Planta

https://doi.org/10.1007/s00425-019-03137-y

REVIEW

Pinoresinol–lariciresinol reductases, key to the lignan synthesis

in plants
Lucija Markulin1 · Cyrielle Corbin1 · Sullivan Renouard1 · Samantha Drouet2 · Laurent Gutierrez2 · Ivan Mateljak1 ·
Daniel Auguin1 · Christophe Hano1 · Elisabeth Fuss3 · Eric Lainé1,4 

Received: 14 January 2019 / Accepted: 12 March 2019

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Main conclusion  This paper provides an overview on activity, stereospecificity, expression and regulation of pinores-
inol–lariciresinol reductases in plants. These enzymes are shared by the pathways to all 8–8′ lignans derived from
pinoresinol.

Pinoresinol–lariciresinol reductases (PLR) are enzymes involved in the lignan biosynthesis after the initial dimerization
of two monolignols. They catalyze two successive reduction steps leading to the production of lariciresinol or secoisola-
riciresinol from pinoresinol. Two secoisolariciresinol enantiomers can be synthetized with different fates. Depending on the
plant species, these enantiomers are either final products (e.g., in the flaxseed where it is stored after glycosylation) or are
the starting point for the synthesis of a wide range of lignans, among which the aryltetralin type lignans are used to semi-
synthesize anticancer drugs such as E ­ toposide®. Thus, the regulation of the gene expression of PLRs as well as the possible
specificities of these reductases for one reduction step or one enantiomer are key factors to fine-tune the lignan synthesis.
Results published in the last decade have shed light on the presence of more than one PLR in each plant and revealed vari-
ous modes of action. Nevertheless, there are not many results published on the PLRs and most of them were obtained in a
limited range of species. Indeed, a number of them deal with wild and cultivated flax belonging to the genus Linum. Despite
the occurrence of lignans in bryophytes, pteridophytes and monocots, data on PLRs in these taxa are still missing and indeed
the whole diversity of PLRs is still unknown. This review summarizes the data, published mainly in the last decade, on the
PLR gene expression, enzymatic activity and biological function.

Keywords  Enantiomer · Isoflavone reductase · Lignan · Pinoresinol reductase · Phenylcoumaran benzylic ether reductase ·
Pinoresinol–lariciresinol reductase

Abbreviations
ABA Abscisic acid
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s0042​5-019-03137​-y) contains DIR Dirigent protein
supplementary material, which is available to authorized users. e.e. Enantiomeric excess
IFR Isoflavone reductase
* Eric Lainé GA Gibberellic acid
eric.laine@univ‑orleans.fr
MeJA Methyl jasmonate
1
LBLGC, INRA USC 1328 Université d’Orléans, Orléans, PCBER Phenylcoumaran benzylic ether reductase
France PLR Pinoresinol–lariciresinol reductase
2
Centre Régional de Ressources en Biologie Moléculaire ROS Reactive oxygen species
(CRRBM), Université Picardie Jules Verne, 33 rue SA Salicylic acid
Saint‑Leu, 80039 Amiens, France SECO Secoisolariciresinol
3
Interfaculty Institute of Biochemistry, Hoppe‑Seyler‑St. 4, SDG Secoisolariciresinol diglucoside
72076 Tübingen, Germany
4
LBLGC, INRA USC 1328 Antenne Scientifique
Universitaire de Chartres, 21 rue de Loigny, 28000 Chartres,
France

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Introduction
Lignans
Lignans are widespread in the plant kingdom
Lignans are a major group of secondary metabolites widely
spread in the plant kingdom. However, unlike lignin they
are not present in all plants. Lignans can be found in non-
vascular plants such as Bryophytes, the hornworts Antho-
ceros punctatus and Megaceros flagellaris (Takeda et al.
1990) and liverworts Lepicolea ochroleuca and Bazzania
trilobata (Cullmann and Becker 1999; Scher et al. 2003)
as well as many Tracheophytes including Pteridophytes
like ferns of the Blechnaceae family (Wada et al. 1992).
They can be found in a number of Spermatophytes belong-
ing to the Gymnosperms, monocots and dicots. All parts
of the plants can contain lignans including knots in trees
(Holmbom et al. 2003), heartwood (Donoso-Fierro et al.
2009) and seeds, e.g., Linum and Sesamum (Moazzami
et al. 2007; Schmidt et al. 2012).
Lignans are diverse
Lignans are dimers of two phenylpropanoid units with a
different degree of oxidation and substitution patterns of
their aromatic moieties (Ward 1999; Teponno et al. 2016).
Structures formed by dimerization between the two central
carbon atoms (8 and 8′) of the side chains of the phenyl-
propanoid units (­ C6C3) form a lignan while other types of
linkages are classified as neolignans. Examples of neolig-
nans are phenylcoumarans in which the two phenylpropa-
noid units are linked by their 8-5′-C atoms (Niculaes et al.
2014) and oxyneolignans where the link between the two
phenylpropanoid units is indirect trough an oxygen atom
(Teponno et al. 2016). Norlignans contain a C16 to C17
core structure and are derived from arylpropane units by
the loss of one or two carbons (Teponno et al. 2016) and
often co-occur with lignans and neolignans.
Lignans are structurally a very diverse group. So far
almost two thousands of them have been described: 1729
lignans were reported in the Dictionary of Natural Prod-
ucts which contains 139,000 entries (Verpoorte 2000).
Based on their carbon skeleton, oxygen incorporation and
cyclization pattern they are classified into eight groups: aryl-
naphthalene (AN), aryltetralin (AT), dibenzocyclooctadiene
(DCO), dibenzylbutane (DB), dibenzylbutyrolactol (DBLL),
dibenzylbutyrolactone (DBL), furan (FR) and furofuran (FF)
group (Umezawa 2003; Teponno et al. 2016). Neolignans are
divided into 15 subgroups (NL1–NL15) (Satake et al. 2013,
2015; Teponno et al. 2016).
13
Lignans are pharmacologically active compounds
The exact biological role of the lignans in planta is not com-
pletely clarified even if their main function is hypothesized
to be protection against herbivores and pathogenic micro-
organisms. This is highly probable as some of the lignans
exhibit a strong toxicity level like podophyllotoxin. They
are mainly described as defensive molecules with antifun-
gal (Céspedes et al. 2006; Akiyama et al. 2007; Cho et al.
2007), antibacterial (Céspedes et al. 2006; Gaafar et al.
2013; Mahendra Kumar and Singh 2015) and antifeedant
(Kraus and Spiteller 1997) properties. Lignans can act as
phytoanticipins or phytoalexins (Naoumkina et al. 2010).
In the latter case, they are often synthesized in response to
fungal attack in various plant species (Gang et al. 1999).
They act as phytoanticipins in heartwood where they are
deposited as a protective postlignification infusion (Gang
et al. 1998) conferring resistance and durability as is the case
in Thuja plicata (Swan et al. 1969). Lignans can participate
in the protection/defense against competitors. There is evi-
dence of inhibitory effect of lignans on the seed germination
(Gutterman et al. 1980; Cutillo et al. 2003) and plant growth
(Elakovich and Stevens 1985; Oliva et al. 2002; Kato-Nogu-
chi et al. 2014) suggesting an allelopathic role of lignans
consistent with their presence at high amount in seeds or
roots of some plants. Additionally, other roles can be hypoth-
esized taking into account their localization and biochemical
properties. In the seeds they could participate in the defense
against reactive oxygen species (ROS) to protect the seed
oil content from oxidation or to maintain seed dormancy
given their antioxidant capacity (Thongphasuk et al. 2004;
Mahendra Kumar and Singh 2015; Li et al. 2016). A defense
against oxidative stress has also been proposed as a role of
oligolignols during the lignification process in poplar (Nicu-
laes et al. 2014). Neolignans could play a role in plant height
as Bruegmann et al. (2018) have shown that the knockdown
of PCBER1 in poplar resulted in increased growth (PCBER
is an enzyme similar to PLR but acting on neolignans). The
chelation ability of SDG reported by Fucassi et al. (2014)
suggests that lignans may play a role in iron (or other metal)
capture to facilitate its intake (phytosiderophore) or internal
transport (phytochelatin). This chelation could also contrib-
ute to the protection against ROS generation by metals in the
cell. A number of biological activities have been reported
for lignans. Many of them have positive impact on human
health and are likely linked to the roles mentioned above.
Cytotoxic lignans, such as podophyllotoxin, have antipro-
liferative activity making them irreplaceable as anticancer
drugs (Canel et al. 2000; Gordaliza et al. 2004). Lignans also
exhibit antiviral (Gordaliza et al. 2004; Allen et al. 2007),
antiangiogenic (Basini et al. 2012), hepatoprotective (Yang
et al. 2014; Al-Jumaily and Al-Azawi 2015), neuroprotective
(Li et al. 2012; In et al. 2015; Zhang et al. 2016), antioxidant
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(Hu et al. 2007; Hano et al. 2017), anti-inflammatory (Kas-
suya et al. 2005; Baumgartner et al. 2011; Zheng et al. 2014)
and antiparasitic (Marcotullio et al. 2014) properties.
Flax lignans have been extensively investigated and their
positive health benefits make flax a functional food. Some
of the favorable effects are the reduction of blood pressure
(Ursoniu et al. 2016), cardioprotection (Zanwar et al. 2011)
and the prevention of hormone-dependent cancers thanks
to their phytoestrogenic features (Lainé et al. 2009; Dik-
shit et al. 2016). Flax lignans are also involved in retarding
development of type 2 diabetes in rats (Prasad 2001) while
dietary lignan supplementation showed positive effect in
type 2 diabetic patients (Pan et al. 2007) which can be due
to an inhibitory effect on alpha-amylase (Hano et al. 2013).
However, epidemiological studies are not always in favor of
the preventive effect (Zamora-Ros et al. 2013). Nevertheless,
we should keep in mind that lignan content of plant food is
still not well known and thus epidemiological studies based
on the calculations of lignan intake should be considered
with caution.
Forsythia, Sesamum and Linum species are model plants
for lignan studies
The model plants used so far for functional genetic studies,
Arabidopsis thaliana and Nicotiana tabacum, are not con-
venient models for the lignan synthesis because of poor or
null lignan contents. Classical model plants do not have, or
contain incomplete, biosynthetic pathway for these special-
ized metabolites. As a consequence, majority of the data
available on this subject come from lignan-rich plants such
as Linum, Forsythia, Sesamum and Podophyllum species for
flowering plants or Thuya, Juniperus, Callitris, Tsuga and
Picea for gymnosperms.
Three genera, Linum, Sesamum and Forsythia, are the
most frequently encountered in published work on lignan
synthesis with about sixty papers for “Forsythia AND
lignan” request on Medline/PubMed, about one hundred
for “Sesamum AND lignan” and almost four hundred for
“Linum AND lignan” (among which a number dedicated
to biological activities). The highest production of lignans
within the non-woody plants is found in cultivated flax.
Indeed, Linum usitatissimum has the highest content of
lignan SDG in seeds (Johnsson et al. 2000; Eliasson et al.
2003), more than 60–700 times higher than in any other
analyzed plant-derived food (Thompson et al. 1991). Lig-
nan diversity in Linum species was investigated by Schmidt
et al. (2010, 2012). Analyses of aerial parts of 41 members
of Linum species show 64 different lignans that are divided
into two major groups, aryltetralines (AT) (sections Sylli-
num, Linopsis and Cathartolinum) and aryldihydronaph-
thalene/arylnaphthalene (ADN/AN) (sections Linum and
Dasylinum). Some members of the section Linum contain
high quantities of dibenzylbutyrolactones (e.g., L. usitatis-
simum), while furofurano lignans are only found in L. bienne
(Schmidt et al. 2010). Seed analyses of 20 Linum species
show similar distribution as in aerial parts in the flowering
stage. Section Syllinum contains AT lignans while Linum
and Dasylinum generally contain ADN/AN lignans. SDG
is found in all but one investigated species of Linum and
Dasylinum. On the contrary, no SDG is detected in sec-
tion Syllinum. Most species contained only 8R,8′R-SDG,
while 8S,8′S-SDG is the predominant form in only three
species (L. usitatissimum, L. decumbens and L. narbonense)
(Schmidt et al. 2012).
Pinoresinol–lariciresinol reductases
The biosynthetic pathway of lignans
The biosynthesis of lignans has been well studied and the
proposed pathway is confirmed and widely accepted (Ford
et al. 2001). Lignans and lignin share a common phenyl-
propanoid precursor, namely the coniferyl alcohol and both
lignin formation and the first step in monolignol polymeriza-
tion are considered to arise via bimolecular phenoxy radical
coupling. A difference being in the stereoselective and the
stereospecific coupling of optically active lignans whereas
the optically inactive lignin is formed in a non-stereospecific
process (Gang et al. 1999). The synthesis of lignans starts
with the coupling of two coniferyl alcohols by an oxidase
(laccase or peroxidase) with the help of a dirigent protein
(DIR) to form pinoresinol (Davin et al. 1997; Dalisay et al.
2015). DIR proteins may have a role in the formation of dif-
ferent pinoresinol enantiomers by holding coniferyl alcohols
in a specific orientation during coupling.
Pinoresinol–lariciresinol reductase (PLR) converts
pinoresinol to lariciresinol and then to SECO. Most of the
characterized PLRs catalyze both reduction steps (von Hei-
mendahl et al. 2005). Known exceptions are A. thaliana
reductases that catalyze only the first step with a reasonable
efficiency and are thus named pinoresinol reductases (PrR).
AtPrR2 has a 35 times lower affinity towards lariciresinol
than for pinoresinol while AtPrR1 does not reduce laricires-
inol at all (Nakatsubo et al. 2008). However, a limited num-
ber of PLRs from different species have been investigated
but genome sequencing allows now speculations that there
are other isozymes still not identified. In L. usitatissimum,
as much as five PLR genes are present in the genome that
encode full PLR proteins (data not shown) but only two L.
usitatissimum PLRs have been studied so far. Therefore, it
is possible that the two reduction steps could in some cases
require actions of different PLRs.
The reduction by PLR is a crucial step in lignan metabo-
lism since its product represents the entry point for the syn-
thesis of various 8–8′-linked lignans: furano, dibenzylbutane,
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dibenzylbutyrolactone and aryltetrahydronaphthalene (Dink-
ova-Kostova et al. 1996). The biosynthetic pathway to arylte-
tralin lignans is the subject of a profound interest as it con-
cerns the biosynthesis of podophyllotoxin, a natural product
used to produce the widely used anticancer drug E ­ toposide®
by semisynthesis. Depending on the plant species and organ,
SECO can be oxidized to form matairesinol by SECO dehy-
drogenase (SDH) (Xia et al. 2001) or SECO can be glyco-
sylated to SDG probably by UGT 74S1 glycosyltransferase
(Ghose et al. 2014; Fofana et al. 2017), as is often the case
in seeds of L. usitatissimum. Like SECO, pinoresinol (Ono
et al. 2010), lariciresinol (Kikuchi and Sugiyama 1993) and
matairesinol (Schmitt and Petersen 2002) can be found in
their glycosylated forms.
The PLRs were most likely present in plants even before
lignin synthesis appeared in the land plants given that lig-
nans are found in bryophytes. The availability of their sub-
strate pinoresinol could be related to the presence of a dozen
DIR genes that are already found in these early land plants
(Corbin et al. 2018). DIR are present in Physcomitrella
patens and induced in response to pathogen attack (Ponce
De León et al. 2012; Reboledo et al. 2015). Moreover, they
might be involved in dimerization of molecules other than
monolignols (Effenberger et al. 2015). Nevertheless, consid-
ering the presence of lignans in these non-spermatophytes
land plants, they should be able to display PLR enzymatic
activity despite no work to attest to that has been published
so far.
Phylogenetic tree of published PLRs and reflections
on evolution
Different reductases are involved in the further conversion
of phenylpropanoid pathway products. PLRs, IFRs (isofla-
vone reductases) and PCBERs (phenylcoumaran benzylic
ether reductases) are phylogenetically linked reductases that
show high sequence similarity and similar catalytic modes of
action while their products share similar roles often involved
in plant defense (Gang et al. 1999). They are NADPH reduc-
tases with conserved “GXXGXXG” sequence at the N-ter-
minal end which form the PIP (PLR/IFR/PCBER) family.
Both PLRs and IFRs have short characteristic amino acid
sequence insertions (von Heimendahl et al. 2005).
Moummou et  al. (2012) performed a genome-wide
inventory of short-chain dehydrogenase superfam-
ily searches of PLR/IFR enzymes in different species.
They identified several PLR/IFR enzymes in Selaginella
moellendorffii (5), Arabidopsis thaliana (8), Populus
trichocarpa (16), Vitis vinifera (14), Glycine max (15),
Oryza sativa (10), Zea mays (7) and Sorghum bicolor
(7). However, none were found in the bryophyte Phy-
scomitrella patens. Note that these numbers do not only
encompass PLRs but include IFRs, vestitone reductase,
13
PCBERs, and eugenol synthase. Note also that the pres-
ence of reductases able to convert pinoresinol is not lim-
ited to plants as a PrR is also found in sphingomonads
bacteria (Fukuhara et al. 2013) thus potentially implicating
horizontal gene transfer (Moummou et al. 2012). Moreo-
ver, the ability to convert pinoresinol to lariciresinol and
SECO has been shown for the gastrointestinal bacteria
Eggerthella lenta, strain SECO-Mt75m3 (Clavel et  al.
2006) and Enterococcus fecalis, strain PDG-1 that is able
to convert (+) pinoresinol to (+) lariciresinol (Xie et al.
2003) which indicates the possible presence of enzymes
similar to pinoresinol–(lariciresinol) reductases.
The bacterial pinZ gene from Sphingobium sp. strain
SYK-6 has been reported to encode for a pinoresinol
reductase able to convert racemic pinoresinol to laricires-
inol, but not lariciresinol to SECO, despite a low per-
centage identity (around 15–21%) with plant pinores-
inol–lariciresinol reductases (Fukuhara et  al. 2013).
Interestingly, heterologous expression of pinZ gene in
transgenic A. thaliana resulted in metabolic alterations of
lignan pathways with increased lariciresinol accumulation
in stems (Tamura et al. 2014).
Genes encoding enzymes involved in the biosynthesis
of secondary metabolites are often more divergent than
housekeeping genes and a clear evolutionary path and
emergence of enantiospecificity in these reductases are not
elucidated; however, there are several hypotheses. As both
PLRs and IFRs use NADPH as a cofactor and abstract the
4R-hydride in a regio- and enantiospecific manner while
PCBERs are only regiospecific, Gang et al. (1999) suggest
that PCBERs, as highly ubiquitous in the plant kingdom
and only regiospecific, are progenitor enzymes of PLRs
and IFRs and that enantiospecificity in PLRs and IFRs has
evolved later. On the other hand, von Heimendahl et al.
(2005) based on the lack of clustering of PIP enzymes
on the level of reaction’s enantiospecificity (PLRs with
the same enantiospecificity do not group together) suggest
that enantiospecificity either evolved several times during
evolution or appeared very early maintaining the residues
for enantiospecificity conserved and changing only non-
essential amino acids during later evolution. Indeed, Thuja
plicata PLRs cluster together despite opposite enantio-
specificity. Fujita et al. (1999) proposed that the reason is
that enzymes are from the same species and that most of
the differences in amino acid sequences do not affect enan-
tiospecificity or that similarity contributes to the ability of
TpPLRs to reduce both pinoresinol enantiomers. Addition-
ally, Nakatsubo et al. (2008) note that angiosperm PLRs
cluster together (FiPLR1, LaPLR1, LuPLR1, LpPLR1,
AtPrR1 and AtPrR2) and gymnosperm PLRs cluster
together (TpPLR1 and TpPLR2) suggesting that there is
an independence between enantiomeric selectivity and the
sequences of PLRs/PrRs.
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Expression patterns and regulation Table 1  Expression pattern of pinoresinol–lariciresinol reductases in

