Lignin biosynthesis studies in plant tissue cultures
Anna Kärkönen1, 2* · Sanna Koutaniemi3
1
Department of Applied Biology, P.O. Box 27 (Latokartanonkaari 7), 00014 University of
Helsinki, Finland
2
MTT Agrifood Research Finland, Tutkimusasemantie 15, 92400 Ruukki, Finland
3
Department of Applied Chemistry and Microbiology, P.O. Box 27 (Latokartanonkaari 11),
00014 University of Helsinki, Finland
*Corresponding author
E-mail: [email protected]
Phone:
+358 9 19157662
Fax:
+358 9 19158727
Abbreviations caffeoyl-CoA O-methyltransferase, CCOMT; cinnamate 4-hydroxylase,
C4H;
cinnamyl
alcohol
dehydrogenase,
CAD;
coniferyl
alcohol,
CA;
5-O-(4-
coumaroyl)shikimate 3’-hydroxylase, C3H; p-coumaryl alcohol, p-CA; dehydrogenation
polymer, DHP; guaiacyl, G; hydroxycinnamoyl:CoA transferase, HCT; p-hydroxyphenyl, H;
phenylalanine ammonia-lyase, PAL; sinapyl alcohol, SA; syringyl, S; tracheary element,
TE
Abstract
Lignin, a phenolic polymer abundant in cell walls of certain cell types, has given
challenges to scientists that study its structure or biosynthesis. In plants lignified tissues
are distributed between other, non-lignified tissues. Characterisation of native lignin in the
cell wall has been difficult due to the highly cross-linked nature of the wall components.
Model systems, like plant tissue cultures with tracheary element differentiation or
extracellular lignin formation, have provided useful information related to lignin structure
and several aspects of lignin formation. For example, many enzyme activities in
phenylpropanoid pathway have been first identified in tissue cultures. This review focuses
on studies where the use of plant tissue cultures has been advantageous in structural and
biosynthesis studies of lignin, and discusses the validity of tissue cultures as models for
lignin biosynthesis.
1
Introduction
Lignin, a phenolic polymer mainly composed of three hydroxycinnamyl alcohols (pcoumaryl (p-CA), coniferyl (CA) and sinapyl alcohol (SA) giving rise to p-hydroxyphenyl
(H), guaiacyl (G) and syringyl (S) type of lignins, respectively), is deposited in the
secondarily thickened cell walls of certain cell types, for example, water-conducting
tracheids and vessels (collectively known as tracheary elements, TEs) and support-giving
schlerenchyma (fibers, stone cells). Lignin provides rigidity and structural support to cell
wall polysaccharides and makes the cell walls water impermeable. In addition to
developmental lignification, lignin synthesis can be induced after wounding or pathogen
attack as a defence response (Vance et al. 1980; Dixon and Paiva 1995). This stressinduced lignin differs structurally from developmental lignins, containing increased
amounts of H units derived from p-CA (Lange et al. 1995).
In a plant, lignified tissues are distributed between other, non-lignified tissues. TEs are
located in xylem, while schlerenchyma is found in xylem, phloem and cortex. In addition,
in certain plant groups, like grasses and cereals, a considerable portion of parenchyma
cells in the outer layers of cortex become lignified during stem development. Lignin acts
as glue and fills in the empty spaces in the cellulose-hemicellulose-pectin network. The
disperse location of lignin-containing tissues and the tight interaction between cell wall
components hinder studies of lignin biosynthesis and lignin structure. It is sometimes
easier to use model systems where the phenomenon to be studied occurs in single cells
or in the culture medium. One of the model systems are plant tissue cultures.
Some tissue cultures of both angiosperms and gymnosperms can synthesise lignin-like
polymers (Table 1). In some systems, a fraction of the cells differentiate into TEs, in which
case lignin is partly or exclusively deposited into the cell wall. Alternatively, extracellular
lignin is formed into the culture medium (Figure 1). In most tissue culture systems lignin
formation is stimulated by a change in growth regulators (Simola et al. 1992; Eberhardt et
al. 1993; Pauwels et al. 2008) or induced by fungal elicitors (Messner and Boll 1993;
Lange et al. 1995) or water stress (Tsutsumi and Sakai 1993). Sucrose has also been
used as an inducer of lignin formation, for example in sycamore (Acer pseudoplatanus)
and loblolly pine (Pinus taeda) cell suspension cultures (Carceller et al. 1971; Nose et al.
