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Effect of lignin content and structure on the anatomical, physical and mechanical properties of genetically engineered aspen trees

ProQuest Dissertations and Theses, 2009
Dissertation
Author: Balazs Horvath
Abstract:
The directed modification of specific traits of trees through genetic engineering provides opportunities for making significant genetic improvements to wood properties in a matter of years instead of extended time frames required for traditional natural selection. An attractive target of forest-tree engineering is the modification of lignin content and its structure which present potential advantages including improved pulping efficiency, lower chemical and energy consumption, and reduced environmental impacts. However, decrease in lignin content and changes in lignin structure could lead to the modifications of wood characteristics that are critical for solid wood. Wild-type and transgenic quaking aspen (Populus tremuloides Michx.) with reduced lignin content, increased syringyl to guaiacyl (S/G) ratio, and both reduced lignin content and increased S/G ratio were investigated in four studies. In the first study , diameter growth and cell morphology of one-year-old transgenic aspen were investigated using quantitative wood anatomy and fiber quality analysis techniques. Similar radial growth and quantitative anatomical properties were observed between the genetic group with reduced lignin content and the wild-type. The genetic group with increased S/G ratio had lower diameter growth, lower vessel lumen diameter, but more numerous vessels. The combined effect of lignin content and structural changes on radial growth and cell morphology seems to be more complex and gave inconsistent results. In the second study , dynamic modulus of elasticity (MOE) using a nondestructive technique (Fakopp Microsecond Timer) and static MOE using micromechanical testing method were investigated for 2.5-year-old transgenic aspen clones. Result showed that a reduction in the lignin content reduce both the dynamic and static MOE. Increase in the syringyl to guaiacyl ratio results in only a slight decrease in the dynamic MOE and static MOE. The combined influence of lignin content and syringyl to guaiacyl ratio changes shows the most obvious negative effect on both the dynamic MOE and static MOE. In the third study , a dynamic mechanical analyzer (DMA) in static bending mode was used to determine the elastic modulus of 2.5-year-old wild-type and transgenic aspen submerged in water and in ethylene glycol. DMA MOE values were compared to those assessed by nondestructive evaluation and micromechanical testing in previous study. All of the measured elastic moduli showed the same trend and detected similar differences across the genetic groups. DMA measurements showed notably lower MOE values than the other techniques. In the fourth study , the in situ lignin glass transition of one-year-old transgenic aspen was investigated using dynamic mechanical analysis and rheometer. Results suggested that an increase in the S/G ratio did not affect the glass transition temperature but a reduction in lignin content decreased the softening temperature of transgenic wood which has practical implication and would be beneficial to pulp and ethanol production. This research revealed that a reduction in lignin content had a more severe negative effect on wood properties than an increase in S/G ratio. However, diameter growth of transgenic trees with increased S/G ratio was lower. The combined effect of reduced lignin content and increased S/G ratio on wood characteristics was inconsistent; therefore it needs further investigation. Although the reduced lignin content genetic group has inferior mechanical properties and cannot be considered for solid and structural applications, the lower lignin content and glass transition temperature make it suitable for pulp and ethanol production, and for composite manufacture.

TABLE OF CONTENTS LIST OF TABLES ............................................................................................... viii

LIST OF FIGURES ...............................................................................................ix

REVIEW OF RELATED LITERATURE ................................................................. 1

Lignin, Lignin Biosynthesis, and Lignin Genetic Engineering ..................... 1

Wood Cell Wall Formation ......................................................................... 8

Anatomical and Chemical Characteristics of Quaking Aspen .................. 11

Anatomical Studies on Aspen and Transgenic Aspen ............................. 15

Nondestructive Evaluation of Wood ......................................................... 20

Dynamical Mechanical Analysis ............................................................... 34

Basics of dynamic mechanical analysis ............................................ 34

Studies on lignin glass transition ....................................................... 38

References .............................................................................................. 45

EFFECT OF LIGNIN GENETIC MODIFICATION ON THE WOOD ANATOMY OF ASPEN TREES .......................................................................... 53

Abstract .................................................................................................... 53

Introduction .............................................................................................. 54

Materials and Methods............................................................................. 57

Materials ............................................................................................ 57

Quantitative wood anatomy ............................................................... 59