different species following different hormone treatments and biotic
and abiotic stresses
Stereochemistry and enantiomeric composition of lignans
vary not only between species but also between different Abscisic acid
plant organs and developmental stages. PLR genes present  ↑ LuPLR1 Renouard et al. (2012), Corbin et al. (2013a, b)
spatio-temporal and organ-specific expression patterns.  ↑ IiPLR1 Xiao et al. (2015)
In Arctium lappa, the lignan composition differs between Gibberellic acid
organs but no PLRs were cloned nor was their expression  ↓ LuPLR1 Corbin et al. (2013a, b)
investigated so far. In flax, the expression of LuPLR1 has Methyl jasmonate
been localized in the seed coat a decade ago (Hano et al.  ↑ LuPLR2 Corbin et al. (2017)
2006b). This localization was logical given the accumulation  ↑ IiPLR1 Xiao et al. (2015)
of the lignan complex in this tissue that contains almost all  ↑ PhPLR Wankhede et al. (2013)
the SECO synthetized by flax in its diglucosylated form. The Salicylic acid
timing of the LuPLR1 gene expression is well coordinated  ↓ LaPLR1 Yousefzadi et al. (2010)
with the timing of SECO production along the different Fungal elicitor
stages of seed development, i.e., the expression of LuPLR1  ↑ LaPLR1 Esmaeilzadeh Bahabadi et al. (2012, 2014),
is increasing at the beginning of the seed development. In Tashackori et al. (2019)
flax, two PLRs, namely LuPLR1 and LuPLR2, are expressed  ↑ LuPLR1 Hano et al. (2006a), Hano et al. (2008)
in the seed and root but only LuPLR2 is also expressed in UV-B
stems and leaves (Hemmati et al. 2010). In Arabidopsis,  ↑ IiPLR1 Xiao et al. (2015)
AtPrR2 is root specific and AtPrP1 is highly expressed in Blue light
root and stem tissue (Nakatsubo et al. 2008). Zhao et al.  ↑ LaPLR1 Yousefzadi et al. (2012)
(2015) suggest an association of AtPrP1 with secondary Wounding
cell wall synthesis in fiber cells. They show that AtPrP2 is  ↑ PhPLR Wankhede et al. (2013)
coexpressed with two genes potentially involved in the for-
mation of the Casparian strip in roots. Further investigations
are necessary to explain the high expression levels of both (van Fürden et al. 2005; Yousefzadi et al. 2010; Esmaeilza-
AtPrP1 and AtPrP2 in roots. deh Bahabadi et al. 2012; Tahsili et al. 2014; Esmaeilzadeh
Stress and hormone-driven expression can also occur. Bahabadi et al. 2014; Tashackori et al. 2018). Esmaeilzadeh
Results in this domain are summarized in Table 1. In flax, Bahabadi et al. (2012, 2014) found that the Fusarium extract
it appears that the expression of LuPLR1 is triggered by the boosted both the podophyllotoxin production and LaPLR
plant hormone ABA which plays a crucial role in the seed expression. Nevertheless, the PLR expression and final
maturation and dormancy (Renouard et al. 2012). Not sur- product synthesis are not necessarily linked. Even though a
prisingly, gibberellic acid (GA) exerted an opposite regula- lignan synthesis improved over three times compared with
tion effect on the expression of LuPLR1 (Corbin et al. 2013a, control cultures following the SA treatment, the LaPLR
b). Working on a cell suspension of flax, Hano et al. (2006a) expression was not found to be stimulated (Yousefzadi et al.
found that expression of the LuPLR1 gene is increased when 2010). These results suggest the existence of a complex reg-
the culture is treated with a Fusarium oxysporum extract. ulation of the whole pathway and a differential control of the
Nevertheless, the lignan accumulation does not increase and gene expression between distinct transduction signals that
even drops in this situation indicating either an incorporation mediate the hormonal control which is confirmed by the
of lignan in the lignin (which was overproduced), a lack of work conducted on flax.
precursor caused by another consequence of this biotic stress The Podophyllum hexandrum gene PhPLR shows an
(it could result from a low DIR availably) or a regulation of increase in expression as a response to wounding stress
the enzyme itself. and MeJA treatment as well as response to UV treatment of
A number of results have been published on Linum album, leaves. Synthesis of podophyllotoxin has been shown to be
a wild flax able to produce podophyllotoxin, the aryltetralin MeJA responsive (Wankhede et al. 2013).
lignan used for the semisynthesis of E ­ toposide®, one of the In situ hybridization conducted on Forsythia inter-
lead anticancer compounds listed as essential medicine by media shows that FiPLR1 mRNA accumulates mainly in
the World Health Organization (2015), for which there is an young leaves followed by young roots and petioles. In stem,
intensive search for alternative sources. Methyl jasmonate FiPLR1 accumulates in the radial parenchyma cells and
(MeJa), salicylic acid (SA) and chitosan or fungal extracts the cambial cells of secondary xylem (Kwon et al. 2001).
(Fusarium or Piriformospora) are able to trigger an over- This result corresponds with an accumulation of secondary
production of podophyllotoxin when applied to the cultures metabolites in the secondary xylem of F. intermedia and

