Abiotic and biotic stresses and changes in the lignin ... - ResearchGate

1
Abiotic and biotic stresses and changes in the lignin content and
composition in plants
Running title: Lignin changes caused by stresses
Jullyana Cristina Magalhães Silva Moura 1 , Cesar Augusto Valencise Bonine 2 , Juliana
de Oliveira Fernandes Viana 1,2 , Marcelo Carnier Dornelas 1 and Paulo Mazzafera 1 *
1 Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de
Campinas, CP 6109, 13083-970, Campinas, SP, Brazil
2 Votorantim Celulose e Papel S.A., Rodovia SP 255, Km 41,2, s/nº, 14210-000, Luis
Antonio, SP, Brazil
*Corresponding author:
email: pmazza@unicamp.br
Tel: +55 19 3521 6358

2
1
2
3
4
5
6
7
8
Abbreviations: PAL - phenylalanine ammonia-lyase; C4H - cinnamate 4-hydroxylase;
C3H - cinnamate 3-hydroxylase or p-coumaroyl shikimic acid/quinic acid 3-
hydroxylase; OMT - O-methyltransferase; HCT - p-hydroxy cinnamoyl-CoA:
shikimate/quinate p-hydroxy cinnamoyl transferase; CCoAOMT - caffeoyl coenzyme A
O-methyltransferase; F5H - ferulate 5-hydroxylase; 4CL - 4-coumarate: coenzyme A
ligase; CCR - cinnamoyl coenzyme A reductase; Cald5H - coniferaldehyde 5-
hydroxylase; AldOMT - 5-hydroxy coniferaldehyde O-methyltransferase; SAD -
sinapyl alcohol dehydrogenase; CAD - cinnamyl alcohol dehydrogenase.
9
10
11
12
13
14
15
16
17
18
19
Abstract: Lignin is a polymer of phenylpropanoid compounds formed through a
complex biosynthesis route, represented by a metabolic grid for which most of the genes
involved have been sequenced in several plants, mainly in the model-plants Arabidopsis
thaliana and Populus. Plants are exposed to different stresses, which may change lignin
content and composition. In many cases, particularly for plant-microbe interactions, this
has been suggested as defence responses of plants to the stress. Thus, understanding
how a stressor modulates expression of the genes related with lignin biosynthesis may
allow us to develop study-models to increase our knowledge on the metabolic control of
lignin deposition in the cell wall. This review focuses on recent literature reporting on
the main types of abiotic and biotic stresses that alter the biosynthesis of lignin in plants.
20
21
22
Key words: disease, drought, light stress, low temperature, mineral nutrition, plant
growth, plant breeding, reaction wood.
23
24
25

3
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Introduction
Lignin (from the Latin word lignum, wood) is a highly branched polymer of
phenylpropanoid compounds, and a component of the plant cell wall. After cellulose,
lignin is the second most abundant organic compound in plants, representing
approximately 30% of the organic carbon in the biosphere (Boerjan et al., 2003).
The lignification process seems to have appeared on Earth 430 million years ago
(Boudet, 2000) among the possible strategies that allowed plants to adapt to terrestrial
life. The functional significance of lignin is associated mainly with the mechanical
support allowing plants to stand, with water transport in the xylem vases and as a
defence against pests and microorganisms (Boudet, 2000). The first two characteristics
are related to the way lignin interacts with other molecules of the cell wall,
strengthening the whole structure.
Despite the importance of lignin in plant evolution and development, it
represents a problem for the agro-industrial application of several plants, particularly in
the papermaking industry. The processes used for the separation of lignin from the
cellulose pulp are the most costly and polluting parts of the whole process (Hatfield and
Fukushima, 2005). Consequently, several studies have investigated how to improve
pulping efficiency (Akgül et al., 2007), as well as alternative pulping methods
(Khristova et al., 2006; Huang et al., 2007).
In addition to the chemical studies trying to find more efficient and
environmentally friendly methods for lignin extraction, there are also several
laboratories around the world seeking to genetically manipulate lignin structure and
content. Genetically modified plants with less lignin and/or with a different structural
lignin composition may allow for improved cellulose yield during pulping and, at the

4
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
same time, reduce the level of pollutants released to the environment. Arabidopsis
thaliana and Populus have been used as model-plants in most of these studies.
Within this context, lignin biosynthesis has been shown to be the result of a
complex genetic network (Rogers and Campbel, 2004), where several enzymes are
involved and may respond differently to biotic and abiotic effects (Figure 1). In this
regard, the literature is rich with examples where lignin content in the plant body is
increased or exhibits a change in chemical composition due to stressors, suggesting
complex genetic and physiological control. It is possible that understanding how a
stressor “modulates” the expression of “lignin genes” would allow us to develop studymodels
to elucidate genetic control of lignin synthesis and its deposition in the cell wall.
Different types of abiotic stresses, such as mineral deficiency, drought, UV-B
radiation and low temperatures, as well as biotic stresses, such as infection by fungi,
bacteria and viruses, cause changes in the lignin contents of several plants. Thus, this
review focuses on the recent literature reporting on the main types of abiotic and biotic
stresses that alter the biosynthesis of lignin in plants.
65
66
Insert Figure 1
67
68
69
70
71
72
73
74
Low temperatures
Low temperature is an important factor limiting the extension of cultivating
areas and therefore the productivity of growing several plants. It is known that, in many
species, a prior exposure to non-freezing low temperatures allows survival following
further exposure to freezing temperatures (Chinnusamy et al., 2007). Understanding
which mechanisms are involved in the acclimation to cold would allow researchers to
produce genetically modified plants with greater resistance to low temperatures. This

5
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
would minimize the impact of cold on the productivity of agriculturally important plants
and would also allow the expansion of agricultural areas in several countries.
The acclimation to cold is a process that involves changes in gene expression
associated with physiological and biochemical changes. During cold acclimation of
Arabidopsis, 3379 genes, including those coding for several transcription factors,
exhibit altered expression patterns (Hannah et al., 2005).
In below-zero temperatures, the membranes are the most injured regions of the
cell. This damage occurs mainly due to dehydration caused by the formation of
extracellular ice (Steponkus, 1984; Steponkus et al., 1993). Other injuries are caused by
reactive oxygen species (Suzuki and Mittler, 2006). Therefore, it may be possible to
improve cold tolerance by examining changes in lipid composition (Thomashow, 1999),
as well as the accumulation of sugars, amino acids and anti-freeze compounds, such as
anti-freeze proteins (Atici and Nalbantoglu, 2003) and antioxidant enzymes and
compounds (Gratão et al., 2005).
Even though we do not know the role of lignin in the acclimation to cold, low
temperatures can cause changes in plant lignin content. Ex vitro poplar (Populus
tremula x P. tremuloides L. cv. Muhs1) seedlings grown at 10 o C showed an increase in
lignin content, but the same was not true for in vitro seedlings (Hausman et al., 2000).
The amount of lignin in latewood Picea abies may be positively correlated with the
annual average temperature (Gindl et al., 2000). Monocots in temperate climate regions
exhibit an increase in the amounts of lignin, cellulose and hemicellulose with increasing
temperature (Ford et al., 1979).
Other studies showed variation in the level of phenylpropanoid lignin precursors
and in the activities of related enzymes in plants exposed to low temperatures. Brassica
napus plants exposed to cold (3 weeks to 2 o C followed by a rapid decline to -5ºC for

6
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
18h, then followed by 6h at 2 o C) did not show any change in the levels of lignin or cell
wall-bound phenols (Solecka et al., 1999). However, there was an increase in the levels
of ρ-coumaric acid, ferulic acid and synapic acid in the cells of the leaf mesophyll, as
well as an increase in the esterified soluble forms of these acids, which may be
associated with an increase in the activity of phenylalanine ammonia-lyase (PAL).
Previous studies showed that B. napus exposed to low temperatures demonstrated
increased PAL activity (Solecka and Kacperska, 1995). The increase in phenolic acid
esterified soluble forms observed by Solecka et al. (1999) might be a mechanism to
avoid the toxic effect on plant cells of free phenols (Whetten and Sederoff, 1995). This
would also allow their transport to the vacuoles (Dixon and Paiva, 1995), where they
could be used as substrates for peroxidase (Takahama, 1988) to protect the cells against
reactive oxygen species, since this enzyme uses peroxide as oxidizing substrate
(Takahama and Oniki, 1997).
Glycine max roots also showed increased PAL activity during acclimation to
cold (10 o C), accompanied by a significant increase in the levels of esterified forms of
ferulic, syringic and p-hydroxybenzoic acids (Janas et al., 2000). Such increases were
also suggested to be a mechanism to prevent cell damage, involving transport to the
vacuole and peroxidase activity as scavenger mechanisms to protect against generated
reactive oxygen species.
Peroxidase activity also increased in wheat (Taşgına et al., 2006; Janda et al.,
2007) and Saccharum spp. hybrid (Jain et al., 2007) exposed to low temperature.
Levels of lignin and soluble phenols were closely related in leaves and roots of
Triticum aestivum plants at 2 o C (Olenichenko and Zagoskina, 2005). While leaves
showed an increase of soluble phenols and a decrease of lignin, the opposite was
observed in roots. The accumulation of soluble phenolic compounds was argued to

7
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
correlate with changes in CO 2 exchange, as there was a smaller reduction in
photosynthesis as compared to respiration, since photosynthesis is less sensitive to low
temperatures. The increase of these compounds was considered a cellular adaptation to
stress, acting as endogenous antioxidant. Other studies with wheat also showed an
increase in the accumulation of soluble phenolic compounds in leaves, but no change in
lignin content was detected (Olenichenko and Zagoskina, 2005). However, lignin
accumulated in tillering nodes; in contrast to other studies where the increased amount
of soluble phenolic compounds was correlated with increased PAL activity (Solecka
and Kacperska, 1995; Janas et al., 2000), in this case less activity was observed in both
tissues with a concomitant increase of free L-phenylalanine.
Curiously, some studies have shown that although no changes in the levels of
lignin or its precursors were observed in plants maintained at low temperatures, there
was an increase in related enzyme activities as well as an increase in gene expression.
During acclimation of Rhododendron to cold, there was an increase in the expression of
the gene coding for C3H, a cytochrome P450-dependent monooxygenase involved in
the biosynthesis of phenylpropanoids and lignin (El Kayal et al., 2006). According to
these authors, increased expression of C3H could result in changes in the composition
of lignin, altering the stiffness of the cell wall.
143
144
145
146
147
148
Water deficit
Water deficit occurs in plants when the water supply is insufficient to maintain
growth, photosynthesis and transpiration (Fan et al., 2006). This is one of the main
problems affecting food production in the world, reducing crop productivity (Vincent et
al., 2005).

