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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 are pharmacologically active compounds

Lignans The exact biological role of the lignans in planta is not com-
pletely clarified even if their main function is hypothesized
Lignans are widespread in the plant kingdom to be protection against herbivores and pathogenic micro-
organisms. This is highly probable as some of the lignans
Lignans are a major group of secondary metabolites widely exhibit a strong toxicity level like podophyllotoxin. They
spread in the plant kingdom. However, unlike lignin they are mainly described as defensive molecules with antifun-
are not present in all plants. Lignans can be found in non- gal (Céspedes et al. 2006; Akiyama et al. 2007; Cho et al.
vascular plants such as Bryophytes, the hornworts Antho- 2007), antibacterial (Céspedes et al. 2006; Gaafar et al.
ceros punctatus and Megaceros flagellaris (Takeda et al. 2013; Mahendra Kumar and Singh 2015) and antifeedant
1990) and liverworts Lepicolea ochroleuca and Bazzania (Kraus and Spiteller 1997) properties. Lignans can act as
trilobata (Cullmann and Becker 1999; Scher et al. 2003) phytoanticipins or phytoalexins (Naoumkina et al. 2010).
as well as many Tracheophytes including Pteridophytes In the latter case, they are often synthesized in response to
like ferns of the Blechnaceae family (Wada et al. 1992). fungal attack in various plant species (Gang et al. 1999).
They can be found in a number of Spermatophytes belong- They act as phytoanticipins in heartwood where they are
ing to the Gymnosperms, monocots and dicots. All parts deposited as a protective postlignification infusion (Gang
of the plants can contain lignans including knots in trees et al. 1998) conferring resistance and durability as is the case
(Holmbom et al. 2003), heartwood (Donoso-Fierro et al. in Thuja plicata (Swan et al. 1969). Lignans can participate
2009) and seeds, e.g., Linum and Sesamum (Moazzami in the protection/defense against competitors. There is evi-
et al. 2007; Schmidt et al. 2012). 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-
Lignans are diverse chi et al. 2014) suggesting an allelopathic role of lignans
consistent with their presence at high amount in seeds or
Lignans are dimers of two phenylpropanoid units with a roots of some plants. Additionally, other roles can be hypoth-
different degree of oxidation and substitution patterns of esized taking into account their localization and biochemical
their aromatic moieties (Ward 1999; Teponno et al. 2016). properties. In the seeds they could participate in the defense
Structures formed by dimerization between the two central against reactive oxygen species (ROS) to protect the seed
carbon atoms (8 and 8′) of the side chains of the phenyl- oil content from oxidation or to maintain seed dormancy
propanoid units (­ C6C3) form a lignan while other types of given their antioxidant capacity (Thongphasuk et al. 2004;
linkages are classified as neolignans. Examples of neolig- Mahendra Kumar and Singh 2015; Li et al. 2016). A defense
nans are phenylcoumarans in which the two phenylpropa- against oxidative stress has also been proposed as a role of
noid units are linked by their 8-5′-C atoms (Niculaes et al. oligolignols during the lignification process in poplar (Nicu-
2014) and oxyneolignans where the link between the two laes et al. 2014). Neolignans could play a role in plant height
phenylpropanoid units is indirect trough an oxygen atom as Bruegmann et al. (2018) have shown that the knockdown
(Teponno et al. 2016). Norlignans contain a C16 to C17 of PCBER1 in poplar resulted in increased growth (PCBER
core structure and are derived from arylpropane units by is an enzyme similar to PLR but acting on neolignans). The
the loss of one or two carbons (Teponno et al. 2016) and chelation ability of SDG reported by Fucassi et al. (2014)
often co-occur with lignans and neolignans. suggests that lignans may play a role in iron (or other metal)
Lignans are structurally a very diverse group. So far capture to facilitate its intake (phytosiderophore) or internal
almost two thousands of them have been described: 1729 transport (phytochelatin). This chelation could also contrib-
lignans were reported in the Dictionary of Natural Prod- ute to the protection against ROS generation by metals in the
ucts which contains 139,000 entries (Verpoorte 2000). cell. A number of biological activities have been reported
Based on their carbon skeleton, oxygen incorporation and for lignans. Many of them have positive impact on human
cyclization pattern they are classified into eight groups: aryl- health and are likely linked to the roles mentioned above.
