2.5

-2

-1.5
MPa

26

-1

-0.5

0

Percent Loss Conductivity (%)

100

Figure 2-6. Xylem pressure at 75 percent loss of conductivity (P75) for 2009 (top) and
2010 (bottom) for wild type (WT), F5H37, and F5H64 lines over time. Rw signifies rewatered treatment. Mean ± SE. n=3.
2009
1

8

Time (days)
22

37

rw

0
-0.2

P75 (MPa)

-0.4
-0.6
-0.8
-1
-1.2
-1.4

WT – 2009
F5H37 – 2009
F5H64 – 2009

-1.6
-1.8
-2

2010

P75 (MPa)

1

14

Time (days)
23

0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
-1.4
-1.6
-1.8
-2

36

WT – 2010
F5H37 – 2010
F5H64 – 2010

27

rw

Physiological Properties
Fv/Fm did not decline until late in the experimental water stress treatment. Fv/Fm
stayed high in all lines until approximately 25 days (Figure 2-7). After this point leaves
began to turn yellow and Fv/Fm values declined. ANOVA results show significant day
effect for all four years. Line effects were significant for 2009 but not for any other year.
Fv'/Fm' and Fs showed a much more linear decline over time (Figure 2-7; Figure 2-8).
Day effects were significant for all four years but only 2010 showed significance from
line effects (Table 2-5).

Figure 2-7. a) 2010 dark acclimated chlorophyll fluorescence (Fv/Fm). b) light
acclimated chlorophyll fluorescence (Fv'/Fm') versus time. Mean ± SE. n=3.
a)
0.900

WT
F5H37
F5H64

0.800
0.700

Fv/Fm

0.600
0.500
0.400
0.300
0.200
0.100
0.000
0

5

10

15

20
25
Time (days)

28

30

35

40

45

50

Figure 2-7 continued.
b)
0.700
WT
F5H37
F5H64

0.600

Fv'/Fm'

0.500
0.400
0.300
0.200
0.100
0.000
0

5

10

15

20
25
30
Time (days)

35

40

45

50

Figure 2-8. 2010 steady-state fluorescence (Fs) versus time. Mean ± SE. n=3.
350
WT
F5H37
F5H64

300
250

Fs

200
150
100
50
0
0

5

10

15

20
25
30
Time (days)
29

35

40

45

50

Table 2-4. ANOVA results for yearly dark acclimated chlorophyll fluorescence (Fv/Fm)
and light acclimated chlorophyll fluorescence (Fv'/Fm') for WT, F5H37 and F5H64 lines
exposed to increasing water stress over time (day). Significant (α<0.05) terms are in
bold.
Response

Year

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

Fv /Fm

2007

0.1988

0.2084

Day
Line
Line*Day
Residuals

7
1
7
80

0.1480
0.0179
0.1219
1.1597

1.46
1.23
1.20

0.1943
0.2701
0.3119

2008

0.7074

<.0001

Day
Line
Line*Day
Residuals

11
1
11
120

9.5599
2.06E-003
0.6136
4.2086

24.78
0.06
1.59

<.0001
0.8091
0.1099

2009

0.7660

<.0001

Day
Line
Line*Day
Residuals

7
2
14
48

5.8358
0.5993
0.7153
2.1841

18.32
6.59
1.12

<.0001
0.0030
0.3635

2010

0.7448

<.0001

Day
Line
Line*Day
Residuals

17
2
34
108

9.3137
0.2105
1.2576
3.6949

16.01
3.08
1.08

<.0001
0.0502
0.3708

2007

0.4525

<.0001

Day
Line
Line*Day
Residuals

8
1
8
90

0.3765
0.0136
0.1185
0.6153

6.88
1.99
2.17

<.0001
0.1622
0.0374

2008

0.7077

<.0001

Day
Line
Line*Day
Residuals

10
1
10
114

4.2571
6.97E-003
0.1235
1.8163

26.72
0.44
0.77

<.0001
0.5096
0.6527

2009

0.8801

<.0001

Day
Line
Line*Day
Residuals

5
2
10
35

1.7628
0.0265
0.0423

48.98
1.84
0.59

<.0001
0.1735
0.8128

2010

0.8271

<.0001

Day
Line
Line*Day
Residuals

17
2
34
108

4.0531
0.0615
0.1777
0.8974

28.69
3.70
0.63

<.0001
0.0279
0.9386

Fv'/Fm '