8
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
Very little is known about the effects of drought on lignin biosynthesis. A
reduction in the amount of ferulic acid and an increase of p-coumaric and caffeic acids
in the xylem sap of maize was observed after 12 days of water upholding (Alvarez et al.,
2008). It was also detected decreased anionic peroxidase activity and increased cationic
peroxidase activity. According to the authors, the increase of free lignin precursors in
the xylem sap as well as the reduced anionic peroxidase activity could be an indication
that drought decreases the biosynthesis of lignin in maize (Alvarez et al., 2008).
Different regions of the maize root may respond differently to drought, as the
deposition of lignin may be greater in a specific region of the root or at certain times of
stress (Fan et al., 2006; Yang et al., 2006; Yoshimura et al., 2008).
It has been shown that the basal part of the roots of maize plants under water
stress exhibit a greater reduction in growth than the apical region (Fan et al., 2006).
Such a reduction was associated with an increased expression of two genes involved in
the biosynthesis of lignin: cinnamoyl-CoA reductase 1 and 2. The reduction was also
associated with increased deposition of lignin, which stiffened cell wall extensibility
and decreased cell wall expansion. In these plants, reduced growth of the basal root
might improve the availability of water, minerals and sugars, factors necessary to
maintain minimum growth and survival of young cells in the most apical portion,
facilitating renewed growth after rehydration (Fan et al., 2006).
Researches have shown an increase in the expression of genes related to cell
growth and extensibility in the roots of rice (Oryza sativa L.) plants during the initial
stages (16 hours) of water stress, enabling root growth in these plants (Yang et al.,
2006). It was also observed that there was an increased expression of genes involved in
lignin biosynthesis during the intermediate and final stages of water stress (from 48 to
72 hours), such as those coding for PAL, C3H, 4CL, CCoAOMT, CAD and peroxidase.

9
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
Similar results were obtained with Citrullus lanatus sp., which shows an extraordinary
resistance to drought (Yoshimura et al., 2008). In the early stages of stress, this plant
showed an increase in root growth, which was associated with the induction of the
synthesis of proteins involved in morphogenesis (actin, α-tubulin and Ran GTPase) as
well as the metabolism of carbon and nitrogen (isoforms of triose-phosphate isomerase,
malate dehydrogenase, α-mannosidase, UDP-sugar pyrophosphorylase, NADP-malic
enzymes, phosphoglucomutase and UDP glucose-6-phosphate dehydrogenase). In the
final stages of the stress, however, there was a reduction in root growth and induction of
lignin biosynthesis, with increasing expression of CCoAOMT and a large number of
isoenzymes comprising class III peroxidases. Growth reduction and tolerance to
desiccation were associated with more lignin in the roots.
Other plant organs such as leaves may show marked physiological changes
during drought stress. Growing leaves of maize plants accumulated more COMT,
mostly at 10 to 20 cm from the point of leaf insertion; drought resulted in a shift of this
region of maximal accumulation toward basal regions (Vincent et al., 2005). The lignin
content in leaves of maize plants subjected to water stress was lower than in the wellwatered
control plants, suggesting an adaptation to drought, since the maintenance of
high levels of lignin in the region of leaf elongation region might negatively affect
growth after rehydration (Vincent et al., 2005).
The biosynthesis of lignin and its functional significance during water stress was
studied in the leaves of Trifolium repens plants subjected to 28 days of drought (Bok-
Rye et al., 2007). Reduced leaf growth occurred concurrently with the increase in lignin
biosynthesis. The activities of enzymes involved in the biosynthesis of lignin revealed
that the enzyme responses to drought might differ, depending on the period for which
plants are exposed to drought. Overall, there was a large increase in the activities of

10
199
200
201
202
203
PAL and ascorbate peroxidase in the early stages of stress (0-14 days), with activity
levels decreasing gradually as the period of stress was extended. On the other hand,
other enzymes such as guaiacol peroxidase, coniferyl alcohol peroxidase and
syringaldazine peroxidase exhibited greater activity during the final stages of stress (14-
28 days).
204
205
Light
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
Light is essential for plants to ensure normal growth. However, an excess of
light has the potential to cause cellular damage, mainly due to the formation of reactive
oxygen species (ROS). To cope with this degenerative stress, plants have evolved
strategies to protect themselves against ROS, such as increased peroxidase activity,
accumulation of anthocyanins and reduction of the antenna systems components to
dissipate the energy of absorbed light (Kimura et al., 2003).
Light itself has an effect on lignin biosynthesis. Ebenus cretica L. seedlings
grown in the light had 2.5 times more lignin than seedlings grown in the dark (Syros et
al., 2005).
The control of lignin biosynthesis by light was investigated in 3-day-old soybean
seedlings, which were divided in three groups: seedlings kept in the dark, seedlings
exposed to light (340 µmol m -2 s -1 ) and seedlings exposed to light (340 µmol m -2 s -1 ,
16h per day) plus diamine oxidase inhibitors (Andersson-Gunneras et al., 2006). Plants
in the light showed increased levels of H 2 O 2 and lignin, as well as higher levels of
diamine oxidase and POD activity. The light plus inhibitor condition reduced the
amount of H 2 O 2 and lignin.
During the differentiation of cultured calli of Pinus radiata into tracheids, it was
found a positive effect of light on the activity of the enzymes PAL and CAD, which

11
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
resulted in an increase in the concentration of lignin when the calli were transferred
from dark to a photoperiod of 16 hours (Möller et al., 2006). Continuous light (16.7 W
m -2 ) increased the activity of anionic and cationic PODs and laccases in mungbean
hypocotyls, resulting in an increase in the lignin content (Chen et al., 2002).
Phalaenopsis orchids maintained for one month at 25ºC ± 2°C at different light
intensities (60, 160 and 300 µmol m -2 s -1 ) tended to increase the lignin content in leaves
and roots with increasing light intensity, which was associated with the induction of
PAL, CAD and POD activities (Akgül et al., 2007). It was concluded that the increase
in the biosynthesis of lignin, probably not only provided cell wall rigidity, but also
provided protection against radiation stress.
In Arabidopsis thaliana, 7,000 genes exhibited altered expression when plants
were submitted to high light intensity. Among these, 110 showed increased expression,
and several of them are involved in the lignin biosynthesis pathway (Kimura et al.,
2003).
The accumulation of gene transcripts coding for enzymes involved in lignin
biosynthesis in A. thaliana varies during the diurnal cycle, stimulated by light provided
that there is a prior period of darkness (Rogers et al., 2005). It was also observed that in
the sex1 mutant, which is deficient in starch metabolism, there was lower expression of
genes involved in lignin synthesis during the day, indicating that the availability of
carbohydrate may regulate lignin biosynthesis. These mutants accumulated less lignin
than wild type; lignin composition was also changed, with a higher proportion of lignintype
S as compared with type G type H (of which there was none). This resulted in
lignin that was easier to extract from the cell wall. It was concluded that at least three
factors could affect the synthesis of lignin in these plants: light, circadian rhythms and
sugar perception.

12
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
UV-B (280-320nm)
In recent years, there has been increased interest in the biological effects of UV
radiation on living organisms, in part due to the reduction of the atmospheric ozone
layer caused by anthropogenic actions, leading to an increase in UV rays reaching the
Earth’s surface.
Plants and animals both undergo morphological, anatomical, physiological and
biochemical changes following exposure to UV rays. Among the changes caused by
UV-B radiation are inhibition of growth, increased thickness of leaf blades, reduced
biomass, damage to the photosynthesis apparatus, altered membrane composition and
modifications to the balance of phytohormones (Zagoskina et al., 2003). Structural
damage to the DNA is also observed, reflected in the formation of cyclobutane
pyrimidine dimers (CDPs) and pyrimidine-(6-4)-pyrimidone (Yamasaki et al., 2007).
Thus, to protect themselves from UV damage, plants produce mechanical
barriers, such as thicker cuticles and trichomes; they also synthesize protective
substances, such as secondary compounds (Yamasaki et al., 2007). UV-B affects the
production of several secondary compounds such as flavonoids, tannins and lignin,
which are thought to have played an important role in the transition of aquatic plants to
land, when they became more exposed to UV-B radiation (Rozema et al., 1997).
UV-B radiation affected the morphogenesis of trichomes in the cotyledons of
Cucumis sativus L. and induced the accumulation of lignin in these structures
(Yamasaki et al., 2007). In a study with two types of Camellia sinensis L. cell cultures
that differed in their capacity to biosynthesize the phenolic compounds ChS1 and ChS-
2, the latter had greater capacity for production of phenolic compounds (Zagoskina et
al., 2003). It was observed that under exposure to UV-B radiation, ChS2 demonstrated

13
274
275
276
277
278
279
280
281
282
283
284
greater accumulation of lignin in the cell walls of the parenchyma of tracheid elements
and in the intercellular space, in addition to the formation of a layer of lignin-likematerial
on the surface of the callus. The ChS2 lineage was also more resistant to UV-
B, suggesting the involvement of lignin in cell protection against this type of stress.
Cotyledons of Chenopodium quinoa exposed to UV-B radiation exhibited increased
epidermal cell wall thickness; this was associated with a large accumulation of lignin
due to increased peroxidase activity (Hilal et al., 2004). Additionally, changes in the
ultrastructure of chloroplasts rendered them similar to the chloroplasts of shaded plants.
It was suggested that such alterations in the chloroplast were caused by reduced light
penetration in the cells of the mesophyll, due to the protective effect of lignin
deposition. Therefore, lignin in epidermal tissue may attenuate radiation.
285
286
287
288
289
290
291
292
293
294
295
296
297
298
Mechanical injuries
Injuries induce specific reactions in the wood which can be restricted to the bark
or might extend to the cambium and xylem (Frankenstein et al., 2006). Several studies,
if not most of them, have focused on characterizing histological and anatomical wood
changes produced by a particular type of injury, and the obtained information is then
used to explain the roles of these changes in adaptation and resistance to stress. In
general, attention has been paid to the changes occurring after insect attack or after
microorganism infection. Some species have defence mechanisms that range from
physical barriers including cuticle formation, lignification, spines and trichomes to the
biosynthesis of toxic compounds such as alkaloids and tannins (Delessert et al., 2004).
Electron microscopy has demonstrated that injuries to the stem of Populus spp.
induce the thickening of the xylem fibre cell wall in three ways: synthesis of an
additional S2 layer, thickening of the existing cell wall or synthesis of a sclereid-like

14
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
sublayer (Frankenstein et al., 2006). The authors also showed, from the UVmicrospectrophotometric
determinations, that cell walls of these xylem modified-fibres
had a higher content of lignin and uneven distribution of this polymer in the middle
lamella and secondary wall. They concluded that the observed changes might enable
these plants to resist stress.
Histochemical (phloroglucinol-HCL) and quantitative (acetyl bromide) studies
on the accumulation of lignin and ferulic acid in the phloem of Chamaecyparis obtusa
exposed to injury (injury of the bark near the base of the stem) reported the formation of
a parenchymal zone and induction of lignification in the necrotic phloem (Kusumoto,
2005) after 7 days. The formation of a ligno-suberized layer between the necrotic tissue
and the parenchymatous area was observed after 14 days. It was also observed the
formation of a callus (due to hyperplasia of cells in the parenchyma and cambium),
which became lignified. The concentration of lignin measured in the cell walls of
necrotic tissues was significantly higher at 28 and 56 days after injury. The
concentration of ferulic acid was significantly higher from the seventh day after injury,
which suggests that the accumulation of ferulic acid occurred before the accumulation
of lignin.
Given the complexity of responses to injury, it is not surprising that genes of
different metabolic pathways are induced. Thus, various studies have aimed at
identifying which genes are involved in this response and many have already
demonstrated the induction of genes related to lignin biosynthesis. The expression of
genes of the At4CL family (At4CL1-i), which codes for the enzyme 4CL, was studied in
Arabidopsis thaliana in response to injury caused by perforation of the leaf with the tip
of a pipette (Soltani et al., 2006). 4CL catalyzes the conversion of hydroxycinnamic
acids in CoA esters, which are subsequently reduced to the corresponding monolignol