naphthalene (AN), aryltetralin (AT), dibenzocyclooctadiene Cytotoxic lignans, such as podophyllotoxin, have antipro-
(DCO), dibenzylbutane (DB), dibenzylbutyrolactol (DBLL), liferative activity making them irreplaceable as anticancer
dibenzylbutyrolactone (DBL), furan (FR) and furofuran (FF) drugs (Canel et al. 2000; Gordaliza et al. 2004). Lignans also
group (Umezawa 2003; Teponno et al. 2016). Neolignans are exhibit antiviral (Gordaliza et al. 2004; Allen et al. 2007),
divided into 15 subgroups (NL1–NL15) (Satake et al. 2013, antiangiogenic (Basini et al. 2012), hepatoprotective (Yang
2015; Teponno et al. 2016). 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- high quantities of dibenzylbutyrolactones (e.g., L. usitatis-
suya et al. 2005; Baumgartner et al. 2011; Zheng et al. 2014) simum), while furofurano lignans are only found in L. bienne
and antiparasitic (Marcotullio et al. 2014) properties. (Schmidt et al. 2010). Seed analyses of 20 Linum species
Flax lignans have been extensively investigated and their show similar distribution as in aerial parts in the flowering
positive health benefits make flax a functional food. Some stage. Section Syllinum contains AT lignans while Linum
of the favorable effects are the reduction of blood pressure and Dasylinum generally contain ADN/AN lignans. SDG
(Ursoniu et al. 2016), cardioprotection (Zanwar et al. 2011) is found in all but one investigated species of Linum and
and the prevention of hormone-dependent cancers thanks Dasylinum. On the contrary, no SDG is detected in sec-
to their phytoestrogenic features (Lainé et al. 2009; Dik- tion Syllinum. Most species contained only 8R,8′R-SDG,
shit et al. 2016). Flax lignans are also involved in retarding while 8S,8′S-SDG is the predominant form in only three
development of type 2 diabetes in rats (Prasad 2001) while species (L. usitatissimum, L. decumbens and L. narbonense)
dietary lignan supplementation showed positive effect in (Schmidt et al. 2012).
type 2 diabetic patients (Pan et al. 2007) which can be due
to an inhibitory effect on alpha-amylase (Hano et al. 2013). Pinoresinol–lariciresinol reductases
However, epidemiological studies are not always in favor of
the preventive effect (Zamora-Ros et al. 2013). Nevertheless, The biosynthetic pathway of lignans
we should keep in mind that lignan content of plant food is
still not well known and thus epidemiological studies based The biosynthesis of lignans has been well studied and the
on the calculations of lignan intake should be considered proposed pathway is confirmed and widely accepted (Ford
with caution. et al. 2001). Lignans and lignin share a common phenyl-
propanoid precursor, namely the coniferyl alcohol and both
Forsythia, Sesamum and Linum species are model plants lignin formation and the first step in monolignol polymeriza-
for lignan studies tion are considered to arise via bimolecular phenoxy radical
coupling. A difference being in the stereoselective and the
The model plants used so far for functional genetic studies, stereospecific coupling of optically active lignans whereas
Arabidopsis thaliana and Nicotiana tabacum, are not con- the optically inactive lignin is formed in a non-stereospecific
venient models for the lignan synthesis because of poor or process (Gang et al. 1999). The synthesis of lignans starts
null lignan contents. Classical model plants do not have, or with the coupling of two coniferyl alcohols by an oxidase
contain incomplete, biosynthetic pathway for these special- (laccase or peroxidase) with the help of a dirigent protein
ized metabolites. As a consequence, majority of the data (DIR) to form pinoresinol (Davin et al. 1997; Dalisay et al.
available on this subject come from lignan-rich plants such 2015). DIR proteins may have a role in the formation of dif-
as Linum, Forsythia, Sesamum and Podophyllum species for ferent pinoresinol enantiomers by holding coniferyl alcohols
flowering plants or Thuya, Juniperus, Callitris, Tsuga and in a specific orientation during coupling.