1995).
2
One of the most studied systems for TE differentiation is Zinnia elegans cell culture,
which, contrary to continuous, subcultivated cell cultures, is a primary cell culture started
freshly from mesophyll cells. Wounding stress caused by isolation of mesophyll cells
together with the stress due to in vitro conditions seem to be essential for activation of TE
differentiation process in a medium with favourable cytokinin-auxin combination (Table 1,
McCann 1997). The isolated mesophyll cells trans-differentiate into TEs synchronously in
a few days, with ca. 40-60 % of cells differentiating (Kohlenbach and Schmidt 1975;
Fukuda and Komamine 1980a, 1982). Secondary cell wall thickenings become visible 4871 h after initiation of the culture and lignification starts some hours later, correlating with
increases in the activities of monolignol biosynthetic enzymes (e.g., Fukuda and
Komamine 1982; Church and Galston 1988; Demura et al. 2002). Inhibitors of
phenylalanine synthesis demonstrated that lignification was regulated separately from
differentiation, as inhibitor treatment did not affect the formation of secondary thickenings
while lignification was prevented (Sato et al. 1993). Thus, this type of in vitro cell culture
system is an efficient model for studies of TE differentiation, and hence, of lignin
formation.
In vitro cultures have the advantage that they allow studies to be conducted in controlled
conditions independent of seasons. Factors related to lignin formation can be studied by
adding the compounds of interest into the culture medium. Culture medium can be
considered as a large apoplastic fluid-filled intercellular space forming a continuum with
the plant cell wall and it is easily isolated for further analysis. This review focuses on
studies related to lignin biosynthesis in in vitro cultures. Differentiation of TEs in vitro has
been reviewed elsewhere (e.g. Fukuda 1997; McCann 1997).
Effect of growth regulators
Tissue culture studies have shown that TE differentiation, and also lignin formation,
depend on plant growth regulators auxin and cytokinin. The optimal combination of these,
however, depends on the tissue culture. For example, auxin alone induced the formation
of tracheids with lignified cell walls in Populus nigra callus cultures (Venverloo 1969),
while in xylogenic Zinnia mesophyll cell cultures only a certain combination of auxin and
cytokinin (and neither growth regulator alone) resulted in TE differentiation (Fukuda and
Komamine 1982; Sato et al. 1993; Table 1). In Zinnia culture auxin directly induced TE
formation, as cell division was not a prerequisite for differentiation (Fukuda and Komamine
3
1980b). On the other hand, elevated levels of endogenous cytokinin led to production of
fiber and tracheid-type cells with lignified cell walls in transgenic Nicotiana tabacum
suspension cultures (Blee et al. 2001). The various requirements for growth regulators
probably reflect differences in the metabolic state of cell and tissue cultures in relation to
endogenous growth hormones.
Formation of extracellular lignin has in most cases been induced by sucrose or elicitor
treatment (Table 1), but growth regulators also have a role. In Picea abies tissue cultures,
for example, cytokinin alone or supplemented with low levels of auxin was optimal for
extracellular lignin formation (Simola et al. 1992). The culture medium was optimised for
spruce tissue cultures and the cells were cultivated in a light-dark rhythm, a condition
likely to be advantageous for lignin production. Spruce cultures did not show
ultrastructural abnormalities common to many tissue cultures of herbs (Simola 1987; L.K.
Simola, unpublished).
There are some reports of the involvement of other hormones in lignin biosynthesis. For
example, addition of gibberellic acid, GA3, to xylogenic Zinnia cultures increased lignin
content in TE cell walls, but the effect was apparently through increased polymerisation of
existing monolignols (Tokunaga et al. 2006). External addition of methyljasmonate, a
signalling molecule related to defence, to Arabidopsis cell cultures led to increased
expression of phenylpropanoid, particularly monolignol, biosynthetic genes (Pauwels et al.
2008). This suggests an active role for jasmonates in the regulation of monolignol
biosynthesis, at least in defence reactions.