15
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
precursors of lignin. The results showed increased expression of At4CL1 and At4CL2
during two distinct phases, at 2.5 hours (early phase) and 48-72 hours (late) after injury.
The level of At4CL4 transcripts also increased during the first 2.5 hours, but was later
reduced to lower levels. The expression of At4CL3 was rapidly reduced in response to
injury, and returned to initial levels after 4 hours; from that point on, expression
gradually increased. The construction of vectors containing At4CL::GUS showed that
At4CL1::GUS and At4CL2::GUS were specific to the vascular regions of the root and
aerial parts of the plant, unlike AT4CL3::GUS and At4CL4::GUS.
The expression of CaF5H1, which codes for F5H, increased in detached leaf
discs of Camptotheca acuminata plants (Kim et al., 2006). F5H is a cytochrome P450-
dependent monooxygenase that catalyzes the hydroxylation of ferulic acid,
coniferaldehyde and coniferyl alcohol, promoting the biosynthesis of syringyl lignin.
The results suggested that such a response represented protection from mechanical
stress.
As a strategy to avoid pathogen infection and water loss, injured leaves of
Arabidopsis thaliana showed increased expression of genes related to lignin
biosynthesis, coding for the enzymes 4CL, CAD and CCR (Delessert et al., 2004). It
was suggested that lignin was formed in the cells surrounding the wound site.
The removal of the stem apex in Eucalyptus gunnii caused an increase in the
activities of CAD and SAD, enzymes involved in the final steps of lignin biosynthesis
(Hawkins and Boudet, 2003). Microscopy and histochemical analyses also detected the
formation of a "barrier zone" where the cell walls exhibited a clear intensification of
lignin deposition. This lignin was poor in S units and differed from the usual quantity of
G-S that has been reported for this species.
348

16
349
350
351
352
353
354
355
356
357
358
Reaction wood: tension wood and compression wood
Reaction wood (RW) is a type of wood that occurs in leaning stems and
branches in order to force them into the normal position. Therefore, in some way
reaction wood may represent a stressing situation.
In angiosperms, RW develops above the leaning region, pulling it up: in this
context, it is called tension wood. In gymnosperms, this wood develops below the
leaning region and it is called compression wood; it pushes the plant up (Paux et al.,
2005). The wood reaction involves a marked reprogramming of the genes involved in
cell wall formation and therefore significantly affects the properties of the wood
(Déjardin et al., 2004).
359
360
361
362
363
364
365
366
367
368
369
370
371
372
Tension wood: A wide variety of characteristics are observed in the tension wood (TW)
of various angiosperms, but the most common type of TW is characterized by having
fewer xylem vessel elements, which have a smaller diameter and a higher proportion of
fibres with thick walls, more cellulose and less lignin (Wilson and White, 1986). In
addition, TW fibres often develop an additional internal layer of the cell wall called the
gelatinous layer or G-layer, with higher amounts of cellulose, low microfibril angles
(MFA: angle that the cellulose microfibrils form with the longitudinal axis of the fibre)
and low levels of lignin (Qiu et al., 2008). Normal wood fibres contain a middle
lamella, a thin primary wall and a thicker secondary wall, which is made of 3 sublayers:
S1, S2 and S3 (Clair et al., 2005). In different trees, TW contains fibres with a
special morphology and chemical composition due to the development of the G-layer,
which replaces the S2-layer and either partially or completely substitutes the S3-layer
(Clair et al., 2005).

17
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
The lower amount of lignin in TW has been attributed mainly to the reduction of
lignin in the G-layer and to changes in the relative proportions of monolignols. During
the development of TW in Eucalyptus viminalis, there is a reduction in the amount of
lignin and an increase in the level of S residues, increasing the ratio of syringyl/guaiacyl
monolignol units (Aoyama et al., 2001). Ultrastructural studies on the distribution of
lignin in cell walls of TW in Populus deltoides using polyclonal antibodies specific to
synthetic polymers of lignin showed that, in these plants, the additional G-layer contains
low amounts of guaiacyl lignin and greater amounts of syringyl lignin (Joseleau et al.,
2004). In contrast, changes in the proportion of monolignols in the lignin have not been
observed in the TW of different eucalyptus species (Rodrigues et al., 2001).
In species that do not have the G layer, the S2-layer of the cell wall has similar
characteristics to those found in the G-layer. Inclined branches of two angiosperm
species of the primitive genus Magnolia, which does not form a gelatinous layer, have
fewer and smaller vessel elements and no S3-layer in the cell walls of xylem tracheids
(Yoshizawa et al., 2000). As expected, the presence of a G-layer was not observed;
however, the more internal layer of secondary cell wall (S2-layer) showed
characteristics of a G-layer, with smaller cellulose microfibril angles. Additionally it
was noted that the amount of lignin stained by the Wiesner reaction was lower in TW
fibres as compared with regions of normal wood, suggesting that the amount of lignin
(mainly the guaiacyl type) would have been reduced. In Liriodendron tulipifera, which
does not form a G-layer in TW, it was observed a decrease in the lignin content of
secondary cell walls, while the syringyl/guaiacyl proportion increased. Additionally, it
was also observed an increase in the amount of cellulose and smaller cellulose
microfibril angles (Yoshida et al., 2002).

18
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
Eucalyptus nitens, which rarely exhibits a G-layer, was bent to 45 o . These plants
displayed a TW with high cellulose content, reduced microfibril angle, and less Klason
lignin (Qiu et al., 2008).
Laeta procera TW has a peculiar polylaminate secondary wall, formed by thin
lignified layers with small microfibril angles alternating with layers that are still thin but
exhibit greater lignification (Ruelle et al., 2007).
Most of the work on TW has focused on anatomical and biochemical
characterization of the wood, failing to consider the physiological and molecular
mechanisms involved. These studies refer only to changes in quantity or quality
(syringyl and guaiacyl) of lignin and do not explore which factors are responsible for
these changes in the lignifications process, whether hormones, proteins or genes.
Auxin was thought to be the main hormone involved in inducing the formation
of reaction wood (Déjardin et al., 2004). For years, experiments with exogenous
supplies of auxin and auxin transport inhibitors showed that TW can be induced by a
deficiency of auxin (Little and Savidge, 1987). However, reaction wood (from both
tension and compression) can be formed without obvious changes in the balance of
auxin. This finding indicated the importance of investigating other factors, such as
mechanisms of auxin perception or even other signalling molecules (Hellgren et al.,
2004). Ethylene appeared as a strong candidate since it was observed that this hormone
is distributed asymmetrically in the TW and that expression of a 1-aminocyclopropane-
1-carboxylate oxidase gene (PttACO1) controls the process (Andersson-Gunnerås et al.,
2003). In addition, there is a connection between ethylene and lignification, as observed
in the phenotype of Arabidopsis mutants, where a mutation in a chitinase gene causes
the ectopic expression of CCoAOMT, ectopic deposition of lignin and the
overproduction of ethylene (Zhong et al., 2002).

19
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
In addition to ethylene, gibberellins also seem to be related to the formation of
reaction wood. It was observed that the application of gibberellins to the stems of some
angiosperms induces the formation of wood with characteristics similar to tension
wood: fibres have inner layers similar to a G-layer, with high cellulose content, small
microfibril angles and low lignin content (Funada et al., 2008).
In addition to studies of plant hormones, important research has characterized
the proteins involved in reaction wood formation. SDS-PAGE comparisons of the
pattern of protein accumulation in TW and normal wood showed the presence of at least
five proteins induced during the formation of TW (Baba et al., 2000). The extraction
conditions used in the protocols led the authors to suggest that these five proteins were
cell wall- or membrane-bound proteins. Additionally, a study on peroxidase activity
showed that the increase in the S/G ratio, generally observed in TW, could be partly
attributed to the increased activity of a syringaldazine peroxidase ionically linked to the
cell wall (Aoyama et al., 2001).
The molecular mechanisms involved in the formation reaction wood and their
relationship to metabolomics data have been investigated. However, due to the
complexity of differential accumulation of lignin in TW, very little is known about the
genes involved in the process.
ESTs obtained from different wood tissues, among them TW and opposite wood
from the poplar (Populus tremula x P. alba), indicated the presence of some genes
specific to these tissues (Déjardin et al., 2004). In tissues under tension, ESTs related to
arabinogalactan protein-like (AGP), fructokinase and sucrose synthase were abundant.
In contrast, the opposite wood exhibited greater CCoA-OMT expression, which is
consistent with the high levels of lignin synthesis observed in this wood.

20
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
The increased expression of fasciclin-like arabinogalactan proteins was also
observed in G-fibres in another poplar hybrid (Populus tremula x P. tremuloides)
(Andersson-Gunneras et al., 2006). In addition, several genes involved in lignin
biosynthesis exhibited reduced expression in the TW: PttPAL1, PttCCoAOMT1, 4CL,
HCT, C3H, CCR, F5H, COMT and CAD. A MYB transcription factor (PttMYB21a)
previously shown to repress biosynthesis of lignin displayed elevated expression levels.
Branches of artificially inclined Eucalyptus globulus showed that the expression
of a cellulose synthase gene (EgCesA) was correlated with the appearance of the G-
layer (Paux et al., 2005). The lignin biosynthesis genes for which expression changed
during TW formation included: 4CL, CCR, CAD, COMT, and CCoAOMT. The response
of these genes was varied and complex, with different patterns of expression for each
gene. The 4CL, CCR and CAD genes exhibited reduced expression during the first 6 h
of stress but the transcript levels were similar to those observed in control plants after a
week. According to the authors, this variation could not explain the reduced amount of
lignin often observed in tension wood; the lower amount of lignin, mainly G-type, was
probably due to the differential expression of CCoAOMT and COMT, genes that encode
methylation enzymes.
463
464
465
466
467
468
469
Compression wood: Compression wood (CW) has small and rounded tracheids with
thicker cell walls, higher lignin content, less cellulose, large microfibril angles, and
typically lacks the S3 layer (McDougall, 2000; Donaldson et al., 2004; Yeh et al., 2005;
Yeh et al., 2006). Additionally, the lignin in this wood has high levels of p-
hydroxyphenylpropane units (Monties, 1989) and alterations in the chemical links
between lignin units, when compared with normal wood (Nimz et al., 1981).