Picea for gymnosperms. Pinoresinol–lariciresinol reductase (PLR) converts
Three genera, Linum, Sesamum and Forsythia, are the pinoresinol to lariciresinol and then to SECO. Most of the
most frequently encountered in published work on lignan characterized PLRs catalyze both reduction steps (von Hei-
synthesis with about sixty papers for “Forsythia AND mendahl et al. 2005). Known exceptions are A. thaliana
lignan” request on Medline/PubMed, about one hundred reductases that catalyze only the first step with a reasonable
for “Sesamum AND lignan” and almost four hundred for efficiency and are thus named pinoresinol reductases (PrR).
“Linum AND lignan” (among which a number dedicated AtPrR2 has a 35 times lower affinity towards lariciresinol
to biological activities). The highest production of lignans than for pinoresinol while AtPrR1 does not reduce laricires-
within the non-woody plants is found in cultivated flax. inol at all (Nakatsubo et al. 2008). However, a limited num-
Indeed, Linum usitatissimum has the highest content of ber of PLRs from different species have been investigated
lignan SDG in seeds (Johnsson et al. 2000; Eliasson et al. but genome sequencing allows now speculations that there
2003), more than 60–700 times higher than in any other are other isozymes still not identified. In L. usitatissimum,
analyzed plant-derived food (Thompson et al. 1991). Lig- as much as five PLR genes are present in the genome that
nan diversity in Linum species was investigated by Schmidt encode full PLR proteins (data not shown) but only two L.
et al. (2010, 2012). Analyses of aerial parts of 41 members usitatissimum PLRs have been studied so far. Therefore, it
of Linum species show 64 different lignans that are divided is possible that the two reduction steps could in some cases
into two major groups, aryltetralines (AT) (sections Sylli- require actions of different PLRs.
num, Linopsis and Cathartolinum) and aryldihydronaph- The reduction by PLR is a crucial step in lignan metabo-
thalene/arylnaphthalene (ADN/AN) (sections Linum and lism since its product represents the entry point for the syn-
Dasylinum). Some members of the section Linum contain thesis of various 8–8′-linked lignans: furano, dibenzylbutane,

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dibenzylbutyrolactone and aryltetrahydronaphthalene (Dink- PCBERs, and eugenol synthase. Note also that the pres-
ova-Kostova et al. 1996). The biosynthetic pathway to arylte- ence of reductases able to convert pinoresinol is not lim-
tralin lignans is the subject of a profound interest as it con- ited to plants as a PrR is also found in sphingomonads
cerns the biosynthesis of podophyllotoxin, a natural product bacteria (Fukuhara et al. 2013) thus potentially implicating
used to produce the widely used anticancer drug E ­ toposide® horizontal gene transfer (Moummou et al. 2012). Moreo-
by semisynthesis. Depending on the plant species and organ, ver, the ability to convert pinoresinol to lariciresinol and
SECO can be oxidized to form matairesinol by SECO dehy- SECO has been shown for the gastrointestinal bacteria
drogenase (SDH) (Xia et al. 2001) or SECO can be glyco- Eggerthella lenta, strain SECO-Mt75m3 (Clavel et  al.
sylated to SDG probably by UGT 74S1 glycosyltransferase 2006) and Enterococcus fecalis, strain PDG-1 that is able
(Ghose et al. 2014; Fofana et al. 2017), as is often the case to convert (+) pinoresinol to (+) lariciresinol (Xie et al.
in seeds of L. usitatissimum. Like SECO, pinoresinol (Ono 2003) which indicates the possible presence of enzymes
et al. 2010), lariciresinol (Kikuchi and Sugiyama 1993) and similar to pinoresinol–(lariciresinol) reductases.