Fiber quality analysis ......................................................................... 59

Experimental data analysis ................................................................ 60

Results and Discussion............................................................................ 62

Conclusions ............................................................................................. 70

References .............................................................................................. 71

ELASTIC MODULUS OF TRANSGENIC ASPEN .............................................. 75

Abstract .................................................................................................... 75

Introduction .............................................................................................. 76

Materials and Methods............................................................................. 78

Dynamic modulus of elasticity measurements ................................... 79

Static modulus of elasticity measurements ........................................ 80

Experimental data analysis ................................................................ 82

Results and Discussion............................................................................ 83

Conclusions ............................................................................................. 88

vi

References .............................................................................................. 89

ELASTIC MODULUS DETERMINATION OF TRANSGENIC ASPEN TREES USING A DYNAMIC MECHANICAL ANALYZER IN STATIC BENDING MODE

................................ ................................ ..................

92

Abstract

................................ ................................ ................................ ....

92

Introduction

................................ ................................ ..............................

93

Materials and Methods ................................ ................................ .............

96

Nondestructive evaluation ................................................................. 96

Dynamic mechanical analyzer in static bending mode ...................... 98

Experimental data analysis ................................................................ 98

Results and Discussion.......................................................................... 100

Conclusions

................................ ................................ ...........................

105

References ............................................................................................ 106

THERMAL SOFTENING OF TRANSGENIC ASPEN ....................................... 109

Abstract .................................................................................................. 109

Introduction ............................................................................................ 110

Materials and Methods........................................................................... 113

Material............................................................................................ 113

Determination of the linear viscoelastic region (LVR) ...................... 114

Submersion three-point bending in a dynamic mechanical analyzer ........................................................................ 114

Parallel plate compression-torsion in a rheometer .......................... 115

Experimental data analysis .............................................................. 117

Results and Discussion.......................................................................... 118

Conclusions ........................................................................................... 125

References ............................................................................................ 126

TRIALS AND TRIBULATIONS ......................................................................... 129

Microfibril Angle Measurements ...................................................... 129

Middle Lamella Strength .................................................................. 130

Shrinkage and Swelling ................................................................... 132

References ............................................................................................ 134

CONCLUSIONS AND RECOMMENDATIONS ................................................. 135

Genetic group with reduced lignin content Ptr4CL .......................... 136

Genetic group with increased S/G ratio PtrCAld5H ......................... 136

vii

Genetic line Ptr4CL/CAld5H-72 with both decreased lignin content and increased S/G ratio ............................................. 137

Genetic line Ptr4CL/CAld5H-141 with both decreased lignin content and increased S/G ratio ............................................. 138

RECOMMENDATION FOR FUTURE WORK ................................................... 140

APPENDIX ....................................................................................................... 141

Appendix A ............................................................................................ 142

Raw data of the different anatomical properties .............................. 142

Appendix B ............................................................................................ 150

Raw data for fiber quality analysis (FQA) ........................................ 150

Appendix C ............................................................................................ 159

ANOVA tables for anatomical properties ......................................... 159

Appendix D ............................................................................................ 169

Raw data for dynamic modulus of elasticity measurements ................................................................................. 169

Appendix E ............................................................................................ 171

Raw data for the static modulus of elasticity measurements ................................................................................. 171

Appendix F ............................................................................................. 173

ANOVA tables of the dynamic and static modulus of elasticity measurements .................................................................. 173

Appendix G ............................................................................................ 179

Raw data for elastic modulus measurements with dynamical mechanical analyzer in static bending mode. ................. 179

Appendix H ............................................................................................ 182

ANOVA table for submersion three-point bending in a dynamical mechanical analyzer ....................................................... 182

Appendix I .............................................................................................. 184

Raw data for submersion three-point bending in a dynamical mechanical analyzer ....................................................... 184

Appendix J ............................................................................................. 186

Raw data for parallel plate compression-torsion in rheometer ........................................................................................ 186

Appendix K ............................................................................................ 187

ANOVA tables for glass transition temperature determination ................................................................................... 187

viii

LIST OF TABLES Table 1. Lignin and cellulose contents of control and transgenic aspen with down-regulated lignin content (Hu et al. 1999). ........................... 5