21
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
As in TW, the chemical and anatomical characteristics of the CW are the results
of various altered molecular and physiological factors. Unravelling these factors will be
necessary to understand the mechanisms involved and elucidate lignin biosynthesis in
wood. Many studies have already identified proteins and genes potentially responsible
for the different characteristics of CW, including the high amount of lignin.
The comparison of proteins in CW and in the opposite normal wood showed that
the presence of a dioxygenase related to the biosynthesis of anthocyanins
(leucoanthocyanidin dioxygenase) might contribute to the colour of this reddish wood
(Gion et al., 2005).
Other proteins abundant in this type of wood are: 1-aminocyclopropane-1-
carboxylate oxidase, an enzyme that catalyzes the conversion of 1-aminocyclopropane-
1-carboxylate to ethylene; glutamine synthetase and fructokinase, enzymes involved in
the assimilation of nitrogen and carbon; malate dehydrogenase, an enzyme of the Krebs
cycle (Plomion et al., 2000); glyceraldehyde-3-phosphate dehydrogenase (G3PDH), an
enzyme related to energy production and metabolism; and α-tubulin, the main
component of cortical microtubules (Le Provost et al., 2003).
Other studies have reported on proteins possibly more directly involved in the
differential accumulation of lignin. A polypeptide corresponding to a laccase-type of
polyphenol oxidase, which is involved in the biosynthesis of lignin, has higher activity
in developing xylem in the CW of Picea sitchensis, as compared to non-compressed
wood (McDougall, 2000).
Twenty-six proteins whose expression was correlated with an increase in the
degree of compression were identified in Pinus pinaster, including two methylating
enzymes involved in lignin biosynthesis - COMT and CCoAOMT – and members of the
S-adenosyl-L-methionine synthetase gene family. In addition to being enzymes

22
495
496
497
498
499
500
501
involved in the response to ethylene, this gene family also plays a role in the
methylation of monolignols during the biosynthesis of lignin (Plomion et al., 2000).
Sequenced cDNAs from CW and normal wood of Pinus taeda showed that the
genes that were most abundant in the biosynthesis of lignin code for the enzymes PAL,
C4H, OMT, 4CL, CAD, diphenol oxidase (laccase) and POD (Allona et al., 1998).
The R2R3-MYB transcription factors, which have been associated with the
biosynthesis of lignin, also display elevated expression in CW (Bedon et al., 2007).
502
503
504
505
506
507
508
Mineral nutrition and heavy metals
Deficiency disability and abnormally high concentrations of a nutrient can both
cause abnormalities in the accumulation of lignin, where this has been documented by
many studies. Most of these, however, do not include a study of the physiological or
molecular causes of their findings. Recent reports have also shown that lignin content
may be affected by toxic heavy metals.
509
510
511
512
513
514
515
516
517
Nitrogen (N): Few studies were conducted to assess the impact of fertilization with N
on the properties of wood (Luo et al., 2005; Liberloo et al., 2006); most of these
investigated grasses used as forage for animal feeding, showing that lignin content was
increased by N due to elevated PAL activity (Pitre et al., 2007).
Moreover, the effect of N seems to vary with the type, degree of development
and tissue examined. In pine (Pinus palustris) seedlings, high-N fertilization reduced
the lignin content in roots but had no effect on the lignin concentration in aerial parts of
the plant (Entry et al., 1998).

23
518
519
520
521
522
523
524
525
526
In red pine (Pinus resinosa), combined nitrogen-phosphorus-potassium (NPK)
fertilization reduced the lignin content of branches (Blodgett et al., 2005) but had an
opposite effect on the main stem of Picea abies (Kostiainen et al., 2004).
Populus plants receiving high concentrations of nitrogen (10 mM NH 4 NO 3 ) had
less lignin, reduced β-O-4 linkage, increased frequency of ρ-hydroxyphenyl units and a
tendency toward low S/G ratios (Pitre et al., 2007).
At the molecular level, the expression of the pot171 gene, which encodes a
protein similar to CCoAOMT, is negatively regulated in response to nitrogen in Populus
trichocarpa x deltoid hybrids (Cooke et al., 2003).
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
Calcium (Ca): The effect of Ca on the biosynthesis of phenylpropanoids is not
sufficiently clear. Some studies have shown that Ca increases the activity of guaiacol-
POD and PAL (Castañeda and Pérez, 1996; Kolupaev et al., 2005); however, a
reduction in the amount of phenolic compounds has also been reported (de Obeso et al.,
2003). Other authors observed a reduction in PAL and POD activity (soluble and cell
wall-bound), as well as reductions in the levels of phenolic compounds, including lignin
(Teixeira et al., 2006). No substantial changes in the amounts of these compounds was
observed (Tomas-Barberan et al., 1997).
In Populus tremula x Populus tremuloides, Ca deficiency was responsible for
wood deformation, which included smaller diameter of xylem vessels, shorter fibre
length and reduced levels of S units in lignin (Lautner et al., 2007). Therefore, it seems
that Ca is necessary for the biosynthesis of lignin in plants. It is important to note that
several PODs are linked to the galacturonic domains of pectin in the presence of Ca
(Penel and Greppin, 1996) and such a link occurs only in the pectin chain that is crosslinked
to Ca, known as Ca-pectates (Carpin et al., 2001). Since the middle lamella and

24
543
544
545
546
cell corners are rich in Ca-pectates (Carpita and Gibeaut, 1993) and are the first places
to be lignified, POD linked to these chains of Ca-pectates might play a role in
controlling the spatial deposition of lignin, and changes in the concentrations of Ca
would define the location of these PODs (Carpin et al., 2001).
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
Copper (Cu): It is known that copper has a positive effect on the biosynthesis of lignin.
Thus, Cu deficiency results in less lignin (Robson et al., 1981), and excess Cu results in
more lignin.
Capsicum annuum hypocotyls grown in a nutrient solution with excess copper
(50 mM CuSO 4 ) showed higher levels of shikimate dehydrogenase and POD activity
and greater accumulation of soluble phenolic compounds and lignin (Diaz et al., 2001).
Roots of Raphanus sativus grown in the presence of Cu (1-10 mM CuSO 4 ) showed
higher levels of cationic and anionic POD activities and higher levels of lignin in
comparison with plants grown in the absence of this element; the increases were dosedependent
(Chen et al., 2002).
Laccases were responsible for the extracellular polymerization of monolignols in
the roots of soybean plants during the early stages of Cu exposure. Later, POD activity
increases, working cooperatively with laccases in the biosynthesis of lignin (Barnes et
al., 1997).
Exposure of Panax ginseng root suspension cultures to increasing concentrations
of Cu led to an enhancement of the activities of glucose-6-phosphate dehydrogenase,
shikimate dehydrogenase, PAL and CAD and to the accumulation of phenolic
compounds and lignin. It also caused an increase in levels of caffeic acid-POD,
polyphenol oxidase and β-glucosidase (Akgül et al., 2007).

25
567
568
569
570
571
572
573
574
575
576
577
578
Thus, lignin accumulates in plant cell walls in the presence of Cu. This process
is correlated with enhanced activities of several enzymes, including POD and laccases,
which are enzymes that carry out the polymerization of monolignol precursors of lignin.
This could be partly explained by the fact that Cu is structurally important for laccases
(Claus, 2004). It appears also that Cu has functional importance in terms of peroxidase
activity, since an amine oxidase containing Cu that generates H 2 O 2 by oxidising
putrescine has been co-located with lignin and with POD activity in tracheid elements
of xylem in Arabidopsis (Møller and McPherson, 1998).
However, Cu also mediates an increase in the activity of other enzymes of the
lignin biosynthesis pathway, such as PAL and CAD. This could be an indirect effect of
this metal, which enhances oxidation and polymerization of monolignols via laccases;
peroxidase would stimulate (by feedback) the synthesis of these monolignols.
579
580
581
582
583
584
585
586
587
588
Boron and Zinc: Boric acid at 5 mM increased the activities of PAL and
syringaldazine-POD and increased the lignin content in soybean (Ghanati et al., 2005).
At high concentrations of Zn, both A. thaliana and Thlaspi caeru lescens showed
increased expression of genes related to the biosynthesis of lignin (van de Mortel et al.,
2006). However, in T. Caerulescens, a Zn hyper-accumulator, the expression of these
genes was even greater. This could be related to the ability of this species to adapt to
higher concentrations of Zn. Among the genes that had higher expression in T.
caerulescens than in A. thaliana were genes related to dirigent proteins, 4CL, CCR,
F5H, CAD, CCoAOMT and laccase.
589
590
591
Metals - Aluminium (Al) and Cadmium: Al is one of the main factors inhibiting plant
growth in acidic tropical soils. A typical symptom of Al toxicity is the inhibition of root

26
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
growth (Tahara et al., 2005). Excess of Al causes increased lignin deposition in the cell
walls of some plant species (Sasaki et al., 1996; Budíková, 1999). In plants susceptible
to Al, lignin accumulation is even higher, dramatically reducing growth; growth
inhibition is strongly correlated with the extent to which lignin is deposited in the cell
wall, independently of the tolerance to this metal (Sasaki et al., 1996).
Among several species of Myrtaceae, only the most sensitive to Al shows
increased lignin content when exposed to high concentrations of aluminium (Tahara et
al., 2005).
Camellia sinensis is know to be highly tolerant to Al, and its growth is
stimulated by high concentrations of this element (Lima et al., 2009). When this species
was grown in high concentrations of Al, there was a reduction in the activities of PAL
and POD bound to the cell wall, as well as a reduction in lignin content, which might
explain enhanced growth of these plants in the presence of Al (Ghanati et al., 2005).
It was also demonstrated that Al induces the expression of genes coding for
enzymes of the lignin biosynthesis pathway. Both resistant and sensitive rice (Oriza
sativa) plants under Al stress exhibited increased expression of 4CL, PAL, CAD and
C3H (Chuanzao et al., 2004). Northern blot analysis indicated that the temporal patterns
of expression differ between the two varieties and, additionally, that transcripts of PAL
accumulated more intensively in the sensitive variety. Al also induces expression of
PAL in wheat (Snowden and Gardner, 1993).
Cd is a heavy metal pollutant, not essential for plants, which enters the
environment primarily through industrial processes and phosphate fertilizers. Among its
many effects on plants, Cd may induce the synthesis of lignin. This was observed in
roots of Phragmites australis (Ederli et al., 2004) and callus tissue culture from the
roots and branches of C. sinensis (Zagoskina et al., 2007).