13
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coincides with DIR expression (mainly in the vascular cam-
bium and ray parenchyma cell initials of stem) (Burlat et al.
2001). However, expression seems to differ over time. PLR
expression appears to alternate between seasons. FiPLR1 is
stably expressed from April to August, but no expression
is detected from September to November. Seasonal expres-
sion of FiPLR1 is consistent with seasonal lignan profiles in
leaves of F. intermedia (pinoresinol, matairesinol and arc-
tigenin synthesis increase from April to June) (Morimoto
and Satake 2013).
Most published data on the expression and regulation of
PLRs refer to two PLRs from L. usitatissimum. LuPLR1
is involved in (+)-SECO synthesis whereas LuPLR2 is
involved in (−)-SECO and eventually to yatein synthesis
(Corbin et al. 2017).
LuPLR1 and LuPLR2 are expressed in the seed and root
but only LuPLR2 is also expressed in stems and leaves
(Hano et al. 2006b; Hemmati et al. 2010). Expression in the
seeds is mainly localized in the seed coat during all devel-
opmental stages of the seed. The expression of LuPLR1 is
increasing at the beginning of the seed development with
a higher expression in earlier developmental stage than in
mature seed. In addition, seed coats of aborted seeds show
no activity of the LuPLR1 gene promoter suggesting a role
of the embryo in the control of PLR gene expression (Hano
et al. 2006b). The timing of the LuPLR1 gene expression is
well coordinated with the timing of SECO production along
the different stages of seed development. This localization
was logical given the accumulation of the lignan complex
in this tissue that contains almost all the SECO synthetized
by flax in its diglucosylated form (SDG).
Analysis of the LuPLR gene promoter regions revealed
several cis-acting elements potentially involved in its regu-
lation including ABRE (ABA-response element), MYB-
PLANT, MYB and W boxes (Hano et al. 2006b) but so far
there is a lack of published results dealing with transcrip-
tion factors implicated in the regulation of PLR expression.
The involvement of a prenylation event in the transcriptional
regulation caused by ABA has been demonstrated by Cor-
bin et al. (2013a). ABRE and MYB2 ABA-response ele-
ments located in the promoter of LuPLR1 are both required
for the LuPLR1 regulation by ABA, but the regulation by
ERA1 (involving protein farnesylation events) is operated
only through the ABRE sequence. AtPrR1, implicated in
lignin distribution in secondary cell wall biosynthesis, is
regulated by the secondary cell wall-related transcription
factors SND1 and MYB46 (Zhao et al. 2015).
Two phytohormones show opposite influence on the
lignan biosynthesis. A strong relationship between ABA,
a key growth regulator and the LuPLR1 gene expression
and SECO synthesis has been shown both in flax seeds
(Renouard et al. 2012) and flax cell suspension cultures
(Corbin et al. 2013b). ABA accumulation in seeds starts in
13
developmental stage 2 (WS2) and reaches its maximum in
WS4 when the near mature seed is filled (Renouard et al.
2012). In WS2 ABA accumulates in the seed coat followed
by accumulation in embryo in WS3 and WS4 (WS4 seed
coat:embryo = 45:55) (Renouard et al. 2012). Exogenous
application of ABA to seeds in WS2 causes an increase of
LuPLR1 gene expression and SECO quantity while inhibi-
tion of ABA biosynthesis causes their decrease (Renouard
et al. 2012).
A negative regulator, gibberellin, causes a decrease in
the LuPLR1 gene expression and SECO synthesis prob-
ably by a direct effect on lignan synthesis and not through
an effect on the ABA biosynthesis or catabolism (Corbin
et al. 2013b). Effect of the ABA is realized through at least
two cis-regulation elements present in the LuPLR1 gene
promoter. Mutation of either ABRE or MYB2 element
decreases ABA-induced LuPLR1 gene expression while
combined mutation of both boxes completely abolishes the
positive effect of the ABA (Corbin et al. 2013b). On the
other hand, down-regulation of LuPLR1 gene expression
influenced by GA is realized through the ABRE element.
Its mutation abolishes negative influence of GA on the
LuPLR1 expression (Corbin et al. 2013b).
Another negative effector of the LuPLR1 gene
expression and SECO synthesis is the LuERA1 protein
(enhanced response to ABA) (Corbin et al. 2013a). It is
a β-subunit of the heterodimeric protein called protein
farnesyltransferase. It makes a post-translational modifi-
cation by transferring a C15 farnesyl unit to the C-ter-
minal end of a cysteine of the target protein. LuERA1 is
expressed in roots, leaves, cell suspension cultures, but
predominantly in the stem and flax seeds. The strong-
est expression is in the seed coat which corresponds to
LuPLR1 gene expression and lignan biosynthesis (Cor-
bin et al. 2013a). The expression overlap of LuPLR1 and
LuERA1 enables LuERA1 to modulate gene expression
of the LuPLR1. LuERA1 probably effects lignan synthe-
sis through negative regulation of ABA signaling. The A.
thaliana era1 mutant shows enhanced sensitivity to ABA
while inhibition of farnesyltransferase positively affects
lignan biosynthesis (Corbin et al. 2013a). LuERA1 action
is probably mediated through ABRE. Indeed, the mutation
of ABRE abolishes the positive effect of the farnesyltrans-
ferase inhibitor on the LuPLR1 gene (Corbin et al. 2013a).
The LuERA1 could possibly inhibit LuPLR1 expression
through farnesylation of the ABI3 (ABA insensitive 3)
transcription factor involved in the ABA signaling (Brady
et al. 2003; Corbin et al. 2013a).
The LuPLR1 gene shows only a weak activation in the
cell suspensions treated with Botrytis cinerea or Fusarium
oxysporum between 8 and 48 h post-elicitation (Hano et al.
2006a). However, total SECO (both aglycone and SDG) con-
tent in a flax cell suspension decreases 96 h post-elicitation
Planta
with F. oxysporum and remains unchanged in cells treated
with B. cinerea.
LuPLR2 is involved in the (−)-SECO biosynthesis which
serves as a precursor of lignans accumulating mainly in
leaves, e.g., yatein, hinokinin, bursehernin, thujaplicatin, and
matairesinol dimethyl ether (von Heimendahl et al. 2005;
Hemmati et al. 2010). Corbin et al. (2017) demonstrated that
LuPLR2 is involved in the biosynthesis of (−)-yatein accu-
mulated in flax leaves. Indeed, in flax, LuPLR2 gene expres-
sion and yatein production increase after MeJA treatment
and wounding, which is in accordance with an involvement
of these lignans in the plant defense response.
Structure of PLRs
The three-dimensional structures of Thuja plicata PLR1
(TpPLR1) were elucidated by Min et al. (2003). Crystal-
lization was performed using the vapor diffusion method
and diffracted X-ray crystal structures were refined to 2.5 Å
resolutions. Sedimentation equilibrium experiments and
MALDI-TOF analysis showed TpPLR1 as a dimer with an
apparent molecular mass of 69.9 ± 0.4. Overall structure
consists of two domains, a cofactor binding N-terminal
domain and a substrate-binding C-terminal domain. The
Fig. 1  2D topological structure plot of pinoresinol–lariciresinol reductase
two domains form a cleft between them and are connected
by five separate peptide segments (Fig. 1).
The larger N-terminal domain forms seven parallel
β-strands surrounded by six α-helices and the smaller C-ter-
minal domain contains two separate β-plated sheets and four
small α-helices. The N-terminal domain of TpPLR1 shows
highest similarity (~ 70%) with IFRs, 43% similarity with
an E. coli UDP-galactose 4-epimerase and several enzymes
belonging to short-chain dehydrogenases/reductases (Min
et al. 2003), but the C-terminal domain shows sequence
similarity only with IFRs and IFRHs. However, some of
the secondary structural elements of the C-terminal domain
appear to be in a similar topological arrangement as those of
UDP-galactose 4-epimerase (Min et al. 2003).
As other NADPH reductases TpPLR1 contains the con-
served GXXGXXG NADPH-binding motif at the N-termi-
nal forming the first β–α–β unit. The nicotinamide ring is
orientated toward the cleft between the two domains in anti-
conformation and is stabilized by a stacking interaction with
the side chain of the conserved Phe.
A PLR-specific amino acid insert seems to be involved
in the formation of the substrate-binding pocket making it
important for PLR-specific activity (Min et al. 2003). The
substrate-binding pocket is mostly hydrophobic made up of
13
 Planta

two β-strands and two α-helices. The substrate-binding site
is adjacent to the cofactor so that the substrate molecules
are orientated to face the nicotinamide ring of NADPH.
Residues involved in cofactor binding are T ­ yr15, ­Ile16, ­Ser41
142
and ­Arg . In the putative active site there is a conserved
­Lys138 whose change to Ala abolishes the ability to convert
pinoresinol suggesting that ­Lys138 is involved in general base
catalysis (Min et al. 2003).
Structure–function studies were performed using
two enantiospecifically opposite PLRs from T. plicata.
Substrate-binding sites differ in the local environment
between TpPLR1 and TpPLR2 that use (−)-pinoresinol
and (+)-pinoresinol as substrates, respectively. In TpPLR1
(−)-pinoresinol fits among the hydrophobic side chains of
­Phe164, ­Val268, and ­Leu272 but in TpPLR2 symmetric sub-
stitution between sites 164 and 272 and change of Val to
Gly causes favoring of (+)-pinoresinol ­(Leu164, ­Gly268 and
­Phe272) (Min et al. 2003).
PLRs could form an active multienzymatic complex with
upstream (DIR) and downstream (SDH or UGT) enzymes
that could explain how opposite enantiomeric forms of
lignans are channeled in one or the other pathway. Examples
of such behavior already exist in the flavonoid biosynthesis
pathway where a cytochrome P450 type enzyme anchored to
the ER membrane that forms a metabolon with dihydrofla-
vonol-4-reductase and leucoanthocyanidin reductase. Dur-
ing biosynthesis, proteins associate transiently and substrates
move from one active site to another via substrate channeling
(Diharce et al. 2016). The substrate needs to diffuse from
one active site to the next one, so only the protein–protein
structures with a possible pathway between cavities should
be considered.
PLRs contain putative phosphorylation sites, some
of them being conserved as shown by multiple sequence
alignment. This feature opens the possibility of regulation
by phosphorylation at the enzymatic level (Gang et al. 1999).
Enzymatic activity
After the first PLRs were cloned and characterized from For-
sythia intermedia and Thuja plicata (Katayama et al. 1993;
Dinkova-Kostova et al. 1996; Fujita et al. 1999) (Table 2),

Table 2  Characteristics of known pinoresinol–lariciresinol reductases (PLR)

Species PLR cDNA (nt) ORF (nt) Protein (aa) Mw (kDa) pI References

Forsythia intermedia FiPLR1 (U81158) 1047 939 312 34.9 7.09 Dinkova-Kostova et al. (1996)
Arabidopsis thaliana AtPrR1 (NM_102944) 1129 954 317 35.5 5.9 Nakatsubo et al. (2008)
AtPrR2 (NM_117440) 1206 954 317 35.4 5.79
Isatis indigotica IiPLR1 (JF264893) 1062 954 317 35.6 5.63 Xiao et al. (2015)
Linum album LaPLR1 (AJ849358) 1482 981 326 36.4 5.65 von Heimendahl et al. (2005)
Linum flavum LfPLR1 (medp_ – – 304a 33.9 595 Shiraishi et al. (2016)
linfl_20101112|16825)
LfPLR2 (medp_ – – 228a 24.9 6.31
linfl_20101112|20619)
Linum corymbulosum LcPLR1 (EU107358) 1244 948 315 35.1 6 Bayindir et al. (2008)
Linum perenne LpPLR1 (EF050530) 1145 945 314 35.2 5.65 Hemmati et al. (2007)
Linum usitatissimum LuPLR1 (AJ849359) – 936 312 35.03 5.34 von Heimendahl et al. (2005)
LuPLR2 (EU029951) 1203 993 330 36.5 6.03 Hemmati et al. (2010)
Podophyllum hexandrum PhPLR (EU855792) 983  936 311 34.8 6.44 Wankhede et al. (2013)
Podophyllum pleianthum PpPLR (AHL21381) – 933 311 34.9 6.32 Kuo et al. (2014)
Taiwania cryptomerioides TcPLR1 (MG264424) – 975 324 36.4 5.82 Chiang et al. (2018)
TcPLR2.2 (MG264425) – 942 313 35.6 5.95
TcPLR3 (MG264426) – 939 312 35.0 5.48
Thuja plicata TpPLR1 (AF242503) 1190 942 313 35.6 6.01 Fujita et al. (1999)
TpPLR2 (AF242504) 1151 939 312 34.8 5.65
TpPLR3 (AF242505) 1308 945 314 35.4 6.34
TpPLR4 (AF242506) 1287 939 312 34.8 5.95
Tsuga heterophylla ThPLR1 (AF242501) 1282 798 265 29.9 5.51 Gang et al. (1999)
ThPLR2 (AF242502) 1314 930 309 34.8 6.62

Protein characteristics (http://web.expas​y.org/protp​aram/)