Table 2. Average velocity [m/s] of the stem measured by Fakopp time of flight (TOF) and WoodSpec (resonance) on logs, green and air- dry boards (Grabianowski et al. 2006). ............................................. 24

Table 3. Studies on glass transition temperatures of wood components. ....... 39

Table 4. Glass transition temperatures of various softwood and hardwood species at different frequencies (Olsson and Salmen (1997). ............................................................................................... 42

Table 5. Chemical composition of wild-type and transgenic aspen. ................ 58

Table 6. Radial growth and anatomical properties of wild-type and transgenic aspen groups using quantitative wood anatomy and fiber quality analysis (FQA). .............................................................. 63

Table 7. Chemical composition of wild-type and transgenic aspen. ................ 78

Table 8. Elastic modulus and selected physical properties of wild-type and transgenic aspen groups. ........................................................... 83

Table 9. Comparison of nondestructive evaluation (dynamic MOE), micromechanical testing (static MOE) and dynamic mechanical analysis in static mode with two different plasticizers (DMA Water MOE, DMA EG MOE). .......................................................... 102

Table 10. Glass transition measurements for wild-type and transgenic aspen clones using dynamic mechanical analyzer in submersion 3-point bending mode. ................................................. 123

Table 11. Glass transition measurements for wild-type and transgenic aspen clones using rheometer in compression-torsion mode. ........ 124

ix

LIST OF FIGURES Figure 1. Monolignol structures (4-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol) and lignin residues (hydroxyphenyl (H units), guaiacyl (G units), and syringyl (S units)) resulted from monolignols (Whetten et al. 1995). .................................................... 2

Figure 2. Phenylpropanoid and monolignol biosynthetic pathways (Boerjan et al. 2003). ......................................................................... 3

Figure 3. Enhanced stem and leaf growth in control and transgenic trees (A6, A9, A11, A8). (Hu et al. 1999). ................................................... 5

Figure 4. Principal biosynthetic pathway for guaiacyl (coniferyl alcohol) and syringyl (sinapyl alcohol) monolignols (Li et al. 2003). ................ 7

Figure 5. A conceptual illustration of relationship between deposition of cell wall polymers and formation of heterogeneous structure of lignin. CC: cell corner, CML: compound middle lamella, G: guaiacyl lignin, H: p-hydroxyphenyl lignin, ML: middle lamella, P: primary wall, S: syringyl lignin, SW, S 1 , S 2 S 3 : secondary wall (Terashima et al. 1993). .............................................................. 9

Figure 6. Microscopic image of iron-alum hematoxylin and safranin stained thin sections of quaking aspen (a) cross section; (b) radial section; (c) tangential section. ................................................ 12

Figure 7. Comparison between transgenic and wild-type hybrid aspen (Tuominen et al. 1995): (a) representative plants of line G1`2`D, wild type and line 58A; (b & d) transverse section of line G1`2`D; (c) tangential section of line G1`2`D; (e & g) transverse section of line 58A; (f) tangential section of line 58A. .......... 16

Figure 8. Scanning electron micrographs of stem transverse sections of (a) control and (b) transgenic line with reduced lignin content (Hu et al. 1999). ............................................................................... 18

Figure 9. Nondestructive test to evaluate wood (Adapted from Galligan (1964) and Ross et al. (1998)). ........................................................ 21

x

Figure 10. Static bolt MOE [GPa] versus dynamic MOE [GPa] (Lindstrom et al. 2002). ...................................................................................... 22

Figure 11. Acoustic MOE [GPa] versus static three-point bending [GPa] (Lindstrom et al. 2002). .................................................................... 23

Figure 12. Average length-weighted fiber length [mm] versus (velocity of sound)2 [km2/s2] for each of the 18 sound classes (Albert et al. 2002). .......................................................................................... 25

Figure 13. Breaking length [km] versus (velocity of sound)2 [km2/s2] for each of the 18 sound classes (Albert et al. 2002). ........................... 25

Figure 14. Breaking length [km] versus (velocity of sound)2 [km2/s2] for each of the 18 sound classes (Albert et al. 2002). ........................... 26

Figure 15. Static MOE [GPa] versus dynamic MOE [GPa] in Norway spruce (triangle) and in sycamore (square) (Spycher et al. 2008). ....... 27