Cell culture lignins
Extracellular lignins are interesting material for research due to the ease of polymer
isolation from the culture medium. This allows studies on polymerisation mechanism and
polymer structure without harsh chemical extractions that are likely to alter lignin structure.
But how representative of native lignin are extracellular lignins? These polymers often
contain higher amounts of H units than wood-derived lignins (Brunow et al. 1990, 1993;
Lange et al. 1995). Hence, more condensed (C-C) linkages and less non-condensed (CO) linkages exist between monomers in extracellular lignins than in lignins isolated from
wood (e.g. Picea abies; Brunow et al. 1990, 1993; Lange et al. 1995). Differences in lignin
composition to normal wood are smaller when TE cell wall lignin is concerned. For
4
example, ratios of H and G units involved in non-condensed linkages of lignin were similar
in cell walls of Pinus radiata tracheids that differentiated in cell culture and those in P.
radiata wood (Möller et al. 2006). Similar observations were made in Pinus taeda, in which
comparison of cell wall lignin produced by natural cinnamyl alcohol dehydrogenase (CAD)
-deficient mutants, either in wood or by cultured cells, showed similarities in lignin
structure (Stasolla et al. 2003).
The main factor that explains these differences between extracellular and cell wall lignins
is likely to be the carbohydrate matrix, in which lignin polymerisation normally takes place.
This matrix affects lignin structure at a macromolecular level, as was shown in P. radiata.
Spherical lignin particles existed in the middle lamella and cell corners, while lamellar
lignin structures were found in the secondary cell walls, corresponding to lamellar
cellulose microfibrils (Donaldson 1994). The effect of carbohydrates on lignin structure
was also demostrated in in vitro (test tube) polymerisation studies of lignin monomers
(production of dehydrogenation polymers, DHPs). Addition of pectic or hemicellulosic
polysaccharides to the reaction induced the formation of lignin-carbohydrate complexes,
increasing the solubility of in vitro lignin and the proportion of non-condensed C-O
linkages, thus making the DHP generated more similar to wood-derived lignins (Cathala
and Monties 2001; Barakat et al. 2007). In this respect it is interesting to note that some
extracellular lignins formed in tissue cultures contain carbohydrates. Neutral or acidic
sugars were attached to extracellular lignins produced in Picea abies tissue cultures after
elicitation (Lange et al. 1995) or by auxin reduction (S. Koutaniemi, unpublished). The
sugar component formed ca. 14% of the dry weight of lignins in both cases. Whether
these carbohydrates can affect lignin polymerisation in the culture medium is a question
that deserves further interest. The possible specificity of lignin-carbohydrate interaction is
another subject for future studies. It has been shown that the extracellular lignin in P.
abies culture is closer in structure to wood lignin than any synthetic DHPs (Kärkönen et al
2002; Koutaniemi et al 2005). This argues for some regulative factors during the synthesis
of extracellular lignin, one of which could be the carbohydrates. Another factor is the rate
of monolignol radical formation (Sarkanen 1971), which is likely to be more controlled in
tissue cultures that secrete monolignols into the culture medium than in in vitro DHP
synthesis with monolignol supplementation. Unfortunately, structural data from most tissue
culture lignins has been obtained using degradative methods, raising a need for
reanalyses with modern day non-destructive methods such as NMR.
5
It is unprobable that extracellular lignins are synthesised in the cell wall and later released
into the culture medium in great amounts through cell lysis. If this were the case also
cellulose should be found attached to extracellular lignins, since polymerisation within the
cell wall anchors lignin tightly into carbohydrate polymers. Great amounts of glucose have
not been reported thus far, but more studies on other cell culture lignins are needed to
verify this. Other observations that support the culture medium as the site of extracellular
lignin polymerisation include the high amounts of lignin found in the culture medium (20
µg/ml; Lange et al. 1995; up to 300 µg/ml; our Picea abies tissue culture line), the high
amount of non-differentiated cells being present with non-lignified primary cell walls (only
ca. 3% of cells differentiate to tracheids in P. abies tissue culture, A. Santanen, Univ. of
Helsinki, unpublished) and the differences observed between the proteomes of the
apoplastic medium and the cytoplasm.