matairesinol (Schmitt and Petersen 2002) can be found in The bacterial pinZ gene from Sphingobium sp. strain
their glycosylated forms. SYK-6 has been reported to encode for a pinoresinol
The PLRs were most likely present in plants even before reductase able to convert racemic pinoresinol to laricires-
lignin synthesis appeared in the land plants given that lig- inol, but not lariciresinol to SECO, despite a low per-
nans are found in bryophytes. The availability of their sub- centage identity (around 15–21%) with plant pinores-
strate pinoresinol could be related to the presence of a dozen inol–lariciresinol reductases (Fukuhara et  al. 2013).
DIR genes that are already found in these early land plants Interestingly, heterologous expression of pinZ gene in
(Corbin et al. 2018). DIR are present in Physcomitrella transgenic A. thaliana resulted in metabolic alterations of
patens and induced in response to pathogen attack (Ponce lignan pathways with increased lariciresinol accumulation
De León et al. 2012; Reboledo et al. 2015). Moreover, they in stems (Tamura et al. 2014).
might be involved in dimerization of molecules other than Genes encoding enzymes involved in the biosynthesis
monolignols (Effenberger et al. 2015). Nevertheless, consid- of secondary metabolites are often more divergent than
ering the presence of lignans in these non-spermatophytes housekeeping genes and a clear evolutionary path and
land plants, they should be able to display PLR enzymatic emergence of enantiospecificity in these reductases are not
activity despite no work to attest to that has been published elucidated; however, there are several hypotheses. As both
so far. PLRs and IFRs use NADPH as a cofactor and abstract the
4R-hydride in a regio- and enantiospecific manner while
Phylogenetic tree of published PLRs and reflections PCBERs are only regiospecific, Gang et al. (1999) suggest
on evolution that PCBERs, as highly ubiquitous in the plant kingdom
and only regiospecific, are progenitor enzymes of PLRs
Different reductases are involved in the further conversion and IFRs and that enantiospecificity in PLRs and IFRs has
of phenylpropanoid pathway products. PLRs, IFRs (isofla- evolved later. On the other hand, von Heimendahl et al.
vone reductases) and PCBERs (phenylcoumaran benzylic (2005) based on the lack of clustering of PIP enzymes
ether reductases) are phylogenetically linked reductases that on the level of reaction’s enantiospecificity (PLRs with
show high sequence similarity and similar catalytic modes of the same enantiospecificity do not group together) suggest
action while their products share similar roles often involved that enantiospecificity either evolved several times during
in plant defense (Gang et al. 1999). They are NADPH reduc- evolution or appeared very early maintaining the residues
tases with conserved “GXXGXXG” sequence at the N-ter- for enantiospecificity conserved and changing only non-
minal end which form the PIP (PLR/IFR/PCBER) family. essential amino acids during later evolution. Indeed, Thuja
Both PLRs and IFRs have short characteristic amino acid plicata PLRs cluster together despite opposite enantio-
sequence insertions (von Heimendahl et al. 2005). specificity. Fujita et al. (1999) proposed that the reason is
Moummou et  al. (2012) performed a genome-wide that enzymes are from the same species and that most of
inventory of short-chain dehydrogenase superfam- the differences in amino acid sequences do not affect enan-
ily searches of PLR/IFR enzymes in different species. tiospecificity or that similarity contributes to the ability of
They identified several PLR/IFR enzymes in Selaginella TpPLRs to reduce both pinoresinol enantiomers. Addition-
moellendorffii (5), Arabidopsis thaliana (8), Populus ally, Nakatsubo et al. (2008) note that angiosperm PLRs
trichocarpa (16), Vitis vinifera (14), Glycine max (15), cluster together (FiPLR1, LaPLR1, LuPLR1, LpPLR1,
Oryza sativa (10), Zea mays (7) and Sorghum bicolor AtPrR1 and AtPrR2) and gymnosperm PLRs cluster
(7). However, none were found in the bryophyte Phy- together (TpPLR1 and TpPLR2) suggesting that there is
scomitrella patens. Note that these numbers do not only an independence between enantiomeric selectivity and the
encompass PLRs but include IFRs, vestitone reductase, 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

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coincides with DIR expression (mainly in the vascular cam- developmental stage 2 (WS2) and reaches its maximum in
bium and ray parenchyma cell initials of stem) (Burlat et al. WS4 when the near mature seed is filled (Renouard et al.