30

Table 2-5. ANOVA results for yearly steady-state chlorophyll fluorescence (Fs) for WT,
F5H37 and F5H64 lines exposed to increasing water stress over time (day). Significant
(α<0.05) terms are in bold.
Response

Year

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

Fs

2007

0.9471

<.0001

Day
Line
Line*Day
Residuals

8
1
8
90

2.18E+006
9.86E+001
1.48E+004
1.23E+005

199.97
0.07
1.36

<.0001
0.7886
0.2271

2008

0.4890

<.0001

Day
Line
Line*Day
Residuals

9
1
9
104

2.94E+005
1.45E+004
3.07E+004
3.50E+005

9.70
4.31
1.01

<.0001
0.0403
0.4334

2009

0.9167

<.0001

Day
Line
Line*Day
Residuals

6
2
12
37

2.59E+005
2.55E+003
9.62E+003
2.52E+004

63.30
1.87
1.18

<.0001
0.1683
0.3340

2010

0.6650

<.0001

Day
Line
Line*Day
Residuals

16
2
32
102

2.66E+005
7.56E+003
3.54E+004
1.56E+005

10.90
2.47
0.72

<.0001
0.0893
0.8500

31

Stomatal conductance and transpiration showed no difference between lines
(Table 2-6). Water stress significantly affected the rates of gs and E over time. Figure
2-9 shows gs over the first 22 days of 2010 experiment. By day 10 stomatal closure
resulted in a significant decline in gs and E.

400
350

WT
F5H37
F5H64

gs (mmol m-2 s-)

300
250
200
150
100
50
0

gs (mmol m-2 s-)

0

5

10

15

20
25
30
Time (days)

35

400
350
300
250
200
150
100
50
0

40

45

50

WT
F5H37
F5H64

4

6

8

10
12
Time (days)

14

16

18

Figure 2-9. 2010 stomatal conductance (gs) for WT (solid line), F5H37 (dashed-dot line),
and F5H64 (dashed line) lines over 22 days (top) and gs for days 5 to 17 (bottom).
Mean ± SE. n=3.
32

Table 2-6. ANOVA results for yearly stomatal conductance (gs) and transpiration (E) for
WT, F5H37 and F5H64 lines exposed to increasing water stress over time (day).
Significant (α<0.05) terms are in bold.
Response

Year

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

gs

2007

0.6943

<.0001

Day
Line
Line*Day
Residuals

7
1
7
74

4.59E+006
1.87E+005
2.76E+005

21.69
6.20
1.30

<.0001
0.0150
0.2603

2009

0.7472

0.0004

Day
Line
Line*Day
Residuals

2
2
4
18

2.58E+005
2.28E+004
2.81E+004
1.05E+005

22.22
1.96
1.21

<.0001
0.1694
0.3411

2010

0.7684

<.0001

Day
Line
Line*Day
Residuals

15
2
30
96

1.37E+006
1.80E+004
3.66E+004
4.30E+005

20.43
2.01
0.27

<.0001
0.1398
0.9999

2007

0.6919

<.0001

Day
Line
Line*Day
Residuals

7
1
7
74

231.2952
7.9924
11.5671

21.82
5.28
1.09

<.0001
0.0244
0.3776

2009

0.3036

0.4813

Day
Line
Line*Day
Residuals

2
2
4
18

3.5688
1.9789
3.7944
21.4242

1.50
0.83
0.80

0.2499
0.4515
0.5426

2010

0.7613

<.0001

Day
Line
Line*Day
Residuals

15
2
30
96

157.4923
2.3332
4.9323
51.6531

19.51
2.17
0.31

<.0001
0.1200
0.9998

E

33

Predawn and midday ψ increased over time as water stress increased. For
2009, F5H37 ψpre was significantly higher from F5H64 but not from the WT. No
difference in lines was detected in ψmid for 2009 (Figure 2-10a). In 2010, both ψpre and
ψmid for F5H37 were significantly lower than WT and F5H64 lines (Figure 2-10b).
ANOVA results also show a significant interaction effect between lines and day effects
for 2010 ψmid (Table 2-7).

Figure 2-10. 2010 mean leaf pressure potential versus time. a) Predawn pressure
potential. b) midday pressure potential. n=3.
a)
8
WT
F5H37
F5H64

7

ψ (MPa)

6
5
4
3
2
1
0
0

10

20
30
Time (days)

34

40

50

Figure 2-10 continued.