nt nucleotides, ORF open reading frame, aa amino acid
a
 Partial sequence

13
Planta
PLRs were well known as reductases catalyzing two sub-
sequent reductions from pinoresinol via lariciresinol to
SECO. These first characterized enzymes seemed to exhibit
enantiospecificity in both reduction steps for either of the
enantiomeric configuration at the 8,8′-C atoms, namely R,R
for (+)-pinoresinol, (+)-lariciresinol and (−)-SECO and S,S
for (−)-pinoresinol, (−)-lariciresinol and (+)-SECO (Fig. 2).
This reflects the complexity of the catalytic action of PLRs
as reductases catalyzing two reduction steps dealing in
total with four substrates taking into account the respec-
tive enantiomers of pinoresinol and lariciresinol as possible
substrates. This may be the reason why not so many PLRs
are in depth characterized up to now. Nevertheless, the exist-
ing data broaden our understanding of these multifunctional
enzymes.
To characterize the identified PLRs with respect to their
catalytic function in more detail some proteins were heter-
ologously expressed in E. coli, namely two enzymes from
Thuja plicata (TpPLR1 and TpPLR2), Forsythia interme-
dia (FiPLR1 and FiPLR), Arabidopsis thaliana (AtPrR1 and
AtPrR2), and Linum usitatissimum (LuPLR1 and 2) and one
from Isatis indigotica (IiPLR1), Linum album (LaPLR1),
Linum perenne (LpPLR1) and Linum corymbulosum
(LcPLR1), respectively (Fig. 2, Table 3).
These experiments allowed to evidence the different
behaviors of the PLR enzymes. Whereas all other PLRs so
far characterized as recombinant purified proteins catalyze
both reductions from pinoresinol to lariciresinol and from
lariciresinol to secoisolariciresinol, the AtPrR1 has an about
35 times lower activity towards lariciresinol than pinoresinol
and the AtPrR2 shows no catalytic activity towards laricires-
inol as substrate at all. For that reason, Umezawa (2003)
named them pinoresinol reductases instead of pinores-
inol–lariciresinol reductases. It is also the AtPrR1 which
shows a very unusual behavior by exhibiting almost no enan-
tiospecificity for either the pinoresinol or lariciresinol enan-
tiomer. The other PLRs characterized so far show at least a
preference for one or the other enantiomer of pinoresinol
and lariciresinol.
The two PLRs cloned and characterized from Forsythia
intermedia by Dinkova-Kostova et al. (1996) were the first
PLRs cloned. They show a marked enantiospecificity prefer-
ring (+)-pinoresinol and (+)-lariciresinol to form (−)-SECO
with comparable Km and Vmax values for pinoresinol of about
25 µM and about 17 µmol h−1 mg−1 and for lariciresinol
of about 122 µM and 27 µmol h−1 mg−1, respectively. The
same is true for the IiPLR1 from Isatis indigotica show-
ing comparable Kcat/Km values for both reduction steps in
the range of 0.9–1.6 µM−1 min−1 although no experiments
with respect to the enantiospecificity of this enzyme were
performed (Xiao et al. 2015).
The PLRs 1 and 2 from Thuja plicata have an opposite
enantiospecificity: TpPLR1 prefers the (−)-enantiomers
of both pinoresinol and lariciresinol whereas the TpPLR2
prefers the (+)-enantiomers of pinoresinol and lariciresinol.
The determination of the kinetic parameters for these two
PLRs with respect to the pinoresinol enantiomers as sub-
strate confirmed this specificity (Davin et al. 2008, Table 3).
The highest catalytic efficiency was observed for the conver-
sion of (+)-pinoresinol by the TpPLR2 with 72 µM−1 min−1.
TpPLR2 converts (−)-pinoresinol with around sevenfold less
catalytic efficiency. The catalytic efficiencies of TpPLR1 for
both of the pinoresinol enantiomers is at least 30 times lower
than the one of TpPLR2, leading the authors to conclude
that the latter is presumably the main PLR used in planta
(Fujita et al. 1999). The two PLRs of Linum usitatissimum
characterized so far also show opposite enantiospecificity
for pinoresinol and lariciresinol (Hemmati et al. 2010). But,
an in-depth investigation of their expression in planta shows
that these PLRs are differentially expressed and, therefore,
are responsible for the enantiomeric composition of the
different lignans accumulated in the different plant organs
(Hano et al. 2006b; Hemmati et al. 2010; Renouard et al.
2012; Corbin et al. 2017). Although PLRs from Arctium
lappa are only investigated from raw extracts from petioles
and leaves, the opposite enantiospecificity observed from the
enzyme extracts of these different plant organs (Suzuki et al.
2002) suggests once more that PLRs with opposite enantio-
specificity are differentially expressed and are responsible
for an organ-specific accumulation of lignans with differ-
ent enantiomeric composition. Therefore, presumably the
Thuja PLRs are differentially expressed as well and may
be involved in different physiological processes in planta.
Although not verified by the determination of kinetic
parameters Km and Vmax, the tracking of the consumption of
one or the other enantiomer of pinoresinol and/or laricires-
inol can give a clue to the substrate specificity of PLRs as
well. When the (±)-pinoresinol consumption until depletion
and (±)-lariciresinol and subsequently (∓)-SECO formation
was followed for the TpPLRs, no (−)-lariciresinol as inter-
mediate was visible with (+)-SECO as product together with
(+)-lariciresinol in the assays with TpPLR1. On the other
hand, with TpPLR2, consumption of the enantiomeric mix-
ture of pinoresinol resulted in an accumulation of both enan-
tiomers of lariciresinol, in which only the (+)-enantiomer
was further converted into (−)-SECO (Fujita et al. 1999).
This observation fits very well to the measured kinetic
parameters as described above. A similar behavior is found
for the two LuPLRs: LuPLR2 converts only (+)-pinoresinol
to (−)-SECO showing no accumulation of (−)-lariciresinol
as intermediate, indicating a higher catalytic efficiency
for the conversion of lariciresinol over pinoresinol. In the
reaction of LuPLR1, the conversion of (+)-pinoresinol to
(+)-lariciresinol seems to be more effective than the further
conversion of the latter to (−)-SECO. In contrast, SECO
production in F. intermedia is only retarded and happens
13
 Table 3  Enantiospecificity and kinetic properties of isolated and characterized pinoresinol–lariciresinol reductases and enzymatic activity of plant protein extract in different plants species
Species PLR PINO LARI SECO Kinetic parameters/notes Expression References

13
Forsythia intermedia FiPLR1 (AAC49608) + > 99:1 + − Km = 27 ± 1.5 (pino) and Young green stem Dinkova-Kostova et al. (1996)
121 ± 5.0 μM (lari) Isolated two isofunctional forms
Vmax = 16.2 ± 0.4 (pino) and
25.2 ± 0.7 μmol h−1 mg−1 (lari)
FiPLR + + − Km = 23 ± 1.3 (pino) and
123 ± 6.0 μM (lari)
Vmax = 17.3 ± 0.5 (pino) and
29.9 ± 0.7 μmol h−1 mg−1 (lari)
Arabidopsis thaliana AtPrR1 (NP_174490) ± ± 35× lower Km ((+)-pino) = 1.6 μM kcat/Km Roots—PrR2; stem Nakatsubo et al. (2008)
act. for ((+)-pino) = 0.77 μM−1 min−1
lari Km ((−)-pino) = 7.3 μM kcat/Km
((−)-pino) = 1.14 μM−1 min−1
AtPrR2 (NP_193102) − − 99% e.e. Ø Km ((−)-pino) = 12.6 μM kcat/Km
((−)-pino) = 0.07 μM−1 min−1
Isatis indigotica IiPLR1 (AEA42007) ND ND ND Km = 65.4 ± 2.76 ((±)pino) and Seedlings; Roots, stem; leaves; Xiao et al. (2015)
2.5 ± 0.14 μM ((±)lari) flowers
kcat/Km = 0.91 ± 0.11 ((±)pino) and
1.59 ± 0.05 μM−1 min−1 ((±)lari)
Role in lariciresinol accumulation
IiPLR2 ND ND ND
IiPLR3 ND ND ND
Linum album LaPLR1 (CAH60857) + 100:0 + − → important for podophyllotoxin Cell suspension von Heimendahl et al. (2005)
synthesis
Linum corymbulosum LcPLR1 (ABW86959) + + − → important for hinokinin syn- Cell suspension, hairy roots Bayindir et al. (2008)
thesis
Linum perenne LpPLR1 (ABM68630) + (−) − (+) + (−) + → − → +Conversion in brackets Cell suspension Hemmati et al. (2007)
is possible but the other one is
preferred—important for Justici-
dine B synthesis
Linum usitatissimum LuPLR1 (CAH60858) − − + → Important for SDG synthesis Cell culture, seeds, roots von Heimendahl et al. (2005)
LuPLR2 (ABW24501) + + − → Important for yatein synthesis Leaves, stem, seeds, roots Hemmati et al. (2010)
Podophyllum hexandrum PhPLR (ACF71492) + ND − Conversion of (−)-pinoresinol was Leaves; rhizome/roots; stem Wankhede et al. (2013), Lau
not investigated and Sattely (2015a, b)
Podophyllum pleianthum PpPLR + + − Km = 19.8 ((+)pino) and 37.3 μM Kuo et al. (2014)
((+)lari)
Vmax = 7.34 ((+)pino) and
3.12 μmol h−1 mg−1 ((+)lari)
Planta
 Table 3  (continued)
Planta

Species PLR PINO LARI SECO Kinetic parameters/notes Expression References

Taiwania cryptomerioides TcPLR1 + + Ø Activity of TcPLRs tested using Cambium, sapwood, heartwood, Chiang et al. (2018)
only (+)-pinoresinol. transition zone of the wood
TcPLR2.2 + + − Cambium, sapwood, heartwood,
transition zone of the wood
TcPLR3 + + − Sapwood
Thuja plicata TpPLR1 (AAF63507) − (+) − + Strictly enantiospecific only at the Young green stem with leaves Fujita et al. (1999)
TpPLR2 (AAF63508) + (−) + − level of lariciresinol to secoiso- attached
lariciresinol conversion
TpPLR3 (AAF63509) ND ND ND
TpPLR4 (AAF63510) ND ND ND
Arctium lappa not isolated + + + → With petiole enzyme prepara- Petioles Suzuki et al. (1996)
tion
not isolated − − − → With seed enzyme preparation Seeds
Wikstroemia sikokiana not isolated ND ND ND → Assayed with crude cell Umezawa et al. (1998)
extract—either enantioselective
or both types of enzymes present
Daphne genkwa not isolated ND − 23% e.e. Ø → Assayed with cell free extract Okunishi et al. (2001)
of D. genkwa

ND not determined, Ø not detected, act activity, e.e. enantiomeric excess

13
 Planta

while conversion of pinoresinol to lariciresinol is still active

LpPLR1
(Katayama et al. 1993). Characteristic of the LaPLR1 and

100
LcPLR1 action is the formation of (−)-SECO only when all
the (+)-pinoresinol is converted to lariciresinol (von Hei-

LuPLR1
mendahl et al. 2005; Bayindir et al. 2008). A similar situ-

81.09
100
ation is found with LpPLR1 where although the enzyme
prefers (+)-pinoresinol, both enantiomers of pinoresinol

IiPLR1
are completely used up before lariciresinol is converted to

63.11
66.88
100
SECO with preference to (−)-lariciresinol (Hemmati et al.

Table 4  Identity matrix (%) created with ClustalOmega using protein sequences of characterized pinoresinol(–lariciresinol) reductases from different plant species
2007). It is important to point out that the LpPLR1 changes

AtPrR2
its enantiospecificity during the conversion of pinoresinol

85.17
61.81
65.59
100
to lariciresinol. In the first reduction step, the enzyme is
specific for pinoresinol with R,R configuration at 8,8′-C

AtPrR1
atom whereas in the conversion of lariciresinol to SECO,

81.07
82.33
64.72
68.17
100
the LpPLR1 is specific for S,S configuration at 8,8′-C atoms.
Taking into account that in the biosynthesis of justicidin B

LcPLR1
the R,R configuration at 8,8′-C atoms is necessary, a second

59.62
60.26
60.26
65.37
66.88
100
PLR specific for the conversion of (+)-lariciresinol to (−)-
SECO may be present in Linum perenne (Hemmati et al.

LaPLR1
2007). This taken together with the sometimes large differ-

88.57
58.86
58.86
59.49
64.72
65.92
100
ences in the catalytic efficiency between the reduction of
pinoresinol to lariciresinol and the reduction of lariciresinol

LuPLR2
to SECO opens up the question whether one and the same

81.62
82.54
61.02
61.02
60.38
63.23
65.71
PLR or two different enzymes are used for both reductive

100
steps in the conversion of pinoresinol to SECO in a bio-
synthetic pathway taking place at a certain time point in a

PhPLR1
73.63
73.31
74.92
61.29
62.26
62.58
65.05
66.88
certain plant tissue.

100
Data on the known PLRs

FiPLR1
75.24
76.21
75.24
74.28
59.68
60.32
59.03
60.52
62.38
100

The data on PLRs studied so far are summarized in Table 2

(genes from different species), Table 3 (protein features),

TpPLR4
Table 4 (protein identity), in Fig. 3 (phylogenetic analysis)
58.71
60.97
60.58
59.16
59.49
56.27
55.31
57.23
60.32
62.06
100

and in Fig. 2 (enantiospecificity of catalysis).

TpPLR2
Forsythia  The first PLR studied was in Forsythia intermedia
94.23
60.32

61.86
59.81
59.81
57.23
55.63

60.97
63.02
62.9

58.2
100

(Chu et al. 1993; Dinkova-Kostova et al. 1996). It converts

(+)-pinoresinol to (+)-lariciresinol and (+)-lariciresinol to
ThPLR2
(−)-SECO. Two isoforms of PLR were purified and char-
77.02
75.08
61.69
60.06
62.46
60.06
60.06
62.01
61.04
62.66
58.77
60.19

acterized from young green stems of the greenhouse-grown

100

F.  intermedia. Two purified forms of PLR catalyzed the

ThPLR1

same reaction with comparable kinetic parameters (Dink-

98.49
77.36
75.85
60.98
59.85
62.64
59.85
59.85
62.12
60.61
63.26
57.95

ova-Kostova et al. 1996). They were capable of converting

100

60

both substrates but show higher affinity towards pinoresinol

(the Km for pinoresinol is almost five times lower than the
TpPLR3
68.94
69.16
69.77
69.13
56.63
57.28
58.52

57.74
55.81
54.84
56.77
55.66
56.77

one for lariciresinol).

57.1
100

Gymnosperms  In gymnosperms, first PLRs were discovered

TpPLR1

by Fujita et al. (1999) in Thuja plicata, the western red cedar

79.81
70.94
70.55
72.44
70.19

58.06
57.05
56.91
57.23
56.59
56.91
57.23
57.74
57.23
57.1
100

that accumulates the lignan plicatic acid in the heartwood.