2001). However, expression seems to differ over time. PLR 2012). In WS2 ABA accumulates in the seed coat followed
expression appears to alternate between seasons. FiPLR1 is by accumulation in embryo in WS3 and WS4 (WS4 seed
stably expressed from April to August, but no expression coat:embryo = 45:55) (Renouard et al. 2012). Exogenous
is detected from September to November. Seasonal expres- application of ABA to seeds in WS2 causes an increase of
sion of FiPLR1 is consistent with seasonal lignan profiles in LuPLR1 gene expression and SECO quantity while inhibi-
leaves of F. intermedia (pinoresinol, matairesinol and arc- tion of ABA biosynthesis causes their decrease (Renouard
tigenin synthesis increase from April to June) (Morimoto et al. 2012).
and Satake 2013). A negative regulator, gibberellin, causes a decrease in
Most published data on the expression and regulation of the LuPLR1 gene expression and SECO synthesis prob-
PLRs refer to two PLRs from L. usitatissimum. LuPLR1 ably by a direct effect on lignan synthesis and not through
is involved in (+)-SECO synthesis whereas LuPLR2 is an effect on the ABA biosynthesis or catabolism (Corbin
involved in (−)-SECO and eventually to yatein synthesis et al. 2013b). Effect of the ABA is realized through at least
(Corbin et al. 2017). two cis-regulation elements present in the LuPLR1 gene
LuPLR1 and LuPLR2 are expressed in the seed and root promoter. Mutation of either ABRE or MYB2 element
but only LuPLR2 is also expressed in stems and leaves decreases ABA-induced LuPLR1 gene expression while
(Hano et al. 2006b; Hemmati et al. 2010). Expression in the combined mutation of both boxes completely abolishes the
seeds is mainly localized in the seed coat during all devel- positive effect of the ABA (Corbin et al. 2013b). On the
opmental stages of the seed. The expression of LuPLR1 is other hand, down-regulation of LuPLR1 gene expression
increasing at the beginning of the seed development with influenced by GA is realized through the ABRE element.
a higher expression in earlier developmental stage than in Its mutation abolishes negative influence of GA on the
mature seed. In addition, seed coats of aborted seeds show LuPLR1 expression (Corbin et al. 2013b).
no activity of the LuPLR1 gene promoter suggesting a role Another negative effector of the LuPLR1 gene
of the embryo in the control of PLR gene expression (Hano expression and SECO synthesis is the LuERA1 protein
et al. 2006b). The timing of the LuPLR1 gene expression is (enhanced response to ABA) (Corbin et al. 2013a). It is
well coordinated with the timing of SECO production along a β-subunit of the heterodimeric protein called protein
the different stages of seed development. This localization farnesyltransferase. It makes a post-translational modifi-
was logical given the accumulation of the lignan complex cation by transferring a C15 farnesyl unit to the C-ter-
in this tissue that contains almost all the SECO synthetized minal end of a cysteine of the target protein. LuERA1 is
by flax in its diglucosylated form (SDG). expressed in roots, leaves, cell suspension cultures, but
Analysis of the LuPLR gene promoter regions revealed predominantly in the stem and flax seeds. The strong-
several cis-acting elements potentially involved in its regu- est expression is in the seed coat which corresponds to
lation including ABRE (ABA-response element), MYB- LuPLR1 gene expression and lignan biosynthesis (Cor-
PLANT, MYB and W boxes (Hano et al. 2006b) but so far bin et al. 2013a). The expression overlap of LuPLR1 and
there is a lack of published results dealing with transcrip- LuERA1 enables LuERA1 to modulate gene expression
tion factors implicated in the regulation of PLR expression. of the LuPLR1. LuERA1 probably effects lignan synthe-
The involvement of a prenylation event in the transcriptional sis through negative regulation of ABA signaling. The A.