ψ (MPa)

b)
8
7
6
5
4
3
2
1
0

WT
F5H37
F5H64

0

10

20
30
Time (days)

35

40

50

Table 2-7. ANOVA results for yearly predawn (ψpre) and midday (ψmid) pressure
potentials for WT, F5H37 and F5H64 lines exposed to increasing water stress over time
(day). Significant (α<0.05) terms are in bold.
Response

Year

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

Ψpre

2007

0.3300

0.0112

Day
Line
Day*Line
Residuals

4
1
4
50

1.21E+003
2.98E+001
8.72E+001
2.69E+003

5.61
0.55
0.41

0.0008
0.4603
0.8036

2008

0.7264

<.0001

Day
Line
Day*Line
Residuals

7
1
7
80

3.84E+004
6.67E-001
1.31E+003
1.50E+004

29.34
0.00
1.00

<.0001
0.9526
0.4396

2009

0.7441

<.0001

Day
Line
Day*Line
Residuals

5
2
10
36

1.77E+004
2.00E+003
2.17E+003
7.53E+003

16.95
4.79
1.04

<.0001
0.0143
0.4347

2010

0.8574

<.0001

Day
Line
Day*Line
Residuals

14
2
28
90

5.37E+004
7.51E+002
3.70E+003
9.68E+003

35.69
3.49
1.23

<.0001
0.0346
0.2309

2007

0.4252

<.0001

Day
Line
Day*Line
Residuals

7
1
7
80

2.19E+003
4.59E+001
1.84E+002
3.27E+003

7.65
1.12
0.64

<.0001
0.2921
0.7196

2008

0.6723

<.0001

Day
Line
Day*Line
Residuals

7
1
7
84

2.72E+004
4.67E+001
1.26E+003
1.41E+004

23.19
0.28
1.08

<.0001
0.5990
0.3857

2009

0.7795

<.0001

Day
Line
Day*Line
Residuals

4
2
8
30

1.53E+004
1.03E+003
1.19E+003
4.95E+003

23.15
3.11
0.90

<.0001
0.0592
0.5255

2010

0.8327

<.0001

Day
Line
Day*Line
Residuals

14
2
28
90

4.16E+004
1.50E+003
5.79E+003
9.82E+003

27.23
6.89
1.90

<.0001
0.0016
0.0125

Ψmid

36

Discussion
Analysis of the ksmax under well watered conditions averaged over the four
experimental years shows an increase in ksmax with increased percent syringyl (Figure
2-1). However, no significant difference was found between the WT and F5H64 lines,
but F5H37 was significantly higher. When data from each year was examined
separately results for ks were contradictory. For 2009, F5H37 was observed to have
lower ksmax than the WT. Both F5H lines were also found to be more vulnerable to
cavitation in 2009 (Figure 2-5). This is in contrast to 2010 where no significant
difference was observed between the WT and F5H lines for both ks and P75 values.
The maximum specific conductivity declined as water stress increased over time
in all lines. Theoretically, gas bubbles present in vessels should be completely removed
after pressurized flushing by de-gassed solution (Sperry, 1988). This inability for
specific conductivity values in stressed plants to return to the levels observed in well
watered plants suggests that vessels are becoming permanently non-functional. This
could be caused by tylosis or gum formation in gas-filled vessels, effectively closing
them off from neighboring functional vessels (Tyree and Zimmerman, 2002). Formation
of tyloses has been previously observed in hybrid Populus with reduced lignin content
(Kitin et al., 2010).
A recent study on the methods of measuring hydraulic conductivity has
questioned the efficacy of using high positive pressure to flush out gas embolism to
obtain stable measurements of maximum conductivity (Espino and Schenk, 2011). If
the solution used for flushing is not thoroughly degassed or if stem samples were not
flushed for long enough gas bubbles may still be present within the xylem reducing
conductivity. For these experiments, the solution used in flushing experiments were
37