Four PLRs are found expressed and isolated from the young
TpPLR1
TpPLR3
ThPLR1
ThPLR2
TpPLR2
TpPLR4

LuPLR2

LpPLR1
LuPLR1
PhPLR1

LaPLR1
LcPLR1
FiPLR1

AtPrR1
AtPrR2
IiPLR1

stem. TpPLR2 (AF242504.1) and TpPLR4 (AF242506.1)

catalyze the same reaction as the FiPLR1 while TpPLR1

13
Planta

AtPrR1 AtPrR2 H
OH OH
O
AtPrR2 O
LuPLR1 H3C
O
S OH
LuPLR1 LpPLR1 S OH
SS O S S O HO
H H
TpPLR1 H H
OH
TpPLR1 H
H3C H3C
O
HO HO
O
O O
H3C H3C OH CH3

S,S-(-)-pinoresinol S,S-(-)-lariciresinol S,S-(+)-secoisolariciresinol

AtPrR1
LuPLR2
LaPLR1 LuPLR2
LcPLR1 LaPLR1
LpPLR1 LcPLR1
FiPLR1 FiPLR1 H
OH O H
O
TpPLR2 O TpPLR2 H3C
O
OH
R
TcPLR1/2.2/3 TcPLR2.2/3 R OH
RR O RR O HO
H H
PpPLR1 H H
OH
PpPLR1 H
H3C H3C
O
HO HO
O
O O
H3C H3C OH CH3

R,R-(+)-pinoresinol R,R-(+)-lariciresinol R,R-(-)-secoisolariciresinol

Fig. 2  Enantiospecificity of characterized pinoresinol–lariciresinol reductases

(AF242503.1) and TpPLR3 (AF242505.1) catalyze conver-
sion of (−)-pinoresinol to (−)-lariciresinol and (−)-laricires-
inol to (+)-SECO. However, unlike FiPLR1, TpPLRs are
not completely enantiospecific, and, based on the enanti-
omers available, exhibit both pinoresinol–lariciresinol and
pinoresinol activity. If the reaction with racemic pinoresinol
is left to run its course, both pinoresinol enantiomers will be
converted albeit formed (+)-lariciresinol and (−)-laricires-
inol will not be converted further by TpPLR1 and TpPLR2,
respectively, meaning that T. plicata PLRs are strictly spe-
cific only on the level of lariciresinol conversion (Fujita
et al. 1999). Recently, new PLRs were described from fam-
ily Cupressaceae expressed in the wood of Taiwania cryp-
tomerioides. Here TcPLR2.2 and TcPLR3 showed pinores-
inol–lariciresinol activity converting (+)-pinoresinol to
(+)-lariciresinol and then to (−)-lariciresinol while TcPLR1
showed pinoresinol activity converting (+)-pinoresinol
only to (+)-lariciresinol (note activity of TcPLRs has been
tested using only (+)-pinoresinol and no data about conver-
sion of (−)-pinoresinol are available) (Chiang et al. 2018).
Two other gymnosperm PLRs, ThPLR1 (AF242501_1) and
ThPLR2 (AAF64185.1), were identified in the young stem
of Tsuga heterophylla (western hemlock) but no investiga-
tion of their specificities has been performed so far (Gang
et al. 1999).
Arabidopsis and  Isatis  Arabidopsis thaliana root MeOH
extracts after β-glucosidase treatment show small amounts
of (−)-lariciresinol in 88% e.e. (enantiomeric excess) that
probably exists only as a glucoside because no lariciresinol
is detected without a β-glucosidase treatment. Pinoresinol,
SECO and matairesinol are absent in the roots and none
of the lignans are detected in aerial parts (Nakatsubo et al.
2008). While most PLRs show the ability to reduce both
pinoresinol and lariciresinol, the same cannot be said for
pinoresinol reductases of A. thaliana. The two character-
ized AtPrR1 (AY065214.1) and AtPrR2 (BT002882.1)
are named pinoresinol reductases since they show no or
a very weak activity towards the reduction of laricires-
inol (Nakatsubo et  al. 2008). Microarray expression data
show that AtPrR1 is expressed in most tissues with highest
expression in stem (lignified 2nd internode) while AtPrR2
has highest expression in roots (Zhao et al. 2015). AtPrR1
and AtPrR2 are expressed in stems and even more in roots
(Nakatsubo et al. 2008). When supplied with both pinores-
inol enantiomers AtPrR2 reduces only (−)-pinoresinol to
(−)-lariciresinol with 96% e.e while AtPrR1 reduces both
pinoresinol enantiomers and forms both lariciresinol enan-
tiomers with comparable kinetic values [6% e.e. in favor of
the (+)-lariciresinol]. Better understanding of lariciresinol
production was obtained from analyses of single and double