regulation caused by ABA has been demonstrated by Cor- thaliana era1 mutant shows enhanced sensitivity to ABA
bin et al. (2013a). ABRE and MYB2 ABA-response ele- while inhibition of farnesyltransferase positively affects
ments located in the promoter of LuPLR1 are both required lignan biosynthesis (Corbin et al. 2013a). LuERA1 action
for the LuPLR1 regulation by ABA, but the regulation by is probably mediated through ABRE. Indeed, the mutation
ERA1 (involving protein farnesylation events) is operated of ABRE abolishes the positive effect of the farnesyltrans-
only through the ABRE sequence. AtPrR1, implicated in ferase inhibitor on the LuPLR1 gene (Corbin et al. 2013a).
lignin distribution in secondary cell wall biosynthesis, is The LuERA1 could possibly inhibit LuPLR1 expression
regulated by the secondary cell wall-related transcription through farnesylation of the ABI3 (ABA insensitive 3)
factors SND1 and MYB46 (Zhao et al. 2015). transcription factor involved in the ABA signaling (Brady
Two phytohormones show opposite influence on the et al. 2003; Corbin et al. 2013a).
lignan biosynthesis. A strong relationship between ABA, The LuPLR1 gene shows only a weak activation in the
a key growth regulator and the LuPLR1 gene expression cell suspensions treated with Botrytis cinerea or Fusarium
and SECO synthesis has been shown both in flax seeds oxysporum between 8 and 48 h post-elicitation (Hano et al.
(Renouard et al. 2012) and flax cell suspension cultures 2006a). However, total SECO (both aglycone and SDG) con-
(Corbin et al. 2013b). ABA accumulation in seeds starts in tent in a flax cell suspension decreases 96 h post-elicitation

13
Planta

with F. oxysporum and remains unchanged in cells treated two domains form a cleft between them and are connected
with B. cinerea. by five separate peptide segments (Fig. 1).
LuPLR2 is involved in the (−)-SECO biosynthesis which The larger N-terminal domain forms seven parallel
serves as a precursor of lignans accumulating mainly in β-strands surrounded by six α-helices and the smaller C-ter-
leaves, e.g., yatein, hinokinin, bursehernin, thujaplicatin, and minal domain contains two separate β-plated sheets and four
matairesinol dimethyl ether (von Heimendahl et al. 2005; small α-helices. The N-terminal domain of TpPLR1 shows
Hemmati et al. 2010). Corbin et al. (2017) demonstrated that highest similarity (~ 70%) with IFRs, 43% similarity with
LuPLR2 is involved in the biosynthesis of (−)-yatein accu- an E. coli UDP-galactose 4-epimerase and several enzymes
mulated in flax leaves. Indeed, in flax, LuPLR2 gene expres- belonging to short-chain dehydrogenases/reductases (Min
sion and yatein production increase after MeJA treatment et al. 2003), but the C-terminal domain shows sequence
and wounding, which is in accordance with an involvement similarity only with IFRs and IFRHs. However, some of
of these lignans in the plant defense response. the secondary structural elements of the C-terminal domain
appear to be in a similar topological arrangement as those of
Structure of PLRs UDP-galactose 4-epimerase (Min et al. 2003).
As other NADPH reductases TpPLR1 contains the con-
The three-dimensional structures of Thuja plicata PLR1 served GXXGXXG NADPH-binding motif at the N-termi-
(TpPLR1) were elucidated by Min et al. (2003). Crystal- nal forming the first β–α–β unit. The nicotinamide ring is
lization was performed using the vapor diffusion method orientated toward the cleft between the two domains in anti-
and diffracted X-ray crystal structures were refined to 2.5 Å conformation and is stabilized by a stacking interaction with
resolutions. Sedimentation equilibrium experiments and the side chain of the conserved Phe.