The origin of extracellular lignins is intriguing. Since their synthesis takes place as a
response to hormonal or abiotic stimuli (Table 1), they could be judged as stress lignins.
This is also supported by structural data, which shows higher amounts of H units, and
hence, C-C linkages, in many extracellular lignins (Brunow et al 1993; Lange et al 1995).
Similar monolignol and linkage compositions are typical also for early developmental
lignins, like that synthesised in middle lamella and cell corners in Pinus thumbergii
(Terashima and Fukushima 1988) and in lignin of compression wood (Timell 1986) which
is produced in response to gravitational stress on the lower sides of leaning stems and
branches of conifers. The methoxylation degree of monolignols increases during later
phases of cell wall lignification in P. thunbergii (Terashima and Fukushima 1988) and
during the transition from juvenility to maturity in P. abies (Lange et al. 1995). Similarily,
lignin in primary-walled in vitro -cultured cells of Populus tremula x tremuloides (hybrid
aspen) consisted of G units only, while lignin in the mature aspen wood is composed of G
and S units in 1:2 ratio (Christiernin et al. 2005). The prevalence of low methoxylation
stage and C-C linkages in lignin could be either a developmentally regulated
phenomenon, or be related to situations where a sudden demand on lignin biosynthesis,
for example during pathogen defence (i.e., elicitation), leads to a preference of
biosynthetically simpler subunits.
Studies of monolignol biosynthesis
Lignin precursors, p-CA, CA and SA, are synthesised through the phenylpropanoid
pathway, starting from phenylalanine ammonia-lyase (PAL) and leading to the three
6
monolignols through a series of hydroxylations, methylations and reductions (Boerjan et
al. 2003; Figure 2). Many of the enzyme activities of the pathway were first identified in
tissue cultures, for example 5-O-(4-coumaroyl)shikimate 3’-hydroxylase (C3H) and
caffeoyl-CoA O-methyltransferase (CCOMT) in elicited parsley (Petroselinum crispum) cell
cultures (Heller and Kühnl 1985; Pakusch et al. 1989). The involvement of CCOMT in
lignin biosynthesis was observed in differentiating Zinnia TEs (Ye et al. 1994). Functional
evidence for the involvement of observed enzyme activities in lignification often requires
the use of transgenic techniques to suppress or knock out the gene expression and the
corresponding enzyme activity. This is a reasonable task in many angiosperms, but most
gymnosperms are less amenable to transformation, in part because of a tedious
regeneration process (Tang and Newton 2003). Therefore, transformable, lignin-forming
tissue cultures would be valuable tools for evaluation of the effects of introduced genes on
cell wall and lignin synthesis. Transformation methods for cultured cells have been
established, for example, for Pinus radiata (Möller et al. 2003), Arabidopsis (Oda et al.
2005) and Zinnia (Endo et al. 2008), and, importantly, in all cases the cells retain the
potential for TE differentiation even after transformation. For example, silencing of P.
radiata hydroxycinnamoyl:CoA transferase (HCT) gene, leading to G units, resulted in a
reduced lignin content in tracheids formed in vitro and in a strong increase in H units with
a concomitant reduction in G units (Wagner et al. 2007). This also proved the evolutionary
conservation of the HCT activity in lignin biosynthesis across gymnosperm and
angiosperm species (Hoffman et al. 2003, 2004; Wagner et al. 2007).
Feeding of phenylpropanoid precursors for cultured cells has been used to study, for
example, the metabolic fluxes in lignin biosynthetic pathway. Feeding of phenylalanine to
cultured cells of Pinus taeda induced the expression of nearly all monolignol biosynthetic
genes and, concomitantly, increased the synthesis and secretion of p-CA relative to CA
(Anterola et al. 1999, 2002). This shows that carbon allocation into different monolignols is
controlled by phenylalanine supply and by differential induction of genes needed for the 3and 4-hydroxylations.