13
 Planta
produce +L and -S
produces -S
produce -L and +S
either not enanospecific (AtPrP1)
or change in enanospecificity (LpPLR1 forms +L and +S)
Fig. 3  Phylogenetic tree of known pinoresinol–lariciresinol reduc-
tases from different plant species created from complete protein
sequence alignment constructed with MUSCLE 3.8.31 using the
maximum likelihood method and bootstrap analysis 500 replicates
AtPrR1 and AtPrR2 mutants. While the lariciresinol con-
tent does not change significantly in single mutants there
is a change in the enantiomeric composition. A knockout
of AtPrR1 brings (−)-lariciresinol to 94–96% e.e. while
knockout plants for AtPrR2 show only 82% e.e. in favor
of (−)-lariciresinol. Contrarily, double mutants contain no
lariciresinol but accumulate pinoresinol [74% e.e. in favor
of the (−)-pinoresinol] (Nakatsubo et al. 2008). The authors
postulate that enantiomeric specificity arises from the action
of pinoresinol reductases along with DIR.
PLRs have also been identified in another member of
the Brassicaceae family, the tinctorial plant Isatis indig-
otica which expresses IiPLR1 (AEA42007.1) responsible
for the accumulation of (±)-lariciresinol from (±)-pinores-
inol (specificity not determined) (Xiao et al. 2015). Gene
silencing of IiPLR1 by RNAi leads to decrease in laricires-
inol quantity while overexpression causes its increase,
which indicates that, as it is the case in A. thaliana PrR
with whom it shares highest identity (82–85%), IiPLR1
in vivo acts mainly as a PrR. Nevertheless, enzymatic assay
showed comparable Kcat/Km values for reduction of both
13
(PhyML v3.0 at http://www.phylo​geny.fr). The protein list is in Sup-
plementary material Table S1. +L (+)-Lariciresinol, −L (−)-laricires-
inol, +S (+)-secoisolariciresinol, −S (−)-secoisolariciresinol, A algae
group, G gymnosperm group, B1 and B2 Brassicaceae groups
(±)-pinoresinol and (±)-lariciresinol. IiPLR1 is expressed
in stems, leaves, flowers and has the highest expression in
roots.
Linum  The genus Linum of the Linaceae family consists of
more than 200 species that are great lignan producers (Gar-
ros et  al. 2018) many of them with importance in human
health (podophyllotoxin, SDG). PLRs with different enan-
tiospecificity were isolated from four Linum species (L.
usitatissimum, L. album, L. perenne and L. corymbulosum)
and share 63–89% identity among each other and 60–76%
identity with FiPLR1 on amino acid level.
Linum usitatissimum, the commonly cultivated flax,
expresses at least two PLRs with opposite enantiospeci-
ficity. First flax PLR was isolated by Lewis et al. (1999).
Later, LuPLR1 (AJ849359.1) was isolated from a cell sus-
pension culture and characterized by von Heimendahl et al.
(2005). LuPLR1 converts (−)-pinoresinol to (+)-SECO via
(−)-lariciresinol. A L. usitatissimum cell culture contains
both pinoresinol enantiomers and pure (+)-SDG formed
from (+)-SECO (von Heimendahl et al. 2005) while the
Planta
seeds contain almost pure (+)-SDG (99% e.e.) (Ford et al.
2001; Sicilia et al. 2003). Transgenic plants with LuPLR1
knocked down produced seeds devoid of lignan which con-
firmed the involvement of LuPLR1 in the lignan synthesis
occurring in the flax seedcoat (Renouard et al. 2014). The
down-regulation of the LuPLR1 gene expression does not
result only in pinoresinol accumulation but the synthesis
of the SECO is partly replaced by the synthesis of the
neolignans with 8-O-4′ or 8-5′ linked monolignols (Ren-
ouard et al. 2014). Aerial parts of L. usitatissimum contain
levorotatory non-polar dibenzylbutyrolactone (DBL) lig-
nans with absolute stereochemistry 8R, 8′R derived from
8R, 8′R-SECO and 8R, 8′R-matairesinol (Schmidt et al.
2006, 2010). With regard to the presence of 8R, 8′R-lig-
nans in aerial parts, a second PLR was isolated from flax
leaves. LuPLR2 (EU029951.1) forms 8R, 8′R-(−)-SECO
by converting (+)-pinoresinol and (+)-lariciresinol (Hem-
mati et al. 2010). Both PLRs are exclusive in which enan-
tiomers they use and produce.
Analysis of the lignan content in different plant parts
and monitoring of the LuPLRs gene expression shows cor-
relation between lignan synthesis and gene expression.
LuPLR2 gene expression was detected in stem, leaves,
roots and seed while LuPLR1 expression was detected
only in roots and seeds (Hemmati et al. 2010). Expression
of LuPLR1 and LuPLR2 in roots is comparable to ones in
the seeds and leaves even though only small amounts of
lignans are detected [traces of 8R, 8′R-(−)-SDG]. Interest-
ingly, coniferin (a glycoside of coniferyl alcohol) seems
to be a major constituent of roots (Hemmati et al. 2010)
and could perhaps serve as an alternative precursor in lig-
nan production (Smollny et al. 1998). Aerial parts of L.
usitatissimum contain non-polar DBL lignans with 8R,
8′R-configuration but their precursor matairesinol is not
detected. 8R, 8′R-configuration corresponds to transcrip-
tional activity of LuPLR2 in stem and leaves (Hemmati
et al. 2010). Transcription levels of LuPLR1 and LuPLR2
in seeds are similar even though (+)-SDG is the predomi-
nant form in the seeds. Hemmati et al. 2010 speculate that
(+)-SECO is the main substrate for glycosylation whereas
(−)-SECO is further converted. In case of L. usitatissi-
mum possible gene duplication enabled development of
two parallel biosynthetic pathways. It is suggested that
gene duplication occurred in Linum bienne, the progenitor
of L. usitatissimum (Hemmati et al. 2010). A search for
LuPLR genes in the flax genome revealed the presence of
at least five LuPLRs. Another case of gene duplication are
pinoresinol reductases of A. thaliana AtPrR1 and AtPrR2
where the authors suggest that enantiomeric specificity is
controlled by the actions of both PrR and DIR (Nakatsubo
et al. 2008). Another example where PLRs from same spe-
cies catalyze the synthesis of opposite SECO enantiomers
are T. plicata TpPLR1 and TpPLR2.
Two other members of the genus Linum, Linum album
and Linum corymbulosum express, respectively LaPLR1
(AJ849358.1) and LcPLR1 (EU107358.1) (both cloned from
cell suspensions), which convert (+)-pinoresinol to (−)-
SECO using (+)-lariciresinol (von Heimendahl et al. 2005;
Bayindir et al. 2008). Formation of the (−)-SECO enanti-
omer corresponds to the accumulation of (−)-hinokinin in
L. corymbulosum (Bayindir et al. 2008) and (−)-podophyl-
lotoxin accumulation in L. album (Smollny et al. 1998).
Silencing of the LcPLR1 shows its role in (−)-hinokinin
synthesis that goes through (−)-matairesinol via either
(−)-haplomyrfolin or (−)-pluviatolide (Bayindir et al. 2008).
In addition to its PLR activity, LcPLR1of Linum corymbulo-
sum can also act as a PCBER (Bayindir et al. 2008). Enan-
tiospecificity assays show no conversion of (−)-pinoresinol.
Characteristic of LaPLR1 action is the formation of (−)-
SECO only when all the (+)-pinoresinol is converted to
lariciresinol (von Heimendahl et al. 2005). A similar situa-
tion is found with LpPLR1 (see above) where (±)-pinores-
inol is completely used up before lariciresinol is converted
to SECO (Hemmati et al. 2007). LaPLR1 is specific in the
step of conversion of pinoresinol to lariciresinol converting
only the (+)-pinoresinol. In contrast, FiPLR1 shows high
preference to (+)-pinoresinol (> 99:1) while two PLRs of T.
plicata are only strictly specific in the lariciresinol to SECO
conversion (Katayama et al. 1993; Fujita et al. 1999; Ford
et al. 2001; von Heimendahl et al. 2005). The down-regula-
tion of the LaPLR1 gene expression in hairy roots of Linum
album leads to pinoresinol and to less extent lariciresinol
accumulation and reduces levels of podophyllotoxin and
6-methoxy podophyllotoxin (Tashackori et al. 2019).
An interesting situation is found in Linum perenne which
accumulates the achiral lignan justicidin B (AN) (Schmidt
et al. 2007). LpPLR1 (EF050530.1) was cloned from a cell
suspension culture of L. perenne Himmelszelt (Hemmati
et al. 2007). Hairy roots lines with suppressed LpPLR1 gene
expression show a decrease in justicidin B quantity impli-
cating LpPLR1 in justicidin B production (Hemmati et al.
2007). The LpPLR1 converts both pinoresinol and laricires-
inol enantiomers but exhibits a preference for (+)-pinores-
inol and (−)-lariciresinol (Hemmati et al. 2007). Unlike
PLRs in F. intermedia, T. plicata, L. album, L. usitatissi-
mum and L. corymbulosum, LpPLR1 is a first PLR to show
opposite enantiospecificity towards pinoresinol and laricires-
inol, i.e., LpPLR1 preferentially converts R,R-pinoresinol
and S,S-lariciresinol.
Other genera  Two PLRs have been cloned from Podophyl-
lum species known for the production of podophyllotoxin. A
putative PLR (PhPLR, EU855792) was identified in a Podo-
phyllum hexandrum (Wankhede et  al. 2013), the current
­ toposide®,
source of the podophyllotoxin used to produce E
the anticancer drug. PhPLR is strongly expressed in leaf
13
 Planta
and at a lower level in rhizome and root. PhPLR expres-
sion increases in response to wounding and MeJa treat-
ment as well as response to UV treatment of leaves which
can be linked to the fact that synthesis of podophyllotoxin
was shown to be MeJa responsive (Wankhede et al. 2013).
There is no extensive biochemical characterization of
PhPLR. However, during investigation of podophyllotoxin
synthesis pathway it was shown that PhPLR is able to con-
vert (+)-pinoresinol to (−)-SECO (no data about laricires-
inol production and conversion) (Lau and Sattely 2015a).
Podophyllotoxin might play a role in the plant response to
wounding in leaves since an increase in PhDPO, PhPLR
and PhSDH is observed in this case (Oliva et  al. 2002).
PpPLR1, identified from Podophyllum pleianthum, shows
enantiospecificity towards conversion of (+)-pinoresinol
to (+)-lariciresinol and then to (−)-lariciresinol (Kuo et al.
2014).
PLR activity was also detected in other plants such as
Arctium lappa (Suzuki et al. 2002), Wikstroemia sikokiana
(Umezawa et al. 1998) and two Daphne species (Okunishi
et al. 2001) but has not been investigated in more detail.
Future prospects
Despite the amount of data provided by researchers dur-
ing the last decade, numerous points remain unclear about
the PLRs and their activity. Moreover, the whole diversity
of PLRs is still to be uncovered as we lack knowledge on
PLRs in bryophytes, pteridophytes and monocots despite
the occurrence of lignans in these taxa. One of the main
questions is the evolution of PLRs. How did PLRs evolve
from a common ancestor enzyme which also gave rise to
the PCBERs and the IFRs? To answer this and other cru-
cial questions, future investigations will need to utilize
genome-wide approaches. Systematic recombinant protein
production will allow further comparison of PLR enzymatic
parameters. Flax is a convenient model for this purpose as it
produces both SECO enantiomers that are either stored as a
diglucoside or further converted in aryltetralin toxic lignans.
PLR enzymatic action was once supposedly clear enough.
However, now that the number of PLRs exceeds the num-
ber of existing enantiomers of their substrate or product,
there are different hypotheses that try to explain this. One
hypothesis is that they exhibit differences in their activity.
Indeed, in many cases it is still unclear if two successive
steps of reductions in planta are always performed by one
sole PLR enzyme. Another hypothesis relies on a possibility
of differential gene expression such as that which exists for
lignin production in response to stress vs vessel formation.
An exhaustive expression data analysis will help to answer
this issue. Additionally, there is a lack of knowledge con-
cerning transcription factors implicated in PLR expression
regulation.
13
Another mostly uninvestigated area is the area of struc-
ture of PLR along with the knowledge of the way PLRs
cope with two different substrates. Another question is do
the PLRs form a complex with other proteins such as DIR
or proteins found downstream in lignan production that
could allow channeling at least from coniferyl alcohol to
SECO? Such a hypothesis can be raised from the data of
co-expression and molecular modeling in silico but further
experiments using recombinant proteins will help shade light
on that.
Expanding the knowledge base in the field of PLRs will
also be of interest for the production of lignans used in can-
cer treatments by biotechnological means (Malik et al. 2014)
or by enzyme-assisted chemistry (Ricklefs et al. 2016).
Author contribution statement  LM, CH, EL and EF designed
the outline of the article, composed the manuscript and fig-
ures. CC, DA, LG, SD and SR provided critical comments
and revised the content. All authors read and approved the
manuscript.
Acknowledgements  Lucija Markulin received a grant from the French
Ministry of Research and Higher Education
Compliance with ethical standard 
Conflict of interest  The authors declare that they have no competing
interest.
References
Akiyama K, Yamauchi S, Nakato T et al (2007) Antifungal activity
of tetra-substituted tetrahydrofuran lignan, (−)-virgatusin, and
its structure-activity relationship. Biosci Biotechnol Biochem
71:1028–1035. https​://doi.org/10.1271/bbb.60696​
Al-Jumaily EF, AL-Azawi AH (2015) antioxidant and hepatoprotec-
tive activity of purelignan from flaxseed (Linum usitatissimum
l.) on acetaminophen induced toxicity in male rabbit. Int J Res
Stud Biosci 3:8–16
Allen KL, Tschantz DR, Awad KS et  al (2007) A plant lignan,
3′-O-methyl-nordihydroguaiaretic acid, suppresses papilloma-
virus E6 protein function, stabilizes p53 protein, and induces
apoptosis in cervical tumor cells. Mol Carcinog 46:564–575.
https​://doi.org/10.1002/mc.20305​
Basini G, Baioni L, Bussolati S et al (2012) Antiangiogenic properties
of an unusual benzo[k, l]xanthene lignan derived from CAPE
(caffeic acid phenethyl ester). Invest New Drugs 30:186–190.
https​://doi.org/10.1007/s1063​7-010-9550-z
Baumgartner L, Sosa S, Atanasov AG et al (2011) Lignan deriva-
tives from Krameria lappacea roots inhibit acute inflammation
in vivo and pro-inflammatory mediators in vitro. J Nat Prod Prod
74:1779–1786. https​://doi.org/10.1021/np200​343t