27
617
618
619
620
621
622
Cd concentrations varying from 0.2 mM to 1 mM reduced the growth of soybean
roots and increased the lignin content (Bhuiyan et al., 2007). This was accompanied by
increased POD (cationic and anionic) activity, elevated laccase activity, and increased
expression of genes related to POD. The authors also noted that the laccases are
responsible for the biosynthesis of lignin during the initial stages of treatment with Cd
and that POD participation become important at later stages.
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
Gases (CO 2 and Ozone)
It is expected that atmospheric concentrations of CO 2 will change from 360
μmol -1 to 550-1000 μmol -1 in the next 100 years. This will probably interfere with the
primary and secondary metabolism of plants (Davey et al., 2004).
With respect to secondary metabolism, it is known that high concentrations of
CO 2 interfere with the biosynthesis of lignin. But it has been observed both increases in
(Koricheva et al., 1998; Richard et al., 2001), reduction in (Knops et al., 2007) or even
no effect on the concentration of lignin (Cotrufo et al., 2005) with increasing rates of
CO 2 . Moreover, the response to high CO 2 concentrations is influenced by fertilization
with N (Matros et al., 2006). Accordingly, high concentrations of CO 2 in the roots of
Nicotiana tabacum cv. Samsun-NN grown in 5 mM of NH 4 NO 3 led to an increase in
lignin content; however, under 8 mM of NH 4 NO 3 , no changes in the levels of lignin
were observed (Matros et al., 2006).
Plantago maritima maintained for a year in an atmosphere of 600 μmol CO 2
638
mol -1
showed an increase in the biomass of stems and roots and changes in
639
640
vascularization and lignification when compared with plants kept in normal
concentrations of CO 2 (Davey et al., 2004). An atmosphere rich in CO 2 resulted in a

28
641
642
643
644
645
646
647
648
higher concentration of caffeic acid in the leaves, while there was an increase of p-
coumaric acid in the roots.
Both the enzyme activity and gene expression of PAL (Paakkonen et al., 1998),
4CL (Booker and Miller, 1998), COMT (Koch et al., 1998) and CAD (Booker and
Miller, 1998; Zinser et al., 1998) are stimulated by exposure to ozone. It was also
observed increased shikimate dehydrogenase activity in leaves of Populus tremula X
alba, as well as elevated lignin content and changes in lignin structure (Cabane et al.,
2004).
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
Biotic stresses (bacteria, fungi and virus)
An increase in lignification is often observed in response to attack by pathogen.
The response is believed to represent one of a plethora of mechanisms designed to block
parasite invasion, thus reducing the susceptibility of the host, since lignin is a nondegradable
mechanical barrier for most microorganisms.
Two genes coding for CCR (AtCCR1 and AtCCR2) in A. thaliana are
differentially expressed upon infection with Xanthomonas campestris. AtCCR1 is
preferentially expressed in the normal development of tissues, while AtCCR2 shows
lower expression. However, this relation is reversed in response to the attack by X.
Campestris, indicating that these genes might participate in the hypersensitive response
to the pathogen.
Agrobacterium induces the production of ferulic acid in wheat. Ferulic acid is a
precursor in lignin biosynthesis, suggesting the existence of a defence response
preventing infection by the bacteria in these plants (Parrott et al., 2002).
No changes occurred in the lignin content of wheat leaves infected with mosaic
virus (Kofalvi and Nassuth, 1995).

29
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
Several authors have shown changes in lignin content due to attack by fungi, but
not all of these studies have sufficiently demonstrated the importance of lignin during
these processes. In addition, fewer still are the works that analyze the changes in plant
metabolism and gene expression responsible for this differential accumulation of lignin.
The infection of Pinus nigra by Sphaeropsis sapinea induces an increase in the
deposition of lignin (Bonello and Blodgett, 2003). Lignification is a defence response in
the hypersensitive reaction of wheat to Puccinia graminis (Moerschbacher et al., 1990).
During such a response in wheat, lignin rich in syringyl units accumulates (Menden et
al., 2007).
The deposition of lignin in necrophylatic periderm in the early stages of
infection by Mycosphaerella explains the greater resistance of E. nitens as compared to
E. globulus, as this response may prevent the spread of toxins and fungal enzymes to the
host, thereby preventing the displacement of water and nutrients from the host cell to
the fungus (Smith et al., 2007).
Metabolic studies on the changes in the xylem tissues of Ulmus minor and
Ulmus minor x Ulmus pumila after inoculation with Ophiostomis new-ulmi showed that
the hybrid has a faster defence response, which is characterized by an increase in the
amount of lignin, diminishing the probability of pathogen invasion (Martin et al., 2007).
Regarding physiological and molecular aspects, the differential accumulation of
lignin and lignans in cell suspension cultures of Linum usitatissimum was studied, in
response to Botrytis cinerea, Fusarium oxysporum and Phoma exigua mycelium
extracts (Hano et al., 2006). In this study, genes coding for PAL, CCR and CAD
showed elevated expression; PAL activity was enhanced as well.
Finally, COMT expression in diploid wheat (Triticum monococcum) is elevated
following inoculation with Blumeria graminis (Bhuiyan et al., 2007).

30
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
Concluding Remarks
Figure 1 shows the main route of lignin biosynthesis in plants and the enzymes
involved in each metabolic step (adapted from Rastogi and Dwivedi, 2008). For most of
the genes coding for these enzymes, transgenic plants have been produced using genesilencing
techniques (Anterola and Lewis, 2002). The aim of these works was to reduce
lignin content or to change the structure of lignin, goals which have economical and
environmental implications: cheaper and more process-amenable trees for pulp and
paper manufacture with less pollution, more readily digestible forage for livestock and
improved feedstock for fuel/chemical production (Anterola and Lewis, 2002).
The potential applications of changing or silencing the expression of these genes
were comprehensively discussed by Anterola and Lewis (2002). Some questions
discussed by these authors were, “why do vascular plants consistently produce various
types of cell walls with lignins from either two or three monolignols, and how does this
occur in vivo? Why are these moieties also differentially deposited into specific regions
of developing cell wall types?” Despite the complexity of the lignin biosynthesis
network, Anterola and Lewis (2002) used accumulated knowledge obtained with
mutants or transgenic plants (in which levels of particular enzymes of the pathway have
been modified in some way) to show that monolignol biosynthesis is under fine
metabolic and transcriptional coordination.
For several of these genes, repressed expression led to diverse and undesirable
impacts on plant physiology due to the multiple products that originate from the
phenylpropanoid pathway. Therefore, the PAL, C4H, C3H and 4CL genes (see Figure 1)
are not good targets for genetic manipulation aiming to reduce lignin, while CCoAOMT,

31
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
COMT and CAD genes can be used to reduce and change lignin composition (Rastogi
and Dwivedi, 2008).
The biosynthesis of monolignols in phenylpropanoid metabolism is a metabolic
grid; that is, different routes may be used to synthesize a compound. This gives plants
the plasticity to produce lignin when a step is inhibited for whatever reason such as
when a specific gene is silenced. In addition, enzymes related to lignin biosynthesis
usually derive from multigene families; depending on the stressing situation (biotic or
abiotic), different genes for different isozymes can be activated. For the sake of
complexity, it is also well known that the relative proportions of the monolignols may
vary due to environmental influence (Boudet, 1998). It has been suggested that plants
are versatile in their ability to tolerate substantial changes in lignin structure and
incorporate phenolic compounds other than the three known monolignols (Ralph et al.,
2004). Recently, it was shown that acylated monolignols could also serve as lignin
precursors (Lu and Ralph, 2003). This can make lignin more recalcitrant to disruption
during pulping in the paper industry, for example (Lapierre et al., 1999). However, it
does not appear that “non-traditional” phenolics, other than the monolignols, are
incorporated into lignin (Anterola and Lewis, 2002).
There are several examples indicating that lignin found in nature may comprise
modified molecules (Lu et al., 2004; Morreel et al., 2004). Phenolic amides may also be
part of the cell wall, as proven by over-expression of the enzyme responsible for their
biosynthesis, which caused an increase in their incorporation into the cell wall matrix
(Hagel and Facchini, 2005). Recent research also showed that acetylation of syringyl
units seems to be an exclusive characteristic of angiosperms, as the process was
observed among 11 angiosperm species but not in 2 gymnosperm species (Del Rio et
al., 2007).

32
740
741
742
743
744
745
746
747
748
This review showed that lignin biosynthesis in nature can increase complexity
depending on the stresses and whether the stressor is acting during plant growth and
development. Unfortunately, little has been done to elucidate how such stressing
situations can change lignin content or alter its composition with regard to the building
blocks called monolignols, or other phenolics entering the polymeric structure. Reaction
wood has proven to be an important model for understanding lignin formation in plants
(Mellerowicz and Sundberg, 2008). Other stresses may also become excellent models
that will yield important clues for understanding such complex metabolic pathways, as
well as the intricacies of genetic and metabolic control.
749
750
751
752
Acknowledgments
The authors (J.C.M.S.M., M.C.D. and P.M.) thank CNPq for research fellowships.
Votorantim Celulose e Papel S.A. is acknowledged for financial support.
753
754
References
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
Akgül M, Çöpür Y, Temiz S (2007). A comparison of kraft and kraft-sodium
borohydrate brutia pine pulps. Building and Environment, 42, 2586-2590.
Allona I, Quinn M, Shoop E, Swope K, St Cyr S, Carlis J, Riedl J, Retzel E,
Campbell MM, Sederoff R, Whetten RW (1998). Analysis of xylem
formation in pine by cDNA sequencing. Proceedings of the National Academy
of Sciences of the United States of America, 95, 9693-9698.
Alvarez S, Marsh EL, Schroeder SG, Schachtman DP (2008). Metabolomic and
proteomic changes in the xylem sap of maize under drought. Plant Cell and
Environment, 31, 325-340.
Andersson-Gunnerås S, Hellgren JM, Björklund S, Regan S, Moritz T, Sundberg
B (2003). Asymmetric expression of a poplar ACC oxidase controls ethylene
production during gravitational induction of tension wood. Plant Journal, 34,
339-349.
Andersson-Gunneras S, Mellerowicz EJ, Love J, Segerman B, Ohmiya Y,
Coutinho PM, Nilsson P, Henrissat B, Moritz T, Sundberg B (2006).
Biosynthesis of cellulose-enriched tension wood in Populus tremula: global
analysis of transcripts and metabolites identifies biochemical and developmental