MALDI-TOF analysis showed TpPLR1 as a dimer with an A PLR-specific amino acid insert seems to be involved
apparent molecular mass of 69.9 ± 0.4. Overall structure in the formation of the substrate-binding pocket making it
consists of two domains, a cofactor binding N-terminal important for PLR-specific activity (Min et al. 2003). The
domain and a substrate-binding C-terminal domain. The substrate-binding pocket is mostly hydrophobic made up of

Fig. 1  2D topological structure plot of pinoresinol–lariciresinol reductase

13
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two β-strands and two α-helices. The substrate-binding site lignans are channeled in one or the other pathway. Examples
is adjacent to the cofactor so that the substrate molecules of such behavior already exist in the flavonoid biosynthesis
are orientated to face the nicotinamide ring of NADPH. pathway where a cytochrome P450 type enzyme anchored to
Residues involved in cofactor binding are T ­ yr15, ­Ile16, ­Ser41 the ER membrane that forms a metabolon with dihydrofla-
142
and ­Arg . In the putative active site there is a conserved vonol-4-reductase and leucoanthocyanidin reductase. Dur-
­Lys138 whose change to Ala abolishes the ability to convert ing biosynthesis, proteins associate transiently and substrates
pinoresinol suggesting that ­Lys138 is involved in general base move from one active site to another via substrate channeling
catalysis (Min et al. 2003). (Diharce et al. 2016). The substrate needs to diffuse from
Structure–function studies were performed using one active site to the next one, so only the protein–protein
two enantiospecifically opposite PLRs from T. plicata. structures with a possible pathway between cavities should
Substrate-binding sites differ in the local environment be considered.
between TpPLR1 and TpPLR2 that use (−)-pinoresinol PLRs contain putative phosphorylation sites, some
and (+)-pinoresinol as substrates, respectively. In TpPLR1 of them being conserved as shown by multiple sequence
(−)-pinoresinol fits among the hydrophobic side chains of alignment. This feature opens the possibility of regulation
­Phe164, ­Val268, and ­Leu272 but in TpPLR2 symmetric sub- by phosphorylation at the enzymatic level (Gang et al. 1999).
stitution between sites 164 and 272 and change of Val to
Gly causes favoring of (+)-pinoresinol ­(Leu164, ­Gly268 and Enzymatic activity
­Phe272) (Min et al. 2003).
PLRs could form an active multienzymatic complex with After the first PLRs were cloned and characterized from For-
upstream (DIR) and downstream (SDH or UGT) enzymes sythia intermedia and Thuja plicata (Katayama et al. 1993;
that could explain how opposite enantiomeric forms of 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- of both pinoresinol and lariciresinol whereas the TpPLR2
sequent reductions from pinoresinol via lariciresinol to prefers the (+)-enantiomers of pinoresinol and lariciresinol.
SECO. These first characterized enzymes seemed to exhibit The determination of the kinetic parameters for these two
enantiospecificity in both reduction steps for either of the PLRs with respect to the pinoresinol enantiomers as sub-
enantiomeric configuration at the 8,8′-C atoms, namely R,R strate confirmed this specificity (Davin et al. 2008, Table 3).
for (+)-pinoresinol, (+)-lariciresinol and (−)-SECO and S,S The highest catalytic efficiency was observed for the conver-
for (−)-pinoresinol, (−)-lariciresinol and (+)-SECO (Fig. 2). sion of (+)-pinoresinol by the TpPLR2 with 72 µM−1 min−1.