degassed overnight until no visible gas bubbles were observed in the solution. To
ensure complete removal of emboli from samples used in these experiments, multiple
flushing events and measurements were performed on samples until the kh value
stopped increasing and reached its maximum. While these precautions were taken to
avoid problems it is still possible some emboli may have remained after flushing (Espino
and Schenk, 2011).
WT and F5H lines were found to be more vulnerable to cavitation in stressed
plants compared to well watered plants. P75 values were observed to become less
negative in all lines as when exposed to water stress (Figure 2-3; Figure 2-6).
Cavitation events may weaken vessels making them subsequently more vulnerable to
cavitation (Hacke et al., 2001) and microfractures in the cell wall (Jacobsen et al.,
2005). Cavitation fatigue in Populus species due to stress has been previously
observed (Hacke et al., 2001). Repair of cavitation fatigued vessels may be
accomplished during the natural refilling of vessels in some plants, such as Helianthus
(Stiller and Sperry, 2002). However, with the exception of the WT line from 2010, P75
values from re-watered treatments showed no significant difference from stressed
treatments (Figure 2-3). This suggests that cavitation fatigue may be a permanent
condition and native processes in intact plants are unable to reverse cavitation fatigue.
The reduction in gs and E corresponded with the decline in ks values around 7 to
14 days after the beginning of the water stress regime. Regulation of stomata in
response to water stress has been observed in a number of studies (Cochard et al.,
2007; Meinzer and Grantz, 1990). This relationship between stomatal closure and
xylem conductivity may be a method of maintaining leaf water status (Cochard et al.,
2007). This could explain the delayed response in Fv/Fm data and the slower decline
38

observed in Fv'/Fm', Fs, ψpre, and ψmid.
Results from these experiments show that increased S:G does not significantly
affect hydraulic efficiency and physiological properties in both healthy plants and those
experiencing water stress. However, increasing syringyl content does appear to have
an effect on vulnerability to embolism. This is likely caused by changes in the
mechanical properties of the vessel cell wall causing reduced ability to withstand
negative pressures induced by movement of the water column.

39

CHAPTER 3
HYDRAULIC RESPONSE TO FREEZE-THAW STRESS IN GENETICALLY MODIFIED
HYBRID POPLAR WITH INCREASED SYRINGYL:GUAIACYL LIGNIN MONOMER
RATIOS

Introduction
The effects of below freezing temperatures on the xylem function are important to
consider when examining the growth and ecology of temperate woody plants. Stress
caused by freezing and thawing of xylem sap can result in increased incidence of xylem
embolism (Sperry, 1993). Xylem embolism can be quite extensive throughout winter
months with up to 80 percent loss of conductivity observed in some deciduous trees
(Sperry, 1993).
Embolism induced by freezing stress occurs when dissolved gas within the sap,
which is insoluble in ice, freezes out, forming bubbles. When the sap thaws, the
tensional forces in the xylem may cause the gas bubbles to expand inducing cavitation
(Sperry and Sullivan, 1992; Tyree and Sperry, 1989).
Xylem structure plays a significant role in the response and vulnerability to
freeze-thaw stress. Increased vulnerability to freezing induced embolism has been
correlated with increased conduit diameter (Ewers, 1985; Sperry and Sullivan, 1992).
Conifers, diffuse-porous, and ring-porous species respond to freeze-thaw stress
differently (Sperry and Sullivan, 1992; Sperry, 1993; Sperry et al., 1994; Hacke and
Sauter, 1996). Diffuse-porous species have been found to refill embolised vessels after
winter freezing. However, ring-porous species are unable to refill their embolised

40

vessels and must develop a new ring of vessels in order to begin conducting water
again in the spring (Sperry and Sullivan, 1992; Sperry, 1993; Sperry et al., 1994).
The response of Populus to freeze-thaw stress has been examined previously
(Sperry and Sullivan, 1992; Sperry et al., 1994; Hacke and Sauter, 1996). Sperry and
Sullivan (1992) observed an increase in percent of embolised vessels throughout the
winter and reaching 90 percent embolism by late March in Populus tremuloides.
Results examining the recovery from winter embolism have been inconsistent in
Populus. Evidence for both embolism reversal and a lack of recovery in Populus has
been observed in experiments from successive years (Sperry et al., 1994; Hacke and
Sauter, 1996). The method of embolism reversal in Populus is still not fully understood.
Positive xylem pressure was not observed in Populus balsamifera (Hacke and Sauter,
1996).
While xylem anatomy has been studied previously, the role of cell wall chemistry,
specifically lignin content, in the response to freeze-thaw stress has not been
characterized. Lignin provides mechanical support to the cell wall and aids in resisting
negative pressures associated with water movement. Since tension increases within
the xylem during leaf expansion in the spring, changes to the mechanical strength of
conduits through modified lignin content may also affect recovery from winter embolism
in the spring.
The goal of this experiment was to quantify the effects of increased syringyl
monomer ratio on the response to freeze-thaw stress in hybrid poplar. The hydraulic
conductivity and vulnerability to cavitation were measured in WT and F5H overexpression lines of hybrid poplar clone 717 (Populus tremula x P. alba) in winter and
early spring. I hypothesized that increasing the syringyl:guaiacyl (S:G) ratio will result in
41