33
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
820
821
822
regulators in secondary wall biosynthesis.(vol 45, pg 144, 2005). Plant Journal,
46, 349-349.
Anterola AM, Lewis NG (2002). Trends in lignin modification: a comprehensive
analysis of the effects of genetic manipulations/mutations on lignification and
vascular integrity Phytochemistry, 61, 221-294.
Aoyama W, Matsumura A, Tsutsumi Y, Nishida T (2001). Lignification and
peroxidase in tension wood of Eucalyptus viminalis seedlings. Journal of Wood
Science, 47, 419-424.
Atici O, Nalbantoglu B (2003). Antifreeze proteins in higher plants. Phytochemistry,
64, 1187-1196.
Baba Ki, Asada T, Hayashi T (2000). Relation between developmental changes on
anatomical structure and on protein pattern in differentiating xylem of tension
wood. Journal of Wood Science, 46, 1-7.
Barnes JD, Bettarini I, Polle A, Slee N, Raines C, Miglietta F, Raschi A, Fordham
M (1997). The impact of elevated CO2 on growth and photosynthesis in
Agrostis canina L. ssp. monteluccii adapted to contrasting atmospheric CO 2
concentrations. Oecologia, 110, 169-178.
Bedon F, Grima-Pettenati J, Mackay J (2007). Conifer R2R3-MYB transcription
factors: sequence analyses and gene expression in wood-forming tissues of white
spruce (Picea glauca). BMC Plant Biology, 7.
Bhuiyan N, Liu W, Liu G, Selvaraj G, Wei Y, King J (2007). Transcriptional
regulation of genes involved in the pathways of biosynthesis and supply of
methyl units in response to powdery mildew attack and abiotic stresses in wheat.
Plant Molecular Biology, 64, 305-318.
Blodgett JT, Herms DA, Bonello P (2005). Effects of fertilization on red pine defense
chemistry and resistance to Sphaeropsis sapinea. Forest Ecology and
Management, 208, 373-382.
Boerjan W, Ralph J, Baucher M (2003). Lignin biosynthesis. Annual Review of Plant
Biology, 54, 519-546.
Bok-Rye L, Kil-Yong K, Woo-Jin J, Jean-Christophe A, Alain O, Tae-Hwan K
(2007). Peroxidases and lignification in relation to the intensity of water-deficit
stress in white clover (Trifolium repens L.). Journal of Experimental Botany, 58,
1271-1271.
Bonello P, Blodgett JT (2003). Pinus nigra - Sphaeropsis sapinea as a model
pathosystem to investigate local and systemic effects of fungal infection of
pines. Physiological and Molecular Plant Pathology, 63, 249-261.
Booker F, Miller J (1998). Phenylpropanoid metabolism and phenolic composition of
soybean [Glycine max (L.) Merr.] leaves following exposure to ozone. Journal
of Experimental Botany, 49, 1191-1202.
Boudet AM (1998). A new view of lignification Trends in Plant Science, 3, 67-71.
Boudet AM (2000). Lignins and lignification: selected issues. Plant Physiology and
Biochemistry, 38, 81-96.
Budíková S (1999). Structural changes and aluminium distribution in maize root
tissues. Biologia Plantarum, 42, 259-266.
Cabane M, Pireaux JC, Leger E, Weber E, Dizengremel P, Pollet B, Lapierre C
(2004). Condensed lignins are synthesized in poplar leaves exposed to ozone.
Plant Physiology, 134, 586-594.
Carpin S, Crevecoeur M, de Meyer M, Simon P, Greppin H, Penel C (2001).
Identification of a Ca2+-pectate binding site on an apoplastic peroxidase. Plant
Cell, 13, 511-520.

34
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
858
859
860
861
862
863
864
865
866
867
868
869
870
871
872
Carpita NC, Gibeaut DM (1993). Structural models of primary-cell walls in flowering
plants - consistency of molecular-structure with the physical-properties of the
walls during growth. Plant Journal, 3, 1-30.
Castañeda P, Pérez LM (1996). Calcium ions promote the response of Citrus limon
against fungal elicitors or wounding. Phytochemistry, 42, 595-598.
Chen EL, Chen YA, Chen LM, Liu ZH (2002). Effect of copper on peroxidase
activity and lignin content in Raphanus sativus. Plant Physiology and
Biochemistry, 40, 439-444.
Chinnusamy V, Zhu J, Zhu J-K (2007). Cold stress regulation of gene expression in
plants. Trends in Plant Science, 12, 444-451.
Chuanzao M, Keke Y, Ling Y, Bingsong Z, Yunrong W, Feiyan L, Ping W (2004).
Identification of aluminium-regulated genes by cDNA-AFLP in rice (Oryza
sativa L.): aluminium-regulated genes for the metabolism of cell wall
components. Journal of Experimental Botany, 55, 137-143.
Clair B, Gril J, Baba K, Thibaut B, Sugiyama J (2005). Precautions for the structural
analysis of the gelatinuous layer in tension wood. IAWA Journal, 26, 189-195.
Claus H (2004). Laccases: structure, reactions, distribution. Micron, 35, 93-96.
Cooke JEK, Brown KA, Wu R, Davis JM (2003). Gene expression associated with N-
induced shifts in resource allocation in poplar. Plant Cell Environment, 26, 757-
770.
Cotrufo MF, DeAngelis P, Polle A (2005). Leaf litter production and decomposition in
a poplar short-rotation coppice exposed to free air CO 2 enrichment (POPFACE)
Global Change Biology, 11, 971-982.
Davey MP, Bryant DN, Cummins I, Ashenden TW, Gates P, Baxter R, Edwards R
(2004). Effects of elevated CO 2 on the vasculature and phenolic secondary
metabolism of Plantago maritima. Phytochemistry, 65, 2197-2204.
de Obeso M, Caparros-Ruiz D, Vignols F, Puigdomenech P, Rigau J (2003).
Characterisation of maize peroxidases having differential patterns of mRNA
accumulation in relation to lignifying tissues. Gene, 309, 23-33.
Déjardin A, Leplé JC, Lesage-Descauses MC, Costa G, Pilate G (2004). Expressed
sequence tags from poplar wood tissues - a comparative analysis from multiple
libraries. Plant Biology, 6, 55-64.
Del Rio JC, Marques G, Rencoret J, Martinez AT, Gutierrez A (2007). Occurrence
of naturally acetylated lignin units. Journal of Agricultural and Food Chemistry,
55, 5461-5468.
Delessert C, Wilson I, Van Der Straeten D, Dennis E, Dolferus R (2004). Spatial and
temporal analysis of the local response to wounding. Plant Molecular Biology,
55, 165-181.
Diaz J, Bernal A, Pomar F, Merino F (2001). Induction of shikimate dehydrogenase
and peroxidase in pepper (Capsicum annuum L.) seedlings in response to copper
stress and its relation to lignification. Plant Science, 161, 179-188.
Dixon RA, Paiva NL (1995). Stress-induced phenylpropanoid metabolism. Plant Cell,
7, 1085-1097.
Donaldson LA, Grace J, Downes GM (2004). Within-tree variation in anatomical
properties of compression wood in radiata pine. Iawa Journal, 25, 253-271.
Ederli L, Reale L, Ferranti F, Pasqualini S (2004). Responses induced by high
concentration of cadmium in Phragmites australis roots. Physiologia Plantarum,
121, 66-74.
El Kayal W, Keller G, Debayles C, Kumar R, Weier D, Teulieres C, Marque C
(2006). Regulation of tocopherol biosynthesis through transcriptional control of

35
873
874
875
876
877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
913
914
915
916
917
918
919
920
tocopherol cyclase during cold hardening in Eucalyptus gunnii. Physiologia
Plantarum, 126, 212-223.
Entry JA, Runion GB, Prior SA, Mitchell RJ, Rogers HH (1998). Influence of CO 2
enrichment and nitrogen fertilization on tissue chemistry and carbon allocation
in longleaf pine seedlings. Plant and Soil, 200, 3-11.
Fan L, Linker R, Gepstein S, Tanimoto E, Yamamoto R, Neumann PM (2006).
Progressive inhibition by water deficit of cell wall extensibility and growth
along the elongation zone of maize roots is related to increased lignin
metabolism and progressive stelar accumulation of wall phenolics. Plant
Physiology, 140, 603-612.
Ford CW, Morrison IM, Wilson JR (1979). Temperature effects on lignin,
hemicellulose and cellulose in tropical and temperate grasses. Australian
Journal of Agricultural Research, 30, 621-633.
Frankenstein C, Schmitt U, Koch G (2006). Topochemical studies on modified lignin
distribution in the xylem of poplar (Populus spp.) after wounding. Annals of
Botany, 97, 195-204.
Funada R, Miura T, Shimizu Y, Kinase T, Nakaba S, Kubo T, Sano Y (2008).
Gibberellin-induced formation of tension wood in angiosperm trees. Planta, 227,
1409-1414.
Ghanati F, Morita A, Yokota H (2005). Deposition of suberin in roots of soybean
induced by excess boron. Plant Science, 168, 397-405.
Gindl W, Grabner M, Wimmer R (2000). The influence of temperature on latewood
lignin content in treeline Norway spruce compared with maximum density and
ring width. Trees - Structure and Function, 14, 409-414.
Gion JM, Lalanne C, Le Provost G, Ferry-Dumazet H, Paiva J, Chaumeil P,
Frigerio JM, Brach J, Barre A, de Daruvar A, Claverol S, Bonneu M,
Sommerer N, Negroni L, Plomion C (2005). The proteome of maritime pine
wood forming tissue. Proteomics, 5, 3731-3751.
Gratão P, Polle A, Lea P, Azevedo R (2005). Making the life of heavy metal-stressed
plants a little easier. Functional Plant Biology, 32, 481-494.
Hagel JM, Facchini PJ (2005). Elevated tyrosine decarboxylase and tyramine
hydroxycinnamoyltransferase levels increase wound-induced tyramine-derived
hydroxycinnamic acid amide accumulation in transgenic tobacco leaves. Planta,
221, 904-914.
Hannah MA, Heyer AG, Hincha DK (2005). A global survey of gene regulation
during cold acclimation in Arabidopsis thaliana. PLoS Genetics, 1, e26.
Hano C, Addi M, Bensaddek L, Crônier D, Baltora-Rosset S, Doussot J, Maury S,
Mesnard F, Chabbert B, Hawkins S, Lainé E, Lamblin F (2006). Differential
accumulation of monolignol-derived compounds in elicited flax (Linum
usitatissimum) cell suspension cultures. Planta, 223, 975-989.
Hatfield R, Fukushima RS (2005). Can lignin be accurately measured? Crop Science,
45, 832-839.
Hausman JF, Evers D, Thiellement H, Jouve L (2000). Compared responses of
poplar cuttings and in vitro raised shoots to short-term chilling treatments. Plant
Cell Reports, 19, 954-960.
Hawkins S, Boudet A (2003). 'Defence lignin' and hydroxycinnamyl alcohol
dehydrogenase activities in wounded Eucalyptus gunnii. European Journal of
Forest Pathology, 33, 91-104.