Bayindir U, Alfermann AW, Fuss E (2008) Hinokinin biosynthesis in
Linum corymbulosum Reichenb. Plant J 55:810–820. https​://doi.
org/10.1111/j.1365-313X.2008.03558​.x
Brady SM, Sarkar SF, Bonetta D, McCourt P (2003) The ABSCI-
SIC ACID INSENSITIVE 3 (ABI3) gene is modulated by
Planta
farnesylation and is involved in auxin signaling and lateral
root development in Arabidopsis. Plant J 34:67–75. https​://doi.
org/10.1046/j.1365-313X.2003.01707​.x
Bruegmann T, Wetzel H, Hettrich K et al (2018) Knockdown of
PCBER1, a gene of neolignan biosynthesis, resulted in
increased poplar growth. Planta. 249:515–525. https​: //doi.
org/10.1007/s0042​5-018-3021-8
Burlat V, Kwon M, Davin LB, Lewis NG (2001) Dirigent proteins
and dirigent sites in lignifying tissues. Phytochemistry 57:883–
897. https​://doi.org/10.1016/S0031​-9422(01)00117​-0
Canel C, Moraes RM, Dayan FE, Ferreira D (2000) Podophyllotoxin.
Phytochemistry 54:115–120. https​: //doi.org/10.1016/S0031​
-9422(00)00094​-7
Céspedes CL, Avila JG, García AM et al (2006) Antifungal and
antibacterial activities of Araucaria araucana (Mol.) K. Koch
heartwood lignans. Zeitschrift fur Naturforsch Sect C J Biosci
61:35–43. https​://doi.org/10.1515/znc-2006-1-207
Chiang N-T, Ma L-T, Lee Y-R et al (2018) The gene expression
and enzymatic activity of pinoresinol–lariciresinol reductase
during wood formation in Taiwania cryptomerioides Hayata.
Holzforschung. https​://doi.org/10.1515/hf-2018-0026
Cho JY, Choi GJ, Son SW et al (2007) Isolation and antifungal activ-
ity of lignans from Myristica fragrans against various plant
pathogenic fungi. Pest Manag Sci 63:935–940. https​: //doi.
org/10.1002/ps.1420
Chu A, Dinkova A, Davin LB et  al (1993) Stereospecificity of
(+)-pinoresinol and (+)-lariciresinol reductases from Forsythia
intermedia. J Biol Chem 268:27026–27033
Clavel T, Borrmann D, Braune A et  al (2006) Occurrence and
activity of human intestinal bacteria involved in the conver-
sion of dietary lignans. Anaerobe 12:140–147. https​: //doi.
org/10.1016/j.anaer​obe.2005.11.002
Corbin C, Decourtil C, Marosevic D et al (2013a) Role of protein
farnesylation events in the ABA-mediated regulation of the
pinoresinol–lariciresinol Reductase 1 (LuPLR1) gene expres-
sion and lignan biosynthesis in flax (Linum usitatissimum L.).
Plant Physiol Biochem 72:96–111. https​://doi.org/10.1016/j.
plaph​y.2013.06.001
Corbin C, Renouard S, Lopez T et al (2013b) Identification and char-
acterization of cis-acting elements involved in the regulation
of ABA- and/or GA-mediated LuPLR1 gene expression and
lignan biosynthesis in flax (Linum usitatissimum L.) cell cul-
tures. J Plant Physiol 170:516–522. https​://doi.org/10.1016/j.
jplph​.2012.11.003
Corbin C, Drouet S, Mateljak I et al (2017) Functional characteriza-
tion of the pinoresinol–lariciresinol reductase-2 gene reveals its
roles in yatein biosynthesis and flax defense response. Planta
243:405–420. https​://doi.org/10.1007/s0042​5-017-2701-0
Corbin C, Drouet S, Markulin L et al (2018) A genome-wide analysis
of the flax (Linum usitatissimum L.) dirigent protein family:
from gene identification and evolution to differential regula-
tion. Plant Mol Biol 97:73–101. https​://doi.org/10.1007/s1110​
3-018-0725-x
Cullmann F, Becker H (1999) Lignans from the liverwort Lepi-
colea ochroleuca. Phytochemistry 52:1651–1656. https​://doi.
org/10.1016/S0031​-9422(99)00372​-6
Cutillo F, D’Abrosca B, DellaGreca M et al (2003) Lignans and neol-
ignans from Brassica fruticulosa: effects on seed germination
and plant growth. J Agric Food Chem 51:6165–6172. https​://
doi.org/10.1021/jf034​644c
Dalisay DS, Kim KW, Lee C, Yang H, Rübel O, Bowen BP, Davin
LB, Lewis NG (2015) Dirigent protein-mediated lignan and
cyanogenic glucoside formation in flax seed: integrated omics
and MALDI mass spectrometry imaging. J Nat Prod 78:1231–
1242. https​://doi.org/10.1021/acs.jnatp​rod.5b000​23
Davin LB, Wang HB, Crowell AL et al (1997) Stereoselective bimolec-
ular phenoxy radical coupling by an auxiliary (dirigent) protein
without an active center. Science 275:362–366
Davin LB, Jourdes M, Patten AM et al (2008) Dissection of lignin
macromolecular configuration and assembly: comparison to
related biochemical processes in allyl/propenyl phenol and lig-
nan biosynthesis. Nat Prod Rep 25:1015. https:​ //doi.org/10.1039/
b5103​86j
Diharce J, Golebiowski J, Fiorucci S, Antonczak S (2016) Fine-tuning
of microsolvation and hydrogen bond interaction regulates sub-
strate channelling in the course of flavonoid biosynthesis. Phys
Chem Chem Phys 18:10337–10345. https​://doi.org/10.1039/
C5CP0​5059F​
Dikshit A, Gao C, Small C et al (2016) Flaxseed and its components
differentially affect estrogen targets in pre-neoplastic hen ovaries.
J Steroid Biochem Mol Biol 159:73–85. https:​ //doi.org/10.1016/j.
jsbmb​.2016.02.028
Dinkova-Kostova AT, Gang DR, Davin LB et al (1996) (+)-Pinores-
inol/(+)-lariciresinol reductase from Forsythia intermedia.
J Biol Chem 271:29473–29482. https ​ : //doi.org/10.1074/
jbc.271.46.29473​
Donoso-Fierro C, Becerra J, Bustos-Concha E, Silva M (2009)
Chelating and antioxidant activity of lignans from Chilean
woods (Cupressaceae). Holzforschung 63:559–563. https​://doi.
org/10.1515/HF.2009.123
Effenberger I, Zhang B, Li L et al (2015) Dirigent proteins from cot-
ton (Gossypium sp.) for the atroposelective synthesis of gossy-
pol. Angew Chemie 54:14660–14663. https​://doi.org/10.1002/
anie.20150​7543
Elakovich SD, Stevens KL (1985) Phytotoxic properties of nordihy-
droguaiaretic acid, a lignan from Larrea tridentata (Creosote
bush). J Chem Ecol 11:27–33. https​://doi.org/10.1007/BF009​
87601​
Eliasson C, Kamal-Eldin A, Andersson R, Aman P (2003) High-per-
formance liquid chromatographic analysis of secoisolariciresinol
diglucoside and hydroxycinnamic acid glucosides in flaxseed by
alkaline extraction. J Chromatogr A 1012:151–159
Esmaeilzadeh Bahabadi S, Sharifi M, Behmanesh M et al (2012) Time-
course changes in fungal elicitor-induced lignan synthesis and
expression of the relevant genes in cell cultures of Linum album.
J Plant Physiol 169:487–491. https​://doi.org/10.1016/j.jplph​
.2011.12.006
Esmaeilzadeh Bahabadi S, Sharifi M, Ahmadian Chashmi N et al
(2014) Significant enhancement of lignan accumulation in hairy
root cultures of Linum album using biotic elicitors. Acta Physiol
Plant 36:3325–3331. https:​ //doi.org/10.1007/s11738​ -014-1700-z
Fofana B, Ghose K, Mccallum J et al (2017) UGT74S1 is the key player
in controlling secoisolariciresinol diglucoside (SDG) formation
in flax. BMC Plant Biol 17:1–35. https​://doi.org/10.1186/s1287​
0-017-0982-x
Ford JD, Huang KS, Wang HB et al (2001) Biosynthetic pathway to
the cancer chemopreventive secoisolariciresinol diglucoside-
hydroxymethyl glutaryl ester-linked lignan oligomers in flax
(Linum usitatissimum) seed. J Nat Prod 64:1388–1397
Fucassi F, Heikal A, Mikhalovska LI et al (2014) Metal chelation by
a plant lignan, secoisolariciresinol diglucoside. J Incl Phenom
Macrocycl Chem 80:345–351. https​://doi.org/10.1007/s1084​
7-014-0411-9
Fujita M, Gang DR, Davin LB, Lewis NG (1999) Recombinant
pinoresinol–lariciresinol reductases from western red cedar
(Thuja plicata) catalyze opposite enantiospecific conversions. J
Biol Chem 274:618–627. https​://doi.org/10.1074/jbc.274.2.618
Fukuhara Y, Kamimura N, Nakajima M et  al (2013) Discovery
of pinoresinol reductase genes in sphingomonads. Enzyme
Microb Technol 52:38–43. https​: //doi.org/10.1016/j.enzmi​
ctec.2012.10.004
13
 Planta
Gaafar AA, Salama ZA, Askar MS et al (2013) In vitro antioxidant
and antimicrobial activities of Lignan flax seed extract (Linum
usitatissimum, L.). Int J Pharm Sci Rev Res 23:291–297
Gang DR, Fujita M, Davin LB, Lewis NG (1998) The “Abnormal
Lignins”: mapping heartwood formation through the lignan
biosynthetic pathway. Lignin and lignan biosynthesis. Ameri-
can Chemical Society Symposium, pp 389–421
Gang DR, Kasahara H, Xia ZZ-Q et al (1999) Evolution of plant
defense mechanisms. J Biol Chem 274:7516–7527. https​://doi.
org/10.1074/jbc.274.11.7516
Garros L, Drouet S, Corbin C et al (2018) Insight into the influence
of cultivar type, cultivation year, and site on the lignans and
related phenolic profiles, and the health-promoting antioxidant
potential of flax (Linum usitatissimum L.) seeds. Molecules.
https​://doi.org/10.3390/molec​ules2​31026​36
Ghose K, Selvaraj K, Mccallum J et al (2014) Identification and func-
tional characterization of a flax UDP-glycosyltransferase glu-
cosylating secoisolariciresinol (SECO) into secoisolaricires-
inol monoglucoside (SMG) and diglucoside (SDG). BMC Plant
Biol 14:1–17. https​://doi.org/10.1186/1471-2229-14-82
Gordaliza M, Garcı́a P, de Miguel Corral J et al (2004) Podophyl-
lotoxin: distribution, sources, applications and new cytotoxic
derivatives. Toxicon 44:441–459. https​: //doi.org/10.1016/j.
toxic​on.2004.05.008
Gutterman Y, Evenari M, Cooper R et al (1980) Germination inhi-
bition activity of a naturally occurring lignan from Aegilops
ovata L. in green and infrared light. Experientia 36:662–663.
https​://doi.org/10.1007/BF019​70124​
Hano C, Addi M, Bensaddek L et al (2006a) Differential accumula-
tion of monolignol-derived compounds in elicited flax (Linum
usitatissimum) cell suspension cultures. Planta 223:975–989.
https​://doi.org/10.1007/s0042​5-005-0156-1
Hano C, Martin I, Fliniaux O et al (2006b) Pinoresinol–lariciresinol
reductase gene expression and secoisolariciresinol digluco-
side accumulation in developing flax (Linum usitatissimum)
seeds. Planta 224:1291–1301. https​://doi.org/10.1007/s0042​
5-006-0308-y
Hano C, Addi M, Fliniaux O, Bensaddek L, Duverger E, Mesnard F
et al (2008) Molecular characterization of cell death induced
by a compatible interaction between Fusarium oxysporum
f. sp. linii and flax (Linum usitatissimum) cells. Plant Phys-
iol Biochem 46:590–600. https​: //doi.org/10.1016/j.plaph​
y.2008.02.004
Hano C, Renouard S, Molinié R et al (2013) Flaxseed (Linum usitatis-
simum L.) extract as well as (+)-secoisolariciresinol diglucoside
and its mammalian derivatives are potent inhibitors of Î ± -amyl-
ase activity. Bioorg Med Chem Lett 23:3007–3012. https​://doi.
org/10.1016/j.bmcl.2013.03.029
Hano C, Corbin C, Drouet D, Quéro A, Rombaut N, Savoire R, Molinié
R, Thomasset B, Mesnard F, Lainé E (2017) The lignan +−secoi-
solariciresinol extracted from flax hulls is an effective protectant
of linseed oil and its emulsion against oxidative damage. Euro-
pean J Lipid Sci Technol 119: 1600219. https​://doi.org/10.1002/
ejlt.20160​0219
Hemmati S, Schmidt TJ, Fuss E (2007) (+)-Pinoresinol/(−)-laricires-
inol reductase from Linum perenne Himmelszelt involved in the
biosynthesis of justicidin B. FEBS Lett 581:603–610. https:​ //doi.
org/10.1016/j.febsl​et.2007.01.018
Hemmati S, von Heimendahl CBI, Klaes M et al (2010) Pinoresinol–
lariciresinol reductases with opposite enantiospecificity deter-
mine the enantiomeric composition of lignans in the different
organs of Linum usitatissimum L. Planta Med 76:928–934. https​
://doi.org/10.1055/s-0030-12500​36
Holmbom B, Eckerman C, Eklund P et al (2003) Knots in trees—a
new rich source of lignans. Phytochem Rev 2:331–340. https​://
doi.org/10.1023/B:PHYT.00000​45493​.95074​.a8
13
Hu C, Yuan YV, Kitts DD (2007) Antioxidant activities of the flaxseed
lignan secoisolariciresinol diglucoside, its aglycone secoisola-
riciresinol and the mammalian lignans enterodiol and enterol-
actone in vitro. Food Chem Toxicol 45:2219–2227. https​://doi.
org/10.1016/j.fct.2007.05.017
In S-J, Seo K-H, Song N-Y et al (2015) Lignans and neolignans from
the stems of Vibrunum erosum and their neuroprotective and
anti-inflammatory activity. Arch Pharm Res 38:26–34. https​://
doi.org/10.1007/s1227​2-014-0358-9
Johnsson P, Kamal-Eldin A, Lundgren LN, Aman P (2000) HPLC
method for analysis of secoisolariciresinol diglucoside in
flaxseeds. J Agric Food Chem 48:5216–5219. https​: //doi.
org/10.1021/JF000​5871
Kassuya CAL, Leite DFP, de Melo LV et al (2005) Anti-inflamma-
tory properties of extracts, fractions and lignans isolated from
Phyllanthus amarus. Planta Med 71:721–726. https​ : //doi.
org/10.1055/s-2005-87125​8
Katayama T, Davin LB, Chu A, Lewis NG (1993) Novel benzylic
ether reductions in lignan biogenesis in Forsythia intermedia.
Phytochemistry 33:581–591. https​: //doi.org/10.1016/0031-
9422(93)85452​-W
Kato-Noguchi H, Kobayashi A, Ohno O et al (2014) Phytotoxic sub-
stances with allelopathic activity may be central to the strong
invasive potential of Brachiaria brizantha. J Plant Physiol
171:525–530. https​://doi.org/10.1016/j.jplph​.2013.11.010
Kikuchi M, Sugiyama M (1993) Characterization of lariciresinol glu-
cosides from Osmanthus asiaticus. Heterocycles 36:117. https​://
doi.org/10.3987/COM-92-6187
Kraus C, Spiteller G (1997) Comparison of phenolic compounds from
galls and shoots of Picea glauca. Phytochemistry 44:59–67. https​
://doi.org/10.1016/S0031​-9422(96)00388​-3
Kuo H-J, Wei Z-Y, Lu P-C et al (2014) Bioconversion of pinoresinol
into matairesinol by use of recombinant Escherichia coli. Appl
Environ Microbiol 80:2687–2692. https​: //doi.org/10.1128/
AEM.03397​-13
Kwon M, Davin LB, Lewis NG (2001) In situ hybridization and immu-
nolocalization of lignan reductases in woody tissues: implica-
tions for heartwood formation and other forms of vascular
tissue preservation. Phytochemistry 57:899–914. https​://doi.
org/10.1016/S0031​-9422(01)00108​-X
Lainé E, Hano C, Lamblin F (2009) Lignans. In: Knasmüller S, De
Marini DM, Johnson I, Gerhäuser C (eds) Phytoestrogens, Wiley,
Weinheim, pp 547–577
Lau W, Sattely E (2015a) Six enzymes from mayapple that complete
the biosynthetic pathway to the Etoposide aglycone. Science
(80-) 349:1224–1228. https​://doi.org/10.1126/scien​ce.aab26​20
Lau W, Sattely ES (2015b) Supplementary materials for six enzymes
from mayapple that complete the biosynthetic pathway to
the etoposide aglycone. Originally 349:1224. https ​ : //doi.
org/10.1126/scien​ce.aac72​02
Lewis NG, Davin LB, Dinkova-kostova AT et al (1999) Recombinant
pinoresinol/lariciresinol reductase, recombinant dirigent protein,
and methods of use. US Patent 62,1094,2