Figure 16. Experimental procedure to evaluate the log properties by longitudinal stress wave, transverse vibration and static bending techniques (Wang et al. 2002). .......................................... 28

Figure 17. Relationship between the dynamic MOE of logs by stress wave measurement (MOEsw) and both the static MOE of logs (MOEs) and the dynamic MOE of logs by transverse vibration measurements (MOEv) (Wang et al. 2002). .................................... 28

Figure 18. Modulus of elasticity (MOE) deviation as a function of log diameter (Wang et al. 2004). ........................................................... 29

Figure 19. Comparison between the dynamic longitudinal and flexural modulus of elasticity and the static modulus of elasticity. Dynamic longitudinal modulus of elasticity static was highly related to static modulus of elasticity (r=0.95). Ilic (2001). ............... 31

xi

Figure 20. Relation between the dynamic longitudinal modulus of elasticity, the static modulus of elasticity and the modulus of rupture. Outlying point represents sample with significant tension wood. Ilic (2001). ................................................................. 31

Figure 21. Stress and strain curve for the dynamic analysis of elastic materials. The strain is in phase with the stress (Menard 1999). .............................................................................................. 35

Figure 22. Stress and strain curve for the dynamic analysis of viscoelastic materials. The strain is out of phase with the stress (Menard 1999). .............................................................................................. 36

Figure 23. Decrease in the storage modulus as temperature increases (Menard 1999). ................................................................................ 37

Figure 24. Main softening temperatures of wood components under completely dry conditions (Back and Salmen 1982). ....................... 40

Figure 25. Correlation between the thermal properties and the condensation degree of milled wood and enzyme lignins isolated from control and transgenic poplars (Baumgartner et al. 2002). .......................................................................................... 43

Figure 26. One-year-old trees and sample stems of wild type and transgenic aspen. ............................................................................ 58

Figure 27. Stem diameter comparison for wild-type quaking aspen PrtWT-271 (a) and transgenic aspen with increased S/G ratio PtrCAld5H-96 (b) using the same magnification under stereo light microscope. .............................................................................. 64

Figure 28. Transverse sections of wild-type aspen PrtWT-271 (a) and transgenic line Prt4CL/CAld5H-72 with both reduced lignin content and increased S/G ratio (b) showing differences in anatomical structure. ....................................................................... 65

xii

Figure 29. Measuring ultrasonic transmission time between two piezo sensors using Fakopp Microsecond Timer. ..................................... 80

Figure 30. Static three-point bending test with a special bearing block............. 81

Figure 31. Dynamic and static modulus of elasticity of wild-type and transgenic aspen clones for the different genetic groups. Whiskers represent the standard deviation. ..................................... 86

Figure 32. Submersion three-point bending clamp in the dynamic mechanical analyzer. ....................................................................... 99

Figure 33. Typical load deflection curve for DMA measurements. .................. 100

Figure 34. Normalized mean elastic modulus of wild-type and transgenic groups using different measurement techniques. Whiskers represent the standard deviation. .................................................. 104

Figure 35. Submersion three-point DMA bending clamp. ............................... 115

Figure 36. Stainless steel 8-mm-diameter parallel plate in compression- torsion mode. ................................................................................. 116

Figure 37. Tan delta curves (a) and representative storage modulus curves (b) from the second heat of submersion three-point bending dynamic mechanical analysis using 0.05Hz frequency and heat rate of 2°C/min. Each point represents the average value of several measurements. Whiskers represent the standard deviation. ........................................................................ 119

Figure 38. Tan delta curves from the second heat of parallel plate compression-torsion dynamic mechanical analysis using 1Hz frequency and a heat rate of 3°C/min. Each point represents the average value of several measurements. Whiskers represent the standard deviation. .................................................. 120

Figure 39. Radial sections of transgenic aspen with small bordered pits on the fiber cell wall. ...................................................................... 130

xiii

Figure 40. Transverse sectioned pulled apart in the radial direction using a tension clamp in a dynamic mechanical analyzer. ...................... 131

Figure 41. Sample in the compression clamp using DMA Q800. .................... 132

Figure 42. Stereomicroscope image of the cross section of wild-type in wet (a) and dry (b) conditions. ....................................................... 133