36
921
922
923
924
925
926
927
928
929
930
931
932
933
934
935
936
937
938
939
940
941
942
943
944
945
946
947
948
949
950
951
952
953
954
955
956
957
958
959
960
961
962
963
964
965
966
967
968
969
970
Hellgren JM, Olofsson K, Sundberg B (2004). Patterns of auxin distribution during
gravitational induction of reaction wood in poplar and pine. Plant Physiology,
135, 212-220.
Hilal M, Parrado MF, Rosa M, Gallardo M, Orce L, Marta Massa E, González JA,
Prado FE (2004). Epidermal lignin deposition in quinoa cotyledons in response
to UV-B radiation. Photochemistry and Photobiology, 79, 205-210.
Huang G, Shi JX, Langrish TAG (2007). A new pulping process for wheat straw to
reduce problems with the discharge of black liquor. BioResource Technology,
98, 2829-2835.
Jain R, Shrivastava A, Solomon S, Yadav R (2007). Low temperature stress-induced
biochemical changes affect stubble bud sprouting in sugarcane ( Saccharum spp.
hybrid). Plant Growth Regulation, 53, 17-23.
Janas KM, Cvikrova M, Palagiewicz A, Eder J (2000). Alterations in
phenylpropanoid content in soybean roots during low temperature acclimation.
Plant Physiology and Biochemistry, 38, 587-593.
Janda T, Szalai G, Leskó K, Yordanova R, Apostol S, Popova LP (2007). Factors
contributing to enhanced freezing tolerance in wheat during frost hardening in
the light. Phytochemistry, 68, 1674-1682.
Joseleau J-P, Imai T, Kuroda K, Ruel K (2004). Detection in situ and
characterization of lignin in the G-layer of tension wood fibres of Populus
deltoides. Planta, 219, 338-345.
Khristova P, Kordsachia O, Patt R, Karar I, Khider T (2006). Environmentally
friendly pulping and bleaching of bagasse. Industrial Crops and Products, 23,
131-139.
Kim YJ, Kim DG, Lee SH, Lee I (2006). Wound-induced expression of the ferulate 5-
hydroxylase gene in Camptotheca acuminata. Biochimica et Biophysica Acta,
1760, 182-190.
Kimura M, Yamamoto YY, Seki M, Sakurai T, Sato M, Abe T, Yoshida S, Manabe
K, Shinozaki K, Matsui M (2003). Identification of Arabidopsis genes
regulated by high light-stress using cDNA microarray. Photochemistry
Photobiology, 77, 226-233.
Knops JMH, Naeemw S, Reich PB (2007). The impact of elevated CO 2 , increased
nitrogen availability and biodiversity on plant tissue quality and decomposition.
Global Change Biology, 13, 1960-1971.
Koch JR, Scherzer AJ, Eshita SM, Davis KR (1998). Ozone sensitivity in hybrid
poplar is correlated with a lack of defense-gene activation Plant Physiology,
118, 1243-1252.
Kofalvi SA, Nassuth A (1995). Influence of wheat streak mosaic virus infection on
phenylpropanoid metabolism and the accumulation of phenolics and lignin in
wheat. Physiological and Molecular Plant Pathology, 47, 365-377.
Kolupaev YE, Akinina GE, Mokrousov AV (2005). Induction of heat tolerance in
wheat coleoptiles by calcium ions and its relation to oxidative stress. Russian
Journal of Plant Physiology, 52, 199-204.
Koricheva J, Larsson S, Haukioja E, Keinanen M (1998). Regulation of woody plant
secondary metabolism by resource availability: hypothesis testing by means of
meta-analysis. Oikos, 83, 212-226.
Kostiainen K, Kaakinen S, Saranpaa P, Sigurdsson BD, Linder S, Vapaavuori E
(2004). Effect of elevated [CO 2 ] on stem wood properties of mature Norway
spruce grown at different soil nutrient availability Global Change Biology, 10,
1526-1538.

37
971
972
973
974
975
976
977
978
979
980
981
982
983
984
985
986
987
988
989
990
991
992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
1012
1013
1014
1015
1016
1017
1018
Kusumoto D (2005). Concentrations of lignin and wall-bound ferulic acid after
wounding in the phloem of Chamaecyparis obtusa. Trees - Structure and
Function, 19, 451-456.
Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leple JC,
Boerjan W, Ferret V, De Nadai V, Jouanin L (1999). Structural alterations of
lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or
caffeic acid O-methyltransferase activity have an opposite impact on the
efficiency of industrial kraft pulping. Plant Physiology, 119, 153-163.
Lautner S, Ehlting B, Windeisen E, Rennenberg H, Matyssek R, Fromm J (2007).
Calcium nutrition has a significant influence on wood formation in poplar. New
Phytologist, 173, 743-752.
Le Provost G, Paiva J, Pot D, Brach J, Plomion C (2003). Seasonal variation in
transcript accumulation in wood-forming tissues of maritime pine (Pinus
pinaster Ait.) with emphasis on a cell wall glycine-rich protein. Planta, 217,
820-830.
Liberloo M, Calfapietra C, Lukac M, Godbold D, Luos ZB, Polle A, Hoosbeek MR,
Kull O, Marek M, Raines C, Rubino M, Taylor G, Scarascia-Mugnozza G,
Ceulemans R (2006). Woody biomass production during the second rotation of
a bio-energy Populus plantation increases in a future high CO 2 world. Global
Change Biology, 12, 1094-1106.
Lima JD, Mazzafera P, Moraes WS, Silva RB (2009). Chá: aspectos relacionados a
qualidade e perspectivas. Ciência Rural, 39, 1270-1278 .
Little CHA, Savidge RA (1987). The role of plant growth regulators in forest tree
cambial growth. Plant Growth Regulation, 6, 137-169.
Lu FC, Ralph J (2003). Non-degradative dissolution and acetylation of ball-milled
plant cell walls: high-resolution solution-state NMR. Plant Journal, 35, 535-
544.
Lu FC, Ralph J, Morreel K, Messens E, Boerjan W (2004). Preparation and
relevance of a cross-coupling product between sinapyl alcohol and sinapyl p-
hydroxybenzoate. Organic and Biomolecular Chemistry, 2, 2888-2890.
Luo Z-B, Langenfeld-Heyser R, Calfapietra C, Polle A (2005). Influence of free air
CO 2 enrichment (EUROFACE) and nitrogen fertilisation on the anatomy of
juvenile wood of three poplar species after coppicing. Trees - Structure and
Function, 19, 109-118.
Martin JA, Solla A, Woodward S, Gil L (2007). Detection of differential changes in
lignin composition of elm xylem tissues inoculated with Ophiostoma novo-ulmi
using fourier transform-infrared spectroscopy. Forest Pathology, 37, 187-191.
Matros A, Amme S, Kettig B, Buck-Sorlin GH, Sonnewald U, Mock H-P (2006).
Growth at elevated CO 2 concentrations leads to modified profiles of secondary
metabolites in tobacco cv. SamsunNN and to increased resistance against
infection with potato virus Y. Plant Cell and Environment, 29, 126-137.
McDougall GJ (2000). A comparison of proteins from the developing xylem of
compression and non-compression wood of branches of Sitka spruce (Picea
sitchensis) reveals a differentially expressed laccase. Journal of Experimental
Botany, 51, 1767-1767.
Mellerowicz EJ, Sundberg B (2008). Wood cell walls: biosynthesis, developmental
dynamics and their implications for wood properties. Current Opinion in Plant
Biology, 11, 293-300.

38
1019
1020
1021
1022
1023
1024
1025
1026
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041
1042
1043
1044
1045
1046
1047
1048
1049
1050
1051
1052
1053
1054
1055
1056
1057
1058
1059
1060
1061
1062
1063
1064
1065
1066
1067
Menden B, Kohlhoff M, Moerschbacher BM (2007). Wheat cells accumulate a
syringyl-rich lignin during the hypersensitive resistance response.
Phytochemistry, 68, 513-520.
Moerschbacher BM, Noll U, Gorrichon L, Reisener H-J (1990). Specific inhibition
of lignification breaks hypersensitive resistance of wheat to stem rust. Plant
Physiology, 93, 465-470.
Möller R, Ball R, Henderson A, Modzel G, Find J (2006). Effect of light and
activated charcoal on tracheary element differentiation in callus cultures of
Pinus radiata D. Don. Plant Cell, Tissue and Organ Culture, 85, 161-171.
Monties B (1989). Lignins. In: Harborne JB, ed, Methods in plant biochemistry, Vol. 1,
Academic Press, London. pp 113-157.
Morreel K, Ralph J, Kim H, Lu FC, Goeminne G, Ralph S, Messens E, Boerjan W
(2004). Profiling of oligolignols reveals monolignol coupling conditions in
lignifying poplar xylem. Plant Physiology, 136, 3537-3549.
Møller SG, McPherson MJ (1998). Developmental expression and biochemical
analysis of the Arabidopsis atao1 gene encoding an H2O2 generating diamine
oxidase. Plant Journal, 13, 781-791.
Nimz HH, Robert D, Faix O, Nemr M (1981). C-13 NMR-spectra of lignins .8.
Structural differences between lignins of hardwoods, softwoods, grasses and
compression wood. Holzforschung, 35, 16-26.
Olenichenko N, Zagoskina N (2005). Response of winter wheat to cold: production of
phenolic compounds and l-phenylalanine ammonia lyase activity. Applied
Biochemistry and Microbiology, 41, 600-603.
Paakkonen E, Seppanen S, Holopainen T, Kokko H, Karenlampi S, L. K,
Kangasjarvi J (1998). Induction of genes for the stress proteins PR-10 and PAL
in relation to growth, visible injuries and stomatal conductance in birch (Betula
pendula) clones exposed to ozone and/or drought New Phytologist, 138, 295-
305.
Parrott DL, Anderson AJ, Carman JG (2002). Agrobacterium induces plant cell
death in wheat (Triticum aestivum L.). Physiological and Molecular Plant
Pathology, 60, 59-69.
Paux E, Carocha V, Marques C, Mendes de Sousa A, Borralho N, Sivadon P,
Grima-Pettenati J (2005). Transcript profiling of Eucalyptus xylem genes
during tension wood formation. New Phytologist, 167, 89-100.
Penel C, Greppin H (1996). Pectin binding proteins: Characterization of the binding
and comparison with heparin. Plant Physiology and Biochemistry, 34, 479-488.
Pitre F, Cooke J, Mackay J (2007). Short-term effects of nitrogen availability on wood
formation and fibre properties in hybrid poplar. Trees - Structure and Function,
21, 249-259.
Plomion C, Pionneau C, Brach J, Costa P, Bailleres H (2000). Compression woodresponsive
proteins in developing xylem of maritime pine (Pinus pinaster Ait.).
Plant Physiology, 123, 959-969.
Qiu D, Wilson IW, Gan S, Washusen R, Moran GF, Southerton SG (2008). Gene
expression in Eucalyptus branch wood with marked variation in cellulose
microfibril orientation and lacking G-layers. New Phytologist, 179, 94-103.
Ralph J, Lundquist K, Brunow G, Lu F, Kim H, Schatz PF, Marita JM, Hatfield
RD, Ralph SA, Christensen JH, Boerjan W (2004). Lignins: natural polymers
from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochemistry
Reviews, 3.