Feeding experiments have also been utilised to study the proposed channelling of
phenylpropanoid intermediate metabolites from one enzyme to the next through
preformed complexes (Winkel-Shirley 1999). Support for the theory of enzyme complex
between the first two enzymes, PAL and cinnamate 4-hydroxylase (C4H), was found,
since cultured tobacco (Nicotiana tabacum) cells could not metabolise cinnamic acid
added to the culture medium; instead, cinnamic acid formed endogenously from supplied
7
phenylalanine was used in the 4-hydroxylation reaction to produce the downstream
metabolite p-coumaric acid (Figure 2; Rasmussen and Dixon 1999). PAL and C4H were
co-localized in microsomes of tobacco plants, an observation that further supported
complex formation (Achnine et al. 2004). Elicitation of the tobacco cell culture or
overexpression of PAL allowed the exogenous cinnamate to be incorporated into pcoumaric acid, suggesting that close association of PAL and C4H isoforms was altered
after elicitation, possibly due to unbalanced concentrations of PAL and C4H (Rasmussen
and Dixon 1999). Alterations in other phenylpropanoid-related metabolites were also
observed, indicating that different isoenzymes are responsible for the biosynthesis of
monolignols and other phenylpropanoids
Monolignol transport
It is not yet resolved how and in which form the monolignols are transported to the cell
wall for lignin polymerisation. As developing wood cells constitute a thin layer inside the
cambium, studying the secretion of monolignols in the developing xylem is difficult.
Tissue-cultured cells like those of Zinnia and Arabidopsis that secrete monolignols into the
culture medium (Hosokawa et al. 2001; Pauwels et al. 2008), and those of conifers with
extracellular lignin formation (Table 1) are excellent tools for monolignol transport studies.
In Zinnia culture, non-differentiating cells seem to contribute in supplying monolignols (CA,
SA, coniferaldehyde) for differentiating TEs (Hosokawa et al. 2001). Also extracellular
dilignols were incorporated into lignin and thus functioned as precursors for polymerisation
(Tokunaga et al. 2005). As stress can induce monolignol synthesis and secretion, care
should be taken when making conclusions since the stress-induced secretion
mechanisms may differ from those occurring during developmental lignification.
Lignin polymerisation
Peroxidases and laccases
After transport into the cell wall, the lignin precursors are polymerised via radical reaction.
The formation of radicals is catalysed by oxidative enzymes, either H2O2-dependent
peroxidases or O2-dependent oxidases/laccases. These enzymes are secreted into the
apoplast where they are either soluble or covalently or ionically bound to the cell wall. The
apoplastic proteome of tissue cultures is relatively easy to isolate (e.g. Blee et al. 2001),
8
potentiating studies of the role of individual enzymes in lignin polymerisation (Gabaldón et
al. 2005; Koutaniemi et al. 2005).
In some conifer species like Pinus taeda (Nose et al. 1995) and Picea abies (Kärkönen et
al. 2002), peroxidases appear to have a crucial role in lignin polymerisation as removal of
H2O2 decreased the extracellular lignin formation in tissue cultures. Soluble culture
medium peroxidases purified from the P. abies culture were efficient in monolignol
oxidation with catalytic preferences towards p-CA and CA (Koutaniemi et al. 2005). Also
the candidate peroxidase genes for lignin polymerization were identified based on
expression profiling studies (Koutaniemi et al. 2007). In angiosperm tissue cultures like
Zinnia, appearance of several peroxidase isoenzymes has correlated with TE
differentiation and lignification (Fukuda and Komamine 1982; Church and Galston 1988;
Sato et al. 1993; López-Serrano et al. 2004; Sato et al. 2006). For example, a cationic
peroxidase bound to the cell walls by ionic interactions was induced during TE
differentiation (Sato et al.1993; López-Serrano et al. 2004). Furthermore, inhibition of TE
differentiation by anti-auxins or tunicamycin repressed the induction of a cationic cell wall
peroxidase (Church and Galston 1988). Also in Populus alba callus, water stress was
shown to stimulate wall-bound peroxidase activity and accumulation of G-type lignin
(Tsutsumi and Sakai 1993). The same isoenzymes that were linked to lignification in
Zinnia cultures were also found from lignifying xylem in Zinnia plants (López-Serrano et al.
2004). In addition, their close homologues were spatially and temporally associated with
lignification in Arabidopsis (Sato et al. 2006). These associations underscore similarities in
developmental lignification in planta and in vivo in cultured cells.