1

REVIEW OF RELATED LITERATURE LIGNIN, LIGNIN BIOSYNTHESIS, AND LIGNIN GENETIC ENGINEERING After cellulose, lignin is the second most abundant class of chemicals found in wood. In general, lignin accounts for 18-25% of the dry weight of hardwoods, and for 20-32% of softwoods (Bowyer et al. 2003). Lignin primarily serves as a binder between wood fibers and gives strength and rigidity to the cell wall (Jeronimidis 1980). It also impacts water transport in terms of vulnerability of hardwood vessels to implosion (Jacobsen et al. 2005) and forms a physico-chemical barrier against microbial attack (Northcote 1989). Lignin is an important regulator for cell wall enzymatic degradation (Goldstein 1991), but often represents a barrier to the utilization of woody biomass in pulp and paper making and ethanol production. During these processes, delignification is highly energy intensive, expensive and requires a complex chemical recovery process. From a chemical point of view, lignin is a complex polymer derived from polymerization of coniferyl alcohol (Whetten and Sederoff 1995). Lignin is formed mainly from three monolignols: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol ( Figure 1 ) (Whetten and Sederoff 1991, Whetten et al. 1998). These monolignols interconnect with each other and with other cell wall polymers via different linkages (ether and carbon-carbon linkages) forming a three dimensional network polymer (Amthor 2003). Softwood lignin is based mainly on guaiacyl subunits, also known as G units or G lignin, polymerized from coniferyl

2

alcohol, and low level of p-hydroxyphenyl units (H units) polymerized from p- coumaryl alcohol (Goldstein 1991, Whetten et al. 1998). On the other hand, hardwood lignin is a copolymer of guaiacyl subunits, syringyl subunits (S units or S lignin) and a low level of p-hydroxyphenyl units. These are polymerized from coniferyl, sinapyl and p-coumaryl alcohol, respectively.

Figure 1. Monolignol structures (4-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol) and lignin residues (hydroxyphenyl (H units), guaiacyl (G units), and syringyl (S units)) resulted from monolignols (Whetten et al. 1995).

3

Figure 2. Phenylpropanoid and monolignol biosynthetic pathways (Boerjan et al. 2003). CAD, cinnamyl alcohol dehydrogenase; 4CL, 4-coumarate:CoA ligase; C3H, p- coumarate 3-hydroxylase; C4H, cinnamate 4-hydroxylase; CCoAOMT, caffeoyl-CoA O- methyltransferase; CCR, cinnamoyl-CoA reductase; COMT, caffeic acid O- methyltransferase; HCT, p-hydroxycinnamoyl-CoA: quinate shikimate p- hydroxycinnamoyltransferase; F5H, ferulate 5-hydroxylase; PAL, phenylalanine ammonia-lyase; SAD, sinapyl alcohol dehydrogenase. ?, conversion demonstrated; ??, direct conversion not convincingly demonstrated; 4CL??, some species have 4CL activity toward sinapic acid; CCR? and F5H?, substrate not tested; others, enzymatic activity shown in vitro (Boerjan et al., 2003)

4

An overview of the main and possible monolignol biosynthesis pathways of wood formation can be seen on Figure 2 (Boerjan et al. 2003). Monolignol biosynthesis involves various enzymatic conversions (Boerjan et al. 2003) where enzyme (protein) control each step in the biosynthesis process. In the last 15 years, many of these enzymes and related genes have been identified and analyzed; and this raised enormous opportunity for lignin genetic engineering (Whetten et al. 1998). It is well known that any biological process starts from DNA, which transcribes to mRNA. This mRNA is further translated into enzyme (protein). This enzyme is the only biomolecule that can convert a substrate into a product in the biosynthesis. Thus, through this enzyme, biosynthetic pathway can only be controlled by the DNA or by part of a DNA (gene). Tremendous progress has been made in understanding and characterizing these genes, which control the monolignol biosynthetic pathways in trees. Lignin genetic engineering is the manipulation of genes encoding enzymes in the biosynthetic pathway (Whetten and Sederoff 1991). Many research efforts have focused on lowering lignin content and increasing lignin reactivity (Whetten et al. 1998). Hu et al. (1999) produced transgenic aspen trees (Populus tremuloides Michx.) with reduced lignin content by antisense inhibition of gene Pt4CL1 (lignin specific 4-coumarate ligase) in the biosynthetic pathway. These transgenic aspen clones were obtained through Agrobacterium-mediated transformation and exhibited a 5-45% reduction in lignin content relative to the

5

conrols, but the chemical structure of the resulting lignin, syringyl/guaiacyl ratio (S/G ratio), remained unchanged. Hu et al. (1999) also reported cellulose accumulation ( Table 1 ) and enhanced stem growth in these transgenic trees ( Figure 3 ).