39
1068
1069
1070
1071
1072
1073
1074
1075
1076
1077
1078
1079
1080
1081
1082
1083
1084
1085
1086
1087
1088
1089
1090
1091
1092
1093
1094
1095
1096
1097
1098
1099
1100
1101
1102
1103
1104
1105
1106
1107
1108
1109
1110
1111
1112
1113
1114
1115
1116
Rastogi S, Dwivedi UN (2008). Manipulation of lignin in plants with special reference
to O-methyltransferase. Plant Science, 174, 264-277.
Richard JN, Cortufo MF, Philip I, Elizabeth GON, Josep GC (2001). Elevated CO 2 ,
litter chemistry, and decomposition: a synthesis. Oecologia, 127, 153-165.
Robson AD, Hartley RD, Jarvis SC (1981). Effect of copper deficiency on phenolic
and other constituents of wheat cell walls. New Phytologist, 89, 361-371.
Rodrigues J, Graça J, Pereira H (2001). Influence of tree eccentric growth on
syringyl/guaiacyl ratio in Eucalyptus globulus wood lignin assessed by
analytical pyrolysis. Journal of Analytical and Applied Pyrolysis, 58, 481-489.
Rogers LA, Campbel MM (2004). The genetic control of lignin deposition during
plant growth and development. New Phytologist, 164, 17-30.
Rogers LA, Dubos C, Cullis IF, Surman C, Poole M, Willment J, Mansfield SD,
Campbell MM (2005). Light, the circadian clock, and sugar perception in the
control of lignin biosynthesis. Journal of Experimental Botany, 56, 1651-1663.
Rozema J, van de Staaij J, Björn LO, Caldwell M (1997). UV-B as an environmental
factor in plant life: stress and regulation. Trends in Ecology & Evolution, 12, 22-
28.
Ruelle J, Yoshida M, Clair B, Thibaut B (2007). Peculiar tension wood structure in
Laetia procera (Poepp.) Eichl. (Flacourtiaceae). Trees - Structure and Function,
21, 345-355.
Sasaki M, Yamamoto Y, Matsumoto H (1996). Lignin deposition induced by
aluminum in wheat (Triticum aestivum) roots. Physiologia Plantarum, 96, 193-
198.
Smith AH, Gill WM, Pinkard EA, Mohammed CL (2007). Anatomical and
histochemical defence responses induced in juvenile leaves of Eucalyptus
globulus and Eucalyptus nitens by Mycosphaerella infection. Forest Pathology,
37, 361-373.
Snowden KC, Gardner RC (1993). Five genes induced by aluminum in wheat
(Triticum aestivum L.) roots. Plant Physiology, 103, 855-861.
Solecka D, Boudet A-M, Kacperska A (1999). Phenylpropanoid and anthocyanin
changes in low-temperature treated winter oilseed rape leaves. Plant Physiology
and Biochemistry, 37, 491-496.
Solecka D, Kacperska A (1995). Phenylalanine ammonia-lyase activity in leaves of
winter oilseed rape plants as affected by acclimation of plants to lowtemperature.
Plant Physiology and Biochemistry, 33, 585-591.
Soltani BM, Ehlting J, Hamberger B, Douglas CJ (2006). Multiple cis-regulatory
elements regulate distinct and complex patterns of developmental and woundinduced
expression of Arabidopsis thaliana 4CL gene family members. Planta,
224, 1226-1238.
Steponkus P (1984). Role of the plasma membrane in freezing injury and cold
acclimation. Annual Review of Plant Physiology, 35, 543-584.
Steponkus PL, Uemura M, Webb MS (1993). Membrane destabilization during
freeze-induced dehydration. Current Topics Plant Physiology, 10, 37-47.
Suzuki N, Mittler R (2006). Reactive oxygen species and temperature stresses: A
delicate balance between signaling and destruction. Physiologia Plantarum, 126,
45-51.
Syros TD, Yupsanis TA, Economou AS (2005). Expression of peroxidases during
seedling growth in Ebenus cretica L. as affected by light and temperature
treatments. Plant Growth Regulation, 46, 143-151.

40
1117
1118
1119
1120
1121
1122
1123
1124
1125
1126
1127
1128
1129
1130
1131
1132
1133
1134
1135
1136
1137
1138
1139
1140
1141
1142
1143
1144
1145
1146
1147
1148
1149
1150
1151
1152
1153
1154
1155
1156
1157
1158
1159
1160
1161
1162
1163
1164
1165
1166
Tahara K, Norisada M, Hogetsu T, Kojima K (2005). Aluminum tolerance and
aluminum-induced deposition of callose and lignin in the root tips of Melaleuca
and Eucalyptus species. Journal of Forest Research, 10, 325-333.
Takahama U (1988). Hydrogen peroxide-dependent oxidation of flavonoids and
hydroxycinnamic acid derivatives in epidermal and guard cells of Tradescantia
virginiana L. Plant and Cell Physiology, 29, 475-481.
Takahama U, Oniki T (1997). A peroxidase/phenolics/ascorbate system can scavenge
hydrogen peroxide in plant cells. Physiologia Plantarum, 101, 845-852
Taşgına E, Atıcıb Ö, Nalbantoğlua B, Popovac LP (2006). Effects of salicylic acid
and cold treatments on protein levels and on the activities of antioxidant
enzymes in the apoplast of winter wheat leaves. Phytochemistry, 67, 710-715.
Teixeira AF, Andrade ADB, Ferrarese-Filho O, Ferrarese MDL (2006). Role of
calcium on phenolic compounds and enzymes related to lignification in soybean
(Glycine max L.) root growth. Plant Growth Regulation, 49, 69-76.
Thomashow MF (1999). Plant cold acclimation: freezing tolerance genes and
regulatory mechanisms. Annual Review of Plant Biology, 50, 571-599.
Tomas-Barberan FA, Gil MI, Castaner M, Artes F, Saltveit ME (1997). Effect of
selected browning inhibitors on phenolic metabolism in stem tissue of harvested
lettuce. Journal of Agricultural and Food Chemistry, 45, 583-589.
van de Mortel JE, Villanueva LA, Schat H, Kwekkeboom J, Coughlan S, Moerland
PD, van Themaat EVL, Koornneef M, Aarts MGM (2006). Large expression
differences in genes for iron and zinc homeostasis, stress response, and lignin
biosynthesis distinguish roots of Arabidopsis thaliana and the related metal
hyperaccumulator Thlaspi caerulescens. Plant Physiology, 142, 1127-1147.
Vincent D, Lapierre C, Pollet B, Cornic G, Negroni L, Zivy M (2005). Water deficits
affect caffeate O-methyltransferase, lignification, and related enzymes in maize
leaves. A proteomic investigation. Plant Physiology, 137, 949-960.
Whetten R, Sederoff R (1995). Lignin biosynthesis. Plant Cell, 7, 1001-1013.
Wilson K, White DJB (1986). Reaction wood: its structure, properties and functions.
In: Wilson K, and White DJB, eds., The anatomy of wood: its diversity and
variability, Stobart and Son Ltd, London, UK. pp 222–250.
Yamasaki S, Noguchi N, Mimaki K (2007). Continuous UV-B irradiation induces
morphological changes and the accumulation of polyphenolic compounds on the
surface of cucumber cotyledons. Journal of Radiation Research, 48, 443-454.
Yang L, Wang CC, Guo WD, Li XB, Lu M, Yu CL (2006). Differential expression of
cell wall related genes in the elongation zone of rice roots under water deficit.
Russian Journal of Plant Physiology, 53, 390-395.
Yeh TF, Braun JL, Goldfarb B, Chang HM, Kadla JF (2006). Morphological and
chemical variations between juvenile wood, mature wood, and compression
wood of loblolly pine (Pinus taeda L.). Holzforschung, 60, 1-8.
Yeh TF, Goldfarb B, Chang HM, Peszlen I, Braun JL, Kadla JF (2005).
Comparison of morphological and chemical properties between juvenile wood
and compression wood of loblolly pine. Holzforschung, 59, 669-674.
Yoshida M, Ohta H, Yamamoto H, Okuyama T (2002). Tensile growth stress and
lignin distribution in the cell walls of yellow poplar, Liriodendron tulipifera
Linn. Trees-Structure and Function, 16, 457-464.
Yoshimura K, Masuda A, Kuwano M, Yokota A, Akashi K (2008). Programmed
proteome response for drought avoidance/tolerance in the root of a C-3
xerophyte (wild watermelon) under water deficits. Plant and Cell Physiology,
49, 226-241.

41
1167
1168
1169
1170
1171
1172
1173
1174
1175
1176
1177
1178
1179
1180
1181
1182
1183
1184
1185
Yoshizawa N, Inami A, Miyake S, Ishiguri F, Yokota S (2000). Anatomy and lignin
distribution of reaction wood in two Magnolia species. Wood Science and
Technology, 34, 183-196.
Zagoskina N, Goncharuk E, Alyavina A (2007). Effect of cadmium on the phenolic
compounds formation in the callus cultures derived from various organs of the
tea plant. Russian Journal of Plant Physiology, 54, 237-243.
Zagoskina NV, Dubravina GA, Alyavina AK, Goncharuk EA (2003). Effect of
ultraviolet (UV-B) radiation on the formation and localization of phenolic
compounds in tea plant callus cultures. Russian Journal of Plant Physiology, 50,
270-275.
Zhong R, Kays SJ, Schroeder BP, Ye Z-H (2002). Mutation of a chitinase-like gene
causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of
ethylene. Plant Cell, 14, 165-179.
Zinser C, Ernst D, Sandermann Jr H (1998). Induction of stilbene synthase and
cinnamyl alcohol dehydrogenase mRNAs in Scots pine (Pinus sylvestris L.)
seedlings. Planta, 204, 169-176.

42
1186
1187
1188
1189
1190
1191
1192
1193
1194
1195
Figure 1. Biosynthesis pathway of lignin in plants (adapted from Rastogi and Dwivedi,
2008). PAL - phenylalanine ammonia-lyase; C4H - cinnamate 4-hydroxylase; C3H -
cinnamate 3-hydroxylase or p-coumaroyl shikimic acid/quinic acid 3-hydroxylase;
OMT - O-methyltransferase; HCT - p-hydroxy cinnamoyl-CoA: shikimate/quinate p-
hydroxy cinnamoyl transferase; CCoAOMT - caffeoyl coenzyme A O-
methyltransferase; F5H - ferulate 5-hydroxylase; 4CL - 4-coumarate: coenzyme A
ligase; CCR - cinnamoyl coenzyme A reductase; Cald5H - coniferaldehyde 5-
hydroxylase; AldOMT - 5-hydroxy coniferaldehyde O-methyltransferase; SAD -
sinapyl alcohol dehydrogenase; CAD - cinnamyl alcohol dehydrogenase.
Cinnamate
PAL
Phenylalanine
C4H
C3H (?) OMT F5H OMT
4-Coumarate Caffeate Ferulate 5-Hydroxyferulate Sinapate
4CL
4CL 4CL 4CL 4CL (?)
CCoAOM
CCoAOM
HCT
C3H
HCT
COMT F5H (?) COMT
4-Coumaryl-CoA 4-Coumaryl Caffeoyl Caffeoyl-CoA Feruloyl-CoA 5-Hydroxyferuolyl-CoA Sinapyl-CoA
shikimic acid / shikimic acid /
4-Coumaryl
Caffeoyl
CCR
quinic acid
quinic acid
CCR CC CC CC
(?)
OMT F5H OMT
4-Coumaraldehyde Caffeoyl aldehyde Coniferaldehyde 5-Hydroxyconiferaldehyde Sinapaldehyde
CAD/SAD
CAD/SAD CAD/SAD CAD/SAD CAD/SAD
(?) OMT
F5H
OMT
Cald5h
AldOM
4-Coumaryl alcohol Caffeoyl alcohol Coniferyl alcohol 5-Hydroxyconiferyl Sinapyl alcohol
alcohol
p-Hydroxypnehyl Guaiacyl Syringil
(H unit) (G unit) (S unit)
R1 = R2 = H p-coumaryl alcohol (p-hydroxy phenyl, H unit)
R1 = H, R2 = OCH 3 coniferyl alcohol (guayacyl, G unit)
R1 = R2 = OCH 3 sinapyl alcohol (syringyl, S unit)