Although roughly two thirds of angiosperm lignin is composed of S units, most studied
peroxidases oxidize SA poorly in in vitro conditions. Interestingly, a cationic peroxidase
ionically bound to cell walls of Populus alba callus oxidized SA efficiently (Aoyama et al.
2002). The same enzyme was also able to oxidize DHP that had been prepared from SA
(Sasaki et al. 2004) and furthermore, was localised to cell walls of lignifying xylem fibers
(Sasaki et al. 2006). This ability to oxidize polymeric lignin, whether enzymatically or via
radical transfer, is a key step in the polymerisation of lignin (Sarkanen 1971). Interestingly,
P. abies tissue culture contains peroxidases that are non-covalently bound to extracellular
lignin, and are thus candidates for polymer oxidation (S. Koutaniemi, T. Warinowski, A.
Kärkönen, T.H. Teeri, unpublished data).
9
The role of laccases in lignin polymerisation is unclear. In Zinnia culture, high expression
of laccase genes existed during the late stages of TE differentiation (Demura et al. 2002).
In P. abies tissue culture, however, a low apoplastic laccase activity and low laccase gene
expression was observed during extracellular lignin formation (Kärkönen et al. 2002;
Koutaniemi et al. 2007). This is in contrast with the situation in lignifying xylem of P. abies
where laccase genes were expressed at a high level both in 1-year old plantlets and adult
trees (Koutaniemi et al. 2007). Since apoplastic laccases could not compensate the
reduction in peroxidase action caused by H2O2 removal in extracellular lignin synthesis
(Kärkönen et al. 2002), it is likely that laccases are involved in processes other than lignin
biosynthesis in this tissue culture.
Apoplastic H2O2 generation
Since peroxidases seem to be crucial for lignin biosynthesis, at least in gymnosperms
(Nose et al. 1995; Kärkönen et al. 2002), H2O2 should be available for peroxidases in the
cell wall during lignin formation. Several possible enzymic systems for apoplastic H2O2
formation exist (reviewed by Quan et al. 2008). Tissue cultures with differentiating TEs or
extracellular lignin formation offer a good model for lignification-related H2O2 formation.
Apoplastic bursts of H2O2 take place in defence and other types of stress responses
(Messner and Boll 1994). The origin of stress-induced H2O2 may be different from that in
lignin formation. Hence, cell cultures in which lignin formation is induced by elicitation or
by an osmotic stress cannot be used when the mechanisms of the apoplastic H2O2
formation related to lignin formation are being studied. It is also well known that transfer of
plant cells to fresh culture medium induces a stress: for example production of reactive
oxygen species occurs (Messner and Boll 1994). Thus, enough time is needed for cells to
acclimatise in in vitro conditions before the experiments are conducted so that the reaction
measured does not represent that of a stress reaction.
In Zinnia cell cultures a steady-state, micromolar H2O2 concentration existed in the culture
medium with some superoxide present throughout the period of TE differentiation (Gómez
Ros et al. 2006). Both developing TEs and undifferentiated cells seemed to take part in
H2O2 production (Gómez Ros et al. 2006), possibly by the action of the plasma
membrane-located NADPH oxidase (Ros Barceló 1998). Addition of diphenylene
iodonium (DPI; an inhibitor of flavine-containing enzymes like NADPH oxidase) or an
10
inhibitor of superoxide dismutase to the medium of Zinnia differentiating TEs supported
the involvement of NADPH oxidase in H2O2 production as the amount of H2O2 was
reduced in cell walls (Karlsson et al. 2005). The development of secondary cell walls was
affected as well: for example lignin content was reduced. Micromolar steady-state H2O2
levels were also present in the culture medium of P. abies cells during extracellular lignin
formation (Kärkönen and Fry 2006; Kärkönen et al. 2009). At least two H2O2 generation
systems were detected in the apoplast (Kärkönen et al. 2009). H2O2 generation was
efficiently inhibited by DPI (at 5 µM) irrespective of elicitation. Azide (at 20 µM), an
inhibitor of haem-containing enzymes like peroxidase, on the contrary, efficiently inhibited
H2O2 production after elicitation but only slightly during constitutive lignin formation
suggesting that elicitor induced additional mechanisms for defence-associated H2O2
production than those present normally during lignin formation.