Table 1. Lignin and cellulose contents of control and transgenic aspen with down- regulated lignin content (Hu et al. 1999). Plant

Lignin Content*

Cellulose Content*

Cellulose - to

Lignin Ratio

Control

A4

A5

A6

A8

A15

A9

A11

A12

21.62 ± 0.30 (100)

12.83 ± 0.28 (60)

13.02 ± 0.28 (60)

11.84 ± 0.08 (55)

12.90 ± 0.04 (60)

18.60 ± 0.18 (86)

19.40 ± 0.27 (89)

20.40 ± 0.10 (94)

20.60 ± 0.08 (95)

44.23 ± 0.43 (100)

48.35 ± 0.60 (109)

49.74 ± 0.45 (112)

50.83 ± 0.26 (115)

48.14 ± 0.29 (109)

45.98 ± 0.83 (104)

47.49 ± 0.30 (107)

45.95 ± 0.28 (104)

46.55 ± 0.04 (105)

2.0

3.8

3.7

4.3

3.8

2.5

2.4

2.2

2.3

Notes: Data are the means ± SD of three independent experiments. Normalized values relative to control are shown in parentheses. Lines A4, 5, 6, 8 exhibit less than 7% residual 4CL activity with 4-coumaric acid, while A15 had 30%, A9 60% and A11 and A12 90% residual 4CL activity compared to the control level. *% of dry wood weight.

Figure 3. Enhanced stem and leaf growth in control and transgenic trees (A6, A9, A11, A8). (Hu et al. 1999).

6

Li et al. (2000, 2001) challenged the traditional monolignols biosynthesis model and stated that three key enzymes are essential to control the biosynthesis and utilization of sinapaldehyde for syringyl monolignol in aspen. These are CAld5H (coniferyl aldehyde 5-hydroxylase), AldOMT (5-hydroxyconiferaldehyde O- methyltransferase) and SAD (sinapyl alcohol dehydrogenase) ( Figure 4 ). CAld5H and AldOMT proteins are absent in gymnosperms (Li et al. 2001), but SAD protein is widely distributed in angiosperms (Li et al. 2000). CAld5H has the lowest enzyme turnover rate compared to the other two enzymes and creates a “bottleneck” in syringyl monolignol biosynthesis. Insertion of sense CAld5H gene diverts the guaiacyl pathway from coniferaldehyde to sinapaldehyde to initiate syringyl monolignols biosynthesis (Chiang 2006). The sense CAld5H gene increases the production ratio of syringyl to guaiacyl lignin. Consequently, sense CAld5H plants achieved an S/G-ratio increase of up to 64% without lignin quantity change through Agrobacterium-mediated transformation by Li et al. (2003). Simultaneous expression of down-regulating 4CL gene and up-regulating CAld5H gene led to lignin quantity reduction and lignin-reactivity augmentation (Li et al. 2003). This combinatorial gene insertion decreased the stem lignin content by 38–52% and increased the lignin S/G ratio by 22–64% in transgenic aspen trees. Similar to the transgenic aspen with reduced lignin content, transgenic aspen with both reduced lignin content and increased S/G ratio were

7

obtained through Agrobacterium-mediated cotransformation and had remarkable increase in cellulose content (Chiang 2006).