reduced hydraulic conductivity in the spring compared to winter treatments and an
increased vulnerability to cavitation due to freeze-thaw embolism in both winter and
spring treatments. As the frozen sap begins to thaw, tensional forces increase within
the vessels. I predict that the increased brittleness of the cell wall caused by the
increase in S:G will be more vulnerable to microfracturing and cavitation as tension
forces increase during thawing events. Bubble manometers were also attached to
excised branches to test for the presence of positive xylem pressure as a source of
embolism reversal.

Methods
The trees used in this experiment consisted of wild type (WT) and the F5H
transgenic lines previously described in Chapter 2 (F5H64 and F5H37). Trees were
grown in pots and moved to an open air courtyard located in the greenhouses at
Michigan State University in 2007. Experiments were performed over two years, during
the winter and spring of 2009 and 2010. In 2009, three trees each of the WT, F5H64,
and F5H37 lines were selected from the courtyard populations for experimentation. To
examine the response of young trees that have not experienced freezing stress, 18 oneyear old, single stemmed trees, six of each line, were taken from the greenhouse and
transplanted into the courtyard in the November, 2009. These trees were added to the
2010 experiment, along with the three trees of each line previously growing in the
courtyard.

Hydraulic conductivity and Vulnerability to Embolism
The trees moved to the courtyard in 2007, all had multiple lateral branches
42

present. For both experiments, two branches were selected each year from each tree
and a stem samples were collected from one branch in February and from the other
branch in May. Since the 18 transplanted one-year old trees were single stemmed with
no branching, samples were collected from only 9 trees, 3 of each line, in February and
May. Branches were cut from the courtyard plants 0.5 to 1m below the previous year’s
terminal bud scale scars. The branches were then quickly moved to a plastic container
filled with water. One segment, 20 to 25 cm long, was then cut from each branch from
the one year old growth underwater to avoid inclusion of embolised vessels. Segments
were stored in the underwater in the plastic container, and transported to the laboratory.
Once in the Laboratory, the samples were cut into 14 cm long separate segments
underwater using razor blade. The excess stem sections were cut down to 4 to 6 cm in
length, perfused with 0.1% crystal violet stain and stored in a freezer for later use in
anatomical measurements. The 14 cm samples were used for measurement of
hydraulic conductivity (kh; kg m-1 s-1 MPa-1) as described by Sperry et al. (1988). The
specific conductivity (ks) values were determined by dividing the kh by the sapwood
2

cross-sectional area (As; m ) of the sample. The As was calculated as the wood crosssectional area subtracted by the pith cross-sectional area. Wood cross-sectional area
was calculated by taking the mean of two perpendicular diameter measurements of the
wood without bark from each end of the stem sample with digital calipers. Pith crosssectional area was calculated by averaging the measurements of the largest diameter of
the pith at each end of the sample. Vulnerability to cavitation and P75 values were
calculated as described in Chapter 2.

Positive Xylem Pressure
43

Manometers were constructed using 0.5 ml glass pipettes sealed at the terminal
end by melting the tip over a Bunsen burner. The pipettes were connected to 2mm 3way polypropylene stopcocks (Bel-Art products) with Nalgene tubing. The manometers
were partially filled with water using vacuum infiltration before attachment to each plant.
Manometers were attached to the branch that was cut for hydraulic conductivity
samples in February, 2010 one hour after the cut was made using a 3cm section of
Nalgene tubing. Adjustable metal clamps were attached to the tubing where it
connected the pipette and the plant to ensure a tight seal. After attachment to the stem,
the 3-way stopcock was opened to vent any pressure created during attachment and
then closed to allow only a 2-way flow between the pipette and the plant stem. To
measure the xylem pressure, the length of the bubble within the pipette (Lx) was
recorded while the stopcock was closed. The stopcock was then opened, and the
length of the bubble at atmospheric pressure (Latm) was measured. Xylem pressure
(Px) was then calculated as: Px = 100*[(Latm/Lx)-1] (Ewers et al., 1997; Fisher et al.,
1997). Xylem pressure was measured daily, between 11am and 3pm, beginning in midFebruary when the winter kh stem samples were collected until May when the spring kh
samples were collected. Temperature data was recorded using a HOBO U10
temperature data logger (Onset Computer Corporation).