Conclusion
Tissue culture systems are particularly useful when they reflect developmental processes
that occur in planta. In certain phenomena that are difficult to be studied in muro, for
example monolignol transport, tissue culture systems are valuable in the primary stage of
analysis to get some indications about the possible mechanisms being involved in the
question studied. Especially, when it is possible to transform cells before induction of TE
differentiation with concomitant lignin formation, cell cultures can be utilised for evaluation
of the function of the gene of interest. As in vitro cultured cells are, however, in artificial
conditions, parallel studies in intact plants are needed to evaluate the processes that
occur in planta.
Acknowledgements
We thank Professor emerita Liisa K. Simola for her comments and suggestions on the
manuscript.
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Table 1. In vitro cultures for lignin formation.
Plant species
Plant part / organ for
culture initiation
Type of lignin
Induction /
stimulation
Reference
Picea abies
zygotic embryo
mainly extracellular
spontaneous,
stimulated by change
in growth regulators
Simola et al. (1992)
Picea abies
seedlings
cell wall, extracellular
elicitor
Messner and Boll (1993)
Picea abies
needles of seedlings
cell wall, extracellular
elicitor
Lange et al. (1995)
Pinus banksiana
root
cell wall
elicitor
Campbell and Ellis (1992a,b)
Pinus elliottii
stem segments
cell wall, extracellular
elicitor
Lesney (1989)
Pinus radiata
xylem strips
cell wall
removal of growth
regulators, activated
charcoal
Möller et al. (2003)
Pinus taeda
young, growing stema
cell wall
change in growth
regulators
Eberhardt et al. (1993)
Pinus taeda
young, growing stema
extracellular
osmoticum
(8% sucrose)
Nose et al. (1995)
Pinus taeda
shoot tips
cell wall
change in growth
regulators
Stasolla et al. (2003)
Gymnosperms:
Angiosperms:
Arabidopsis thaliana
roots of seedlings
Arabidopsis thaliana
cell wall
brassinosteroid
Mathur et al. (1998)
Oda et al. (2005)
extracellular
spontaneous,
stimulated by methyljasmonate
Pauwels et al. (2008)
Populus alba
cambial zone of
2-y old branches
cell wall
spontaneous,
Tsutsumi and Sakai (1993)
stimulated by osmoticum
(5% or 8% mannitol)
Populus tremula
x P. tremuloides
stem of a plantlet
cell wall
spontaneous
Zinnia elegans
Christiernin et al. (2005)
leaf mesophyll
cell wall
0.44-4.4 µM BAP +
Fukuda & Komamine (1980a,
of seedlings
0.5 µM NAA
1982)
_____________________________________________________________________________________________________________
a
N.G. Lewis, personal communication
Legends for the Figures
Figure 1. Cell suspension culture of Norway spruce (Picea abies) (A) after transfer of callus cells
into liquid medium. (B) Four days later extracellular lignin is visible as a white precipitate in the
culture medium. Photograph courtesy of Sami Holmström.
Figure 2. Schematic representation of lignin biosynthesis. Lignin monomers, p-coumaryl, coniferyl
and sinapyl alcohol (collectively known as monolignols), are synthesised through the
phenylpropanoid pathway, starting from phenylalanine, which is converted to monolignols through
several hydroxylations, methylations and reductions. After secretion into the apoplast, the
monolignols are activated to radicals, which couple to form the lignin polymer. Radical formation is
catalysed by peroxidases and/or laccases. PAL, phenylalanine ammonia-lyase; C4H, cinnamate-4hydroxylase; 4CL, 4-coumarate:CoA ligase; HCT, hydroxycinnamoyl:CoA transferase; C3H, 5-O(4-coumaroyl)shikimate 3’-hydroxylase; CCoAOMT, caffeoyl-CoA O-methyl transferase; CCR,
cinnamoyl-CoA reducatase; CAD, cinnamyl alcohol dehydrogenase, F5H, ferulate/coniferaldehyde
5-hydroxylase; COMT, caffeate/5-hydroxyconiferaldehyde O-methyltransferase.
A
B