Li XB, Yang ZX, Yang L et al (2012) Neuroprotective effects of flax
lignan against NMDA-induced neurotoxicity in vitro. CNS Neu-
rosci Ther 18:927–933. https​://doi.org/10.1111/cns.12003​
Li DQ, Wang D, Zhou L et al (2016) Antioxidant and cytotoxic lignans
from the roots of Bupleurum chinense. J Asian Nat Prod Res
19:519–527. https​://doi.org/10.1080/10286​020.2016.12344​56
Mahendra Kumar C, Singh SA (2015) Bioactive lignans from sesame
(Sesamum indicum L.): evaluation of their antioxidant and
antibacterial effects for food applications. J Food Sci Technol
52:2934–2941. https​://doi.org/10.1007/s1319​7-014-1334-6
Malik S, Bíba O, Grúz J et al (2014) Biotechnological approaches for
producing aryltetralin lignans from Linum species. Phytochem
Rev 13:893–913. https​://doi.org/10.1007/s1110​1-014-9345-5
Planta
Marcotullio MC, Pelosi A, Curini M (2014) Hinokinin, an emerg-
ing bioactive lignan. Molecules 19:14862–14878. https​://doi.
org/10.3390/molec​ules1​90914​862
Min T, Kasahara H, Bedgar DL et al (2003) Crystal structures of
pinoresinol–lariciresinol and phenylcoumaran benzylic ether
reductases and their relationship to isoflavone reductases. J Biol
Chem 278:50714–50723. https​://doi.org/10.1074/jbc.M3084​
93200​
Moazzami AA, Haese SL, Kamal-Eldin A (2007) Lignan contents in
sesame seeds and products. Eur J Lipid Sci Technol 109:1022–
1027. https​://doi.org/10.1002/ejlt.20070​0057
Morimoto K, Satake H (2013) Seasonal alteration in amounts of lig-
nans and their glucosides and gene expression of the relevant
biosynthetic enzymes in the forsythia suspense leaf. Biol Pharm
Bull 36:1519–1523. https​://doi.org/10.1248/bpb.13-00437​
Moummou H, Kallberg Y, Tonfack LB et al (2012) The Plant short-
chain dehydrogenase (SDR) superfamily: genome-wide inven-
tory and diversification patterns. BMC Plant Biol 12:219. https​
://doi.org/10.1186/1471-2229-12-219
Nakatsubo T, Mizutani M, Suzuki S et al (2008) Characterization
of Arabidopsis thaliana pinoresinol reductase, a new type of
enzyme involved in lignan biosynthesis. J Biol Chem 283:15550–
15557. https​://doi.org/10.1074/jbc.M8011​31200​
Naoumkina MA, Zhao Q, Gallego-Giraldo L et al (2010) Genome-wide
analysis of phenylpropanoid defence pathways. Mol Plant Pathol
11:829–846. https​://doi.org/10.1111/j.1364-3703.2010.00648​.x
Niculaes C, Morreel K, Kim H et al (2014) Phenylcoumaran benzylic
ether reductase prevents accumulation of compounds formed
under oxidative conditions in poplar xylem. Plant Cell 26:3775–
3791. https​://doi.org/10.1105/tpc.114.12526​0
Okunishi T, Umezawa T, Shimada M (2001) Isolation and enzymatic
formation of lignans of Daphne genkwa and Daphne odora. J
Wood Sci 47:383–388. https​://doi.org/10.1007/BF007​66790​
Oliva A, Moraes RM, Watson SB et al (2002) Aryltetralin lignans
inhibit plant growth by affecting the formation of mitotic micro-
tubular organizing centers. Pestic Biochem Physiol 72:45–54.
https​://doi.org/10.1006/pest.2002.2582
Ono E, Kim HJ, Murata J et  al (2010) Molecular and functional
characterization of novel furofuran-class lignan glucosyltrans-
ferases from Forsythia. Plant Biotechnol 27:317–324. https:​ //doi.
org/10.5511/plant​biote​chnol​ogy.27.317
Pan A, Sun J, Chen Y et al (2007) Effects of a flaxseed-derived lig-
nan supplement in type 2 diabetic patients: a randomized,
double-blind, cross-over trial. PLoS ONE 2:e1148. https​://doi.
org/10.1371/journ​al.pone.00011​48
Ponce De León I, Schmelz EA, Gaggero C et al (2012) Physcomitrella
patens activates reinforcement of the cell wall, programmed cell
death and accumulation of evolutionary conserved defence sig-
nals, such as salicylic acid and 12-oxo-phytodienoic acid, but not
jasmonic acid, upon Botrytis cinerea infection. Mol Plant Pathol
13:960–974. https​://doi.org/10.1111/j.1364-3703.2012.00806​.x
Prasad K (2001) Secoisolariciresinol diglucoside from flaxseed delays
the development of type 2 diabetes in Zucker rat. J Lab Clin Med
138:32–39. https​://doi.org/10.1067/mlc.2001.11571​7
Reboledo G, Del Campo R, Alvarez A et al (2015) Physcomitrella
patens activates defense responses against the pathogen Colle-
totrichum gloeosporioides. Int J Mol Sci 16:22280–22298. https​
://doi.org/10.3390/ijms1​60922​280
Renouard S, Corbin C, Lopez T et al (2012) Abscisic acid regulates
pinoresinol–lariciresinol reductase gene expression and secoiso-
lariciresinol accumulation in developing flax (Linum usitatissi-
mum L.) seeds. Planta 235:85–98. https​://doi.org/10.1007/s0042​
5-011-1492-y
Renouard S, Tribalatc M-A, Lamblin F et al (2014) RNAi-medi-
ated pinoresinol lariciresinol reductase gene silencing in flax
(Linum usitatissimum L.) seed coat: consequences on lignans
and neolignans accumulation. J Plant Physiol 171:1372–1377.
https​://doi.org/10.1016/j.jplph​.2014.06.005
Ricklefs E, Girhard M, Urlacher VB (2016) Three-steps in one-
pot: whole-cell biocatalytic synthesis of enantiopure (+)- and
(−)-pinoresinol via kinetic resolution. Microb Cell Fact 15:78.
https​://doi.org/10.1186/s1293​4-016-0472-0
Satake H, Ono E, Murata J (2013) Recent advances in the metabolic
engineering of lignan biosynthesis pathways for the production
of transgenic plant-based foods and supplements. J Agric Food
Chem 61:11721–11729. https​://doi.org/10.1021/jf400​7104
Satake H, Koyama T, Bahabadi SE et al (2015) Essences in meta-
bolic engineering of lignan biosynthesis. Metabolites 5:270–
290. https​://doi.org/10.3390/metab​o5020​270
Scher JM, Zapp J, Becker H (2003) Lignan derivatives from the liv-
erwort Bazzania trilobata. Phytochemistry 62:769–777
Schmidt TJ, Hemmati S, Fuss E, Alfermann AW (2006) A combined
HPLC-UV and HPLC-MS method for the identification of lig-
nans and its application to the lignans of Linum usitatissimum
L. and L. bienne Mill. Phytochem Anal 17:299–311. https​://
doi.org/10.1002/pca.918
Schmidt TJ, Vössing S, Klaes M, Grimme S (2007) An aryldihy-
dronaphthalene lignan with a novel type of ring system and
further new lignans from Linum perenne L. Planta Med
73:1574–1580. https​://doi.org/10.1055/s-2007-99374​8
Schmidt TJ, Hemmati S, Klaes M et al (2010) Lignans in flowering
aerial parts of Linum species—chemodiversity in the light of
systematics and phylogeny. Phytochemistry 71:1714–1728.
https​://doi.org/10.1016/j.phyto​chem.2010.06.015
Schmidt TJ, Klaes M, Sendker J (2012) Lignans in seeds of Linum
species. Phytochemistry 82:89–99. https​://doi.org/10.1016/j.
phyto​chem.2012.07.004
Schmitt J, Petersen M (2002) Influence of methyl jasmonate and
coniferyl alcohol on pinoresinol and matairesinol accumula-
tion in a Forsythia × intermedia suspension culture. Plant Cell
Rep 20:885–890. https​://doi.org/10.1007/s0029​9-001-0414-z
Shiraishi A, Murata J, Matsumoto E, Matsubara S, Ono E, Satake H
(2016) De novo transcriptomes of Forsythia koreana using a
novel assembly method: insight into tissue- and species-spe-
cific expression of lignan biosynthesis-related gene. PLoS One
11: e0164805. https​://doi.org/10.1371/journ​al.pone.01648​05
Sicilia T, Niemeyer HB, Honig DM et al (2003) Identification and
stereochemical characterization of lignans in flaxseed and
pumpkin seeds. J Agric Food Chem 51:1181–1188. https​://
doi.org/10.1021/jf020​7979
Smollny T, Wichers H, Kalenberg S et al (1998) Accumulation of
podophyllotoxin and related lignans in cell suspension cul-
tures of Linum album. Phytochemistry 48:975–979. https​://
doi.org/10.1016/S0031​-9422(97)00957​-6
Suzuki S, Umezawa T, Shimada M (2002) Stereochemical diversity
in lignan biosynthesis of Arctium lappa L. Biosci Biotechnol
Biochem 66:1262–1269
Swan EP, Jiang KS, Gardner JAF (1969) The lignans of thuja plicata
and the sapwood–heartwood transformation. Phytochemistry
8:345–351. https​://doi.org/10.1016/S0031​-9422(00)85430​-8
Tahsili J, Sharifi M, Safaie N et al (2014) Induction of lignans and
phenolic compounds in cell culture of Linum album by culture
filtrate of Fusarium graminearum. J Plant Interact 9:412–417.
https​://doi.org/10.1080/17429​145.2013.84641​9
Takeda R, Hasegawa J, Shinozaki M (1990) The first isolation of
lignans, megacerotonic acid and anthocerotonic acid, from
non-vascular plants, anthocerotae (hornworts). Tetrahedron
Lett 31:4159–4162
Tamura M, Tsuji Y, Kusunose T et al (2014) Successful expres-
sion of a novel bacterial gene for pinoresinol reductase and
its effect on lignan biosynthesis in transgenic Arabidopsis
13
 Planta
thaliana. Appl Microbiol Biotechnol 98:8165–8177. https​://
doi.org/10.1007/s0025​3-014-5934-x
Tashackori H, Sharifi M, Chashmi NA et al (2018) Piriformospora
indica cell wall modulates gene expression and metabolite profile
in Linum album hairy roots. Planta 248:1289–1306. https​://doi.
org/10.1007/s0042​5-018-2973-z
Tashackori H, Sharifi M, Ahmadian Chashmi N et al (2019) RNAi-
mediated silencing of pinoresinol lariciresinol reductase in
Linum album hairy roots alters the phenolic accumulation in
response to fungal elicitor. J Plant Physiol 232:115–126. https​://
doi.org/10.1016/j.jplph​.2018.11.005
Teponno RB, Kusari S, Spiteller M (2016) Recent advances in
research on lignans and neolignans. Nat Prod Rep 1:1. https​://
doi.org/10.1039/c6np0​0021e​
Thompson LU, Robb P, Serraino M, Cheung F (1991) Mammalian
lignan production from various foods. Nutr Cancer 16:43–52.
https​://doi.org/10.1080/01635​58910​95141​39
Thongphasuk P, Suttisri R, Bavovada R, Verpoorte R (2004) Anti-
oxidant lignan glucosides from Strychnos vanprukii. Fitoterapia
75:623–628. https​://doi.org/10.1016/j.fitot​e.2004.04.013
Umezawa T (2003) Diversity in lignan biosynthesis. Phytochem Rev
2:371–390. https:​ //doi.org/10.1023/B:PHYT.000004​ 5487.​ 02836​
.32
Umezawa T, Okunishi T, Shimada M (1998) Stereochemical differ-
ences in lignan biosynthesis between Arctium lappa, Wikstro-
emia sikokiana, and Forsythia spp. ACS Symp Ser 697:377–388.
https​://doi.org/10.1021/bk-1998-0697.ch024​
Ursoniu S, Sahebkar A, Andrica F et al (2016) Effects of flaxseed
supplements on blood pressure: a systematic review and meta-
analysis of controlled clinical trial. Clin Nutr 35:615–625. https​
://doi.org/10.1016/j.clnu.2015.05.012
van Fürden B, Humburg A, Fuss E (2005) Influence of methyl jas-
monate on podophyllotoxin and 6-methoxypodophyllotoxin accu-
mulation in Linum album cell suspension cultures. Plant Cell Rep
24:312–317. https​://doi.org/10.1007/s0029​9-005-0954-8
Verpoorte R (2000) Pharmacognosy in the new millennium: leadfind-
ing and biotechnology. J Pharm Pharmacol 52:253–262. https​://
doi.org/10.1211/00223​57001​77393​1
von Heimendahl CBI, Schäfer KM, Eklund P et al (2005) Pinores-
inol–lariciresinol reductases with different stereospecificity
from Linum album and Linum usitatissimum. Phytochemistry
66:1254–1263. https:​ //doi.org/10.1016/j.phytoc​ hem.2005.04.026
Wada H, Kido T, Tanaka N et al (1992) Chemical and chemotaxonomi-
cal studies of ferns. LXXXI. Characteristic lignans of blechna-
ceous ferns. Chem Pharm Bull 40:2099–2101
Wankhede DP, Biswas DK, Rajkumar S, Sinha AK (2013) Expressed
sequence tags and molecular cloning and characterization of
gene encoding pinoresinol/lariciresinol reductase from Podo-
phyllum hexandrum. Protoplasma 250:1239–1249. https​://doi.
org/10.1007/s0070​9-013-0505-z
Ward RS (1999) Lignans, neolignans and related compounds. Nat Prod
Rep 16:75–96. https​://doi.org/10.1039/a7059​92b
WHO (2015) WHO model list of essential medicines: 19th list. http://
www.who.int/medic​ines/publi​catio​ns/essen​tialm​edici​nes/en/.
Accessed 25 Oct 2016
13
Xia Z-Q, Costa MA, Pélissier HC et al (2001) Secoisolariciresinol
dehydrogenase purification, cloning, and functional expres-
sion. implications for human health and protection. J Biol Chem
276:12614–12623. https​://doi.org/10.1074/jbc.M0086​22200​
Xiao Y, Ji Q, Gao S et al (2015) Combined transcriptome and metab-
olite profiling reveals that Ii PLR1 plays an important role
in lariciresinol accumulation in Isatis indigotica. J Exp Bot
66:6259–6271. https​://doi.org/10.1093/jxb/erv33​3
Xie L-H, Akao T, Hamasaki K et  al (2003) Biotransformation of
pinoresinol diglucoside to mammalian lignans by human intes-
tinal microflora, and isolation of Enterococcus faecalis strain
PDG-1 responsible for the transformation of (+)-pinoresinol to
(+)-lariciresinol. Chem Pharmaceutical Bull 51:508–515
Yang Y-N, Huang X-Y, Feng Z-M et al (2014) Hepatoprotective activity
of twelve novel 7′-hydroxy lignan glucosides from Arctii fruc-
tus. J Agric Food Chem 62:9095–9102. https​://doi.org/10.1021/
jf501​859x
Yousefzadi M, Sharifi M, Behmanesh M et al (2010) Salicylic acid
improves podophyllotoxin production in cell cultures of Linum
album by increasing the expression of genes related with
its biosynthesis. Biotechnol Lett 32:1739–1743. https​://doi.
org/10.1007/s1052​9-010-0343-4
Yousefzadi M, Sharifi M, Behmanesh M, Ghasempour A, Moyano
E, Palazon J (2012) The effect of light on gene expression and
podophyllotoxin biosynthesis in Linum album cell culture. Plant
Physiol Biochem 56:41–46. https​: //doi.org/10.1016/j.plaph​
y.2012.04.010
Zamora-Ros R, Forouhi NG, Sharp SJ et al (2013) The association
between dietary flavonoid and lignan intakes and incident type
2 diabetes in European populations: the EPIC-InterAct study.
Diabetes Care 36:3961–3970. https:​ //doi.org/10.2337/dc13-0877
Zanwar AA, Hegde MV, Bodhankar SL (2011) Cardioprotective activ-
ity of flax lignan concentrate extracted from seeds of Linum usi-
tatissimum in isoprenalin induced myocardial necrosis in rats.
Interdiscip Toxicol 4:90–97. https​://doi.org/10.2478/v1010​
2-011-0016-8
Zhang J, Guo S, Chan KW, et al (2016) New lignans with neuropro-
tective activity from Adelostemma gracillimum. Phytochem Lett
16:1–7. https​://doi.org/10.1016/j.phyto​l.2016.02.011
Zhao Q, Zeng Y, Yin Y et al (2015) Pinoresinol reductase 1 impacts
lignin distribution during secondary cell wall biosynthesis
in Arabidopsis. Phytochemistry 112:170–178. https ​ : //doi.
org/10.1016/j.phyto​chem.2014.07.008
Zheng C-J, Zhang X-W, Han T et al (2014) Anti-inflammatory and
anti-osteoporotic lignans from Vitex negundo seeds. Fitoterapia
93:31–38. https​://doi.org/10.1016/j.fitot​e.2013.12.006
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