Data Analysis
The response of ksinitial, ksmax, and P75 were analyzed using a two-way ANOVA
®

with line, treatment, and their interaction effects as factors using SAS software (SAS
Institute Inc.). Tukey’s HSD was performed to compare means at each factor level. For
positive xylem pressure, Px, a two-way ANOVA was performed using time (day) and line
44

as factors, and Tukey’s HSD was performed on each factor in SAS.

Results
The response of specific conductivity to winter and spring treatments for F5H and
wt lines was inconsistent between both years of experiments. No significant difference
was found for initial ks for line, treatment, or interaction effects during either year. In
2009 the average maximum ks increased for all lines from winter to spring treatments.
Mean values for ksmax for wt, F5H37 and F5H64 lines were 3.7619, 3.7701, and
2.8553, respectively, in winter and 4.0759, 4.5234, and 4.1694, respectively, in the
spring (Figure 3-1). However, the 2010 data showed a declining trend in mean ksmax
from winter to spring treatments. Mean values of ksmax for all lines ranged from 2.7 –
3.27 in the winter and 1.51 – 2.0 in the spring (Figure 3-1). ANOVA results for ksinitial
and ksmax showed no significant difference between line or treatment effects for 2009,
but treatment effects were significant (α < 0.05) for 2010 data (Table 3-1; Table 3-2).
Figure 3-1 shows mean ksmax for winter and spring treatments plotted against percent
syringyl content.

45

Table 3-1. ANOVA results for yearly initial specific conductivity (ksinitial) for WT, F5H37,
and F5H64 lines for winter and spring treatments (Trt). 2010 data includes analysis on
effects of transplanted versus non-transplanted plants (tran). Significant (α<0.05) terms
are in bold.
Response

Year

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

ksinitial

2009

0.4522

0.3452

Line

2

1.5949

1.21

0.3473

Trt

0.8245

<.0001

1.7148

2.60

0.1453

2

0.1268

0.10

0.9093

Residuals
2010

1

Line*Trt

8

5.2707

Line

2

0.1668

0.13

0.8819

Trt

1

1.0824

1.64

0.2125

tran

1

70.3011

106.53

<.0001

Line*Trt

2

0.6835

0.52

0.6023

Line*tran

2

0.9021

0.68

0.5144

Trt*tran

1

0.4975

0.75

0.3939

Line*Trt*tran
Residuals

2
24

0.7478
15.8379

0.57

0.5749

Table 3-2. ANOVA results for yearly maximum specific conductivity (ksmax) for WT,
F5H37, and F5H64 lines for winter and spring treatments (Trt). 2010 data includes
analysis on effects of transplanted versus non-transplanted plants (tran).Significant
(α<0.05) terms are in bold.
Response

Year

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

ksmax

2009

0.2984

0.6511

Line

2

0.8145

0.36

0.7077

Trt

1

1.7014

1.51

0.2542

Line*Trt

2

0.7518

0.33

0.7260

Residuals

8

9.0215

Line

2

0.5686

0.38

0.6895

Trt

1

14.2018

18.86

0.0002

tran

1

70.1044

93.11

<.0001

2010

0.8288

<.0001

Line*Trt

2

1.3386

0.89

0.4242

Line*tran

4

0.2096

0.14

0.8708

Trt*tran

2

0.5439

0.72

0.4038

Line*Trt*tran
Residuals

4
24

0.5243
18.0706

0.35

0.7095

46

Figure 3-1. Linear regression of maximum specific conductivity (ksmax) against syringyl
content (%) for winter (solid line) and spring (dashed line) treatments from 2009 (top)
and 2010 (bottom). For 2009, WT and F5H64 n=3; F5H37 n=1. For 2010, WT, F5H37,
and F5H64 n=3.
2009
6.00

ks (kg m- s- MPa-)

5.00
4.00
3.00
winter
spring

2.00
1.00
0.00
60

65

70

75
80
% Syringyl

85

90

95

100

2010
6.00

ks (kg m- s- MPa-)