Figure 4. Principal biosynthetic pathway for guaiacyl (coniferyl alcohol) and syringyl (sinapyl alcohol) monolignols (Li et al. 2003). C4H, cinnamate 4-hydroxylase; C3H, 4-coumarate 3-hydroxylase; 4CL, 4- coumarate:CoA ligase; CCoAOMT, Caffeoyl-CoA O-methyltransfe

8

WOOD CELL WALL FORMATION The lignified plant cell wall is composed of different cell wall polymers, such as cellulose, hemicelluloses, and lignin that are physically and chemically bound to each other in a biochemically regulated manner (Terashima et al. 1993). The first step in cell wall formation is the development of the primary wall (PW), consisting of cellulose microfibrils (CMF) and hemicelluloses. CMF form a twisted honeycomb structure where the distance between the CMF determined by the hemicelluloses as spacer. After the PW has formed but before secondary wall (S1) formation begins, lignification starts in the cell corner region and in the middle lamella (Bailey 1954, Terashima et al. 1993). Both the cell corner region and the middle lamella are highly lignified and contain condensed p- hydroxyphenyl (H) and guaiacyl (G) units (Mellerowicz et al. 2001). In the secondary wall, the S1 layer has a flat, almost transverse microfibril angle (MFA). This MFA gradually changes clockwise to a longitudinal arrangement that can be found in the S2 layer. The S3 layer is formed after an abrupt reorientation of the microfibrils, resulting in a transverse MFA similar to S1. In the secondary wall CMF, the hemicelluloses and lignin matrix forms lamellae. Lignin composition changes during the differentiation of the secondary wall. In the early stages more H and G units are deposited, while in later stages syringyl (S) units are involved (Terashima et al. 1979).

9

Figure 5. A conceptual illustration of the relationship between the deposition of cell wall polymers and the formation of the heterogeneous structure of lignin. CC: cell corner, CML: compound middle lamella, G: guaiacyl lignin, H: p-hydroxyphenyl lignin, ML: middle lamella, P: primary wall, S: syringyl lignin, SW, S 1 , S 2 S 3 : secondary wall (Terashima et al. 1993).

The three different kinds of monolignol units are incorporated at different stages of cell wall formation in the order of: H, G, and S. Lignification starts in the middle lamella first, where highly lignified and highly condensed type H and G

10

units are deposited (Fukushima and Terashima 1991). As lignification progresses, the lignifying region moves from the outer to the inner wall resulting in more and more G and S units and more uncondensed type lignin (Terashima and Fukushima 1988). In poplars, the vessels contain more condensed H and G type lignin since vessel elements lignify earlier than fibers ( Figure 5 ). Mostly, uncondensed S type lignin is deposited in fibers during the middle/late stages of the lignification process. These are mainly located in the secondary cell wall.

11

ANATOMICAL AND CHEMICAL CHARACTERISTICS OF QUAKING ASPEN Quaking aspen or trembling aspen (Populus tremuloides) is the most widely distributed tree in North America (Rauscher et al. 1995). It is usually a tall tree with an average height of 20-25m and an average diameter of 20-80cm at maturation (Mitton and Grant 1996). It often serves as a model species for tree improvement programs due to the fact that it is easy to propagate, it is fast growing and most of the biochemical functions of its various genes are characterized (Chiang et al. 2006). Quaking aspen is a diffuse porous hardwood species with relatively soft and light colored wood (Mackes and Lynch 2001). As typical of hardwoods, aspen has basically two different longitudinal cell types: vessels and fibers. Vessels, which are mainly responsible for water conduction, are relatively small (~80μm) and evenly distributed throughout the growth ring ( Figure 6 a) (Matyas and Peszlen 1997). Perforation plates between vessel elements are simple, which allow for rapid water conduction. Fibers are mainly responsible for the strength of the wood. There is a slight diameter and cell wall thickness difference between earlywood fibers and latewood fibers (density gradient is small). Therefore overall texture is uniform. The average fiber lumen diameter is 16μm and the average fiber length is 1.1 mm at maturation (Matyas and Peszlen 1997). With regard to pulp and paper production, quaking aspen has an excellent fiber length-to-diameter ratio with a thin to medium fiber cell wall thickness (Mitton and

12

Grant 1996). Rays are hardly visible, even with a hand lens, because they are usually one cell wide ( Figure 6 c).