This reflects the complexity of the catalytic action of PLRs TpPLR2 converts (−)-pinoresinol with around sevenfold less
as reductases catalyzing two reduction steps dealing in catalytic efficiency. The catalytic efficiencies of TpPLR1 for
total with four substrates taking into account the respec- both of the pinoresinol enantiomers is at least 30 times lower
tive enantiomers of pinoresinol and lariciresinol as possible than the one of TpPLR2, leading the authors to conclude
substrates. This may be the reason why not so many PLRs that the latter is presumably the main PLR used in planta
are in depth characterized up to now. Nevertheless, the exist- (Fujita et al. 1999). The two PLRs of Linum usitatissimum
ing data broaden our understanding of these multifunctional characterized so far also show opposite enantiospecificity
enzymes. for pinoresinol and lariciresinol (Hemmati et al. 2010). But,
To characterize the identified PLRs with respect to their an in-depth investigation of their expression in planta shows
catalytic function in more detail some proteins were heter- that these PLRs are differentially expressed and, therefore,
ologously expressed in E. coli, namely two enzymes from are responsible for the enantiomeric composition of the
Thuja plicata (TpPLR1 and TpPLR2), Forsythia interme- different lignans accumulated in the different plant organs
dia (FiPLR1 and FiPLR), Arabidopsis thaliana (AtPrR1 and (Hano et al. 2006b; Hemmati et al. 2010; Renouard et al.
AtPrR2), and Linum usitatissimum (LuPLR1 and 2) and one 2012; Corbin et al. 2017). Although PLRs from Arctium
from Isatis indigotica (IiPLR1), Linum album (LaPLR1), lappa are only investigated from raw extracts from petioles
Linum perenne (LpPLR1) and Linum corymbulosum and leaves, the opposite enantiospecificity observed from the
(LcPLR1), respectively (Fig. 2, Table 3). enzyme extracts of these different plant organs (Suzuki et al.
These experiments allowed to evidence the different 2002) suggests once more that PLRs with opposite enantio-
behaviors of the PLR enzymes. Whereas all other PLRs so specificity are differentially expressed and are responsible
far characterized as recombinant purified proteins catalyze for an organ-specific accumulation of lignans with differ-
both reductions from pinoresinol to lariciresinol and from ent enantiomeric composition. Therefore, presumably the
lariciresinol to secoisolariciresinol, the AtPrR1 has an about Thuja PLRs are differentially expressed as well and may
35 times lower activity towards lariciresinol than pinoresinol be involved in different physiological processes in planta.
and the AtPrR2 shows no catalytic activity towards laricires- Although not verified by the determination of kinetic
inol as substrate at all. For that reason, Umezawa (2003) parameters Km and Vmax, the tracking of the consumption of
named them pinoresinol reductases instead of pinores- one or the other enantiomer of pinoresinol and/or laricires-
inol–lariciresinol reductases. It is also the AtPrR1 which inol can give a clue to the substrate specificity of PLRs as
shows a very unusual behavior by exhibiting almost no enan- well. When the (±)-pinoresinol consumption until depletion
tiospecificity for either the pinoresinol or lariciresinol enan- and (±)-lariciresinol and subsequently (∓)-SECO formation
tiomer. The other PLRs characterized so far show at least a was followed for the TpPLRs, no (−)-lariciresinol as inter-
preference for one or the other enantiomer of pinoresinol mediate was visible with (+)-SECO as product together with
and lariciresinol. (+)-lariciresinol in the assays with TpPLR1. On the other
The two PLRs cloned and characterized from Forsythia hand, with TpPLR2, consumption of the enantiomeric mix-
intermedia by Dinkova-Kostova et al. (1996) were the first ture of pinoresinol resulted in an accumulation of both enan-
PLRs cloned. They show a marked enantiospecificity prefer- tiomers of lariciresinol, in which only the (+)-enantiomer
ring (+)-pinoresinol and (+)-lariciresinol to form (−)-SECO was further converted into (−)-SECO (Fujita et al. 1999).
with comparable Km and Vmax values for pinoresinol of about This observation fits very well to the measured kinetic
25 µM and about 17 µmol h−1 mg−1 and for lariciresinol parameters as described above. A similar behavior is found
of about 122 µM and 27 µmol h−1 mg−1, respectively. The for the two LuPLRs: LuPLR2 converts only (+)-pinoresinol
same is true for the IiPLR1 from Isatis indigotica show- to (−)-SECO showing no