5.00
4.00
3.00
2.00
winter
1.00

spring

0.00
60

65

70

75
80
85
% Syringyl

47

90

95

100

Vulnerability to cavitation also differed for each year of experiments. The
vulnerability curve for 2009 shows spring treatments more vulnerable to cavitation at
lower pressures compared to the winter treatment (Figure 3-2a). In 2010, however,
winter treatments were more vulnerable to cavitation than the spring treatment (Figure
3-2b). P75 values for the winter treatment were similar for both years ranging from -1.15
to -0.86 MPa (Figure 3-3). P75 values for spring treatment in 2009 show a trend of
decreasing P75 values as syringyl content increases with mean P75 for WT and F5H37
lines reaching 75 percent embolism at higher pressures than the winter treatments
(Figure 3-3a). In 2010 mean P75 values for all lines were significantly lower than winter
treatments and no clear trend can be observed between lines (Figure 3-3b). ANOVA
results show no significant effects for 2009, but the treatment effect was significantly
different in 2010 (Table 3-3).

Table 3-3. ANOVA results for yearly xylem pressure at 75% loss of conductivity (P75) for
WT, F5H37, and F5H64 lines for winter and spring treatments (Trt).Significant (α<0.05)
terms are in bold.

Response

Year

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

P75

2009

0.2296

0.7848

Line

2

0.1277

0.23

0.8019

Trt

1

0.0918

0.33

0.5837

Line*Trt

2

0.4198

0.75

0.5047

Residuals

8

2.2519

Line

2

0.0109

0.06

0.9415

Trt

1

2.7474

30.37

<.0001

Line*Trt
Residuals

2
30

0.0115

0.06

0.9386

2010

0.5051

0.0005

48

Figure 3-2. Percent loss of conductivity versus xylem pressure (Px) for winter (solid) and
spring (empty) treatments from a) 2009 and b) 2010. For 2009, WT and F5H64 n=3;
F5H37 n=1. For 2010, WT, F5H37, and F5H64 n=3.
a)

90
80
70
60
50
40
WT – w

F5H37 – w

WT – s

F5H37 – s

30

F5H64 – w
F5H64 – s

20
10
0
-2.5

-2

-1.5

MPa

-1

-0.5

Loss of Hydraulic Conductivity (%)

100

0

b)

90
80
70
60
50
40
30
WT – w

F5H37 – w

F5H64 – w

20

WT – s

F5H37 – s

F5H64 – s

10
0

-3.0

-2.5

-2.0

-1.5
MPa
49

-1.0

-0.5

0.0

Loss of Hydraulic Conductivity (%)

100

Figure 3-3. Regression of xylem pressure at 75% loss of conductivity (P75) against
syringyl content (%) for winter (solid line) and spring (dashed line) treatments from 2009
(top) and 2010 (bottom). For 2009, WT and F5H64 n=3; F5H37 n=1. For 2010, WT,
F5H37, and F5H64 n=3.
2009

P75 (MPa)

60

65

70

% Syringyl
75
80
85

0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
-1.4
-1.6
-1.8

90

95

100

95

100

winter
spring

2010

P75 (MPa)

60

65

70

% Syringyl
75
80
85

0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
-1.4
-1.6
-1.8

90

winter
spring

50

Positive xylem pressure was not detected in any of the lines throughout the 2010
experiment. ANOVA results showed no significant difference in xylem pressure over
time or between lines (Table 3-4).

Table 3-4. ANOVA results for positive pressure manometer measurements of xylem
pressure (Px) for WT, F5H37, and F5H64 lines. Significant (α<0.05) terms are in bold.

Response

Adj R2

F (p-value)

Term

Df

Type III SS

F-value

P-value

Px

0.2598

0.3216

day

50

25.5861

1.15

0.2341

Line

2

0.9208

1.03

0.3566

Line*day
Residuals

100
459

45.2663
204.4966

1.02

0.4462

Discussion
Based on these results we can conclude that increasing syringyl content has no
significant effect on the plant's response to freeze-thaw stress and spring recovery. All
lines showed similar responses in both ks and P75 values. The lack of significant
difference between ks values between winter and spring treatments in 2009 suggests
that embolism reversal did not occur. In 2010, spring ks values were significantly lower
than the winter treatment. Samples in the spring were collected as leaves were
beginning to expand. The increase in tension created by transpiration returning as
leaves become active could be causing the decreased ks values observed in 2010. The
inconsistent response of xylem embolism in poplar from one year to the next has been
observed previously (Sperry et al., 1994; Hacke and Sauter, 1996).