Figure 6. Microscopic image of iron-alum hematoxylin and safranin stained thin sections of quaking aspen (a) cross section; (b) radial section; (c) tangential section. (Source: http://micro.magnet.fsu.edu/trees/pages/quakingaspen.html)

a

b

c

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Like other hardwoods, aspen lignin contains both syringyl and guaiacyl lignin and has a 2.2 syringyl to guaiacyl ratio (Li et al. 2003). A review by Campbell and Sederoff (1996) stated that variation in lignin content, composition, and location is likely to affect essential processes such as the resistance of xylem to compressive stresses imposed by water transport and by the mass of the plants which is important to growth and development and resistance to degradation by most microorganisms serving an important function in plant defense. Investigating the distribution of lignin in different cell types, Fergus and Goring (1970) used UV microscopy to identify the guaiacyl and syringyl lignified tissues in birch and found that vessels contain predominately guaiacyl lignin, whereas the fiber and ray parenchyma secondary walls have a large proportion of syringyl lignin. In addition, Saleh et al. (1967) showed that guaiacyl lignin was deposited in the middle lamella of aspen. These distribution of different lignin types were also confirmed by Musha and Goring (1975b) in the different morphological regions of fourteen hardwood species. Formation of syringyl and guaiacyl lignin is active in different stages of the xylem differentiation. Guaiacyl lignin is deposited on the vessel wall in the early stage followed by the deposition of syringyl lignin on the fiber cell wall (Terashima et al. 1986). Lignin deposition does not occur randomly but chronologically from the external part of the wall to the inside of the cell (Terashima et al. 1998). Lignification occurs at the cell corners and middle

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Abstract: The directed modification of specific traits of trees through genetic engineering provides opportunities for making significant genetic improvements to wood properties in a matter of years instead of extended time frames required for traditional natural selection. An attractive target of forest-tree engineering is the modification of lignin content and its structure which present potential advantages including improved pulping efficiency, lower chemical and energy consumption, and reduced environmental impacts. However, decrease in lignin content and changes in lignin structure could lead to the modifications of wood characteristics that are critical for solid wood. Wild-type and transgenic quaking aspen (Populus tremuloides Michx.) with reduced lignin content, increased syringyl to guaiacyl (S/G) ratio, and both reduced lignin content and increased S/G ratio were investigated in four studies. In the first study , diameter growth and cell morphology of one-year-old transgenic aspen were investigated using quantitative wood anatomy and fiber quality analysis techniques. Similar radial growth and quantitative anatomical properties were observed between the genetic group with reduced lignin content and the wild-type. The genetic group with increased S/G ratio had lower diameter growth, lower vessel lumen diameter, but more numerous vessels. The combined effect of lignin content and structural changes on radial growth and cell morphology seems to be more complex and gave inconsistent results. In the second study , dynamic modulus of elasticity (MOE) using a nondestructive technique (Fakopp Microsecond Timer) and static MOE using micromechanical testing method were investigated for 2.5-year-old transgenic aspen clones. Result showed that a reduction in the lignin content reduce both the dynamic and static MOE. Increase in the syringyl to guaiacyl ratio results in only a slight decrease in the dynamic MOE and static MOE. The combined influence of lignin content and syringyl to guaiacyl ratio changes shows the most obvious negative effect on both the dynamic MOE and static MOE. In the third study , a dynamic mechanical analyzer (DMA) in static bending mode was used to determine the elastic modulus of 2.5-year-old wild-type and transgenic aspen submerged in water and in ethylene glycol. DMA MOE values were compared to those assessed by nondestructive evaluation and micromechanical testing in previous study. All of the measured elastic moduli showed the same trend and detected similar differences across the genetic groups. DMA measurements showed notably lower MOE values than the other techniques. In the fourth study , the in situ lignin glass transition of one-year-old transgenic aspen was investigated using dynamic mechanical analysis and rheometer. Results suggested that an increase in the S/G ratio did not affect the glass transition temperature but a reduction in lignin content decreased the softening temperature of transgenic wood which has practical implication and would be beneficial to pulp and ethanol production. This research revealed that a reduction in lignin content had a more severe negative effect on wood properties than an increase in S/G ratio. However, diameter growth of transgenic trees with increased S/G ratio was lower. The combined effect of reduced lignin content and increased S/G ratio on wood characteristics was inconsistent; therefore it needs further investigation. Although the reduced lignin content genetic group has inferior mechanical properties and cannot be considered for solid and structural applications, the lower lignin content and glass transition temperature make it suitable for pulp and ethanol production, and for composite manufacture.