51

Lack of positive xylem pressures in Populus is in agreement with observations by
Hacke and Sauter (1996). Refilling of embolised vessels in Populus could be influenced
by the high occurrence of vessels in contact with each other. This increases the
probability of embolised vessels located beside several water-filled vessels which could
aid in dissolution of gas bubbles (Hacke and Sauter, 1996).
In summary, increased S:G does not affect the response of hybrid poplars to
freeze-thaw stress. Both the response to winter freeze-thaw induced embolism and
spring recovery are not affected by lignin monomer content and are likely influenced by
xylem anatomical structure as reported by previous studies (Ewers, 1985; Sperry and
Sullivan, 1992).

52

CHAPTER 4
GENERAL CONCLUSIONS

The overall objective of this research was twofold: to experimentally evaluate the
function of lignin monomers with respect to water transport in hybrid poplars and how
genetic modification of lignin monomer ratios, specifically increasing S:G, affects water
transport and physiological response to drought and freezing stress. Results from these
experiments can also be used to assess the viability of using transgenic hybrid poplars
with increased S:G in economic forest plantations.
It was hypothesized that increased S:G would result in an increased hydraulic
efficiency, i.e. increased ks. While ksinitial and ksmax was observed to have a positive
trend with increased S:G when averaged across all four experimental years, this trend
was not statistically significant. It may be valuable to incorporate RNAi transgenic lines
that have a reduced S:G ratio in future experiments. This positive trend observed, while
not significant here, may be only part of the picture, and data from transgenic lines with
lower S:G may help illuminate the broader role that S and G lignin play in the hydraulic
functions of xylem. No significant difference in hydraulic conductivity was calculated
between lines for well watered treatments, thus the hypothesis that increased S:G will
increase ks can be rejected. P75 values show a positive relationship with increasing
S:G. This is consistent with the hypothesis that increasing S:G would increase
vulnerability to cavitation. F5H37 showed significantly less negative P75 than WT and
F5H64 suggesting reduced resistance to embolism. It is unclear why F5H37 is more
vulnerable to cavitation than F5H64 at this time. Further research that incorporates a

53

larger number of transgenic lines with a gradient of multiple S:G values, as well as,
RNAi lines with decreased S:G is needed to better understand how lignin monomers
fully affect the function of vessels with respect to vulnerability to cavitation. The
increased vulnerability to cavitation is likely caused by increased brittleness and
susceptibility to microfracture in the cell wall as a result of changes to the mechanical
properties of vessel element cell walls and neighboring fiber cells (Jacobsen et al.,
2005).
Water transport and physiological properties were all significantly reduced by
water stress. However, the response of F5H lines to water stress was not significantly
different from the WT for both ks and P75 values. Plants that underwent the re-watered
treatment were unable to restore ksmax values to those observed in well watered
treatments.
Physiological properties were not significantly different between lines under both
well watered and stressed treatments. From a whole plant perspective, this shows that
any changes to xylem functioning caused by increased S:G were not significant enough
to effect leaf gas exchange and photosynthesis.
Response to winter freeze-thaw stress was not affected by S:G. Results for ks
and P75 for winter and spring treatments were inconsistent between study years. This
lack of significant difference between F5H and WT lines and inconsistency between
years suggests that other factors may be involved in the response to freeze-thaw stress
and spring recovery. Xylem structure, specifically vessel lumen diameter, most likely
has a stronger affect on determining the plant's response to freeze-thaw stress as
reported in previous literature (Ewers, 1985; Sperry and Sullivan, 1992).
Overall, these results show that modification of S:G in hybrid poplars will reduce
54

xylem resistance to embolism but has no significant effect on leaf physiology, efficiency
of water transport, and response to drought and winter freezing stress at the tissue
level. Further research is needed, however, to fully evaluate the effects of changes to
S:G on xylem functioning at the cellular level. Taken all together, these results show that
increasing S:G does not significantly alter xylem functioning and physiological
properties. Thus, the use of transgenic hybrid poplars with increased S:G will not have
reduced biomass production compared to the WT and should be considered as a viable
alternative for use in economic tree plantations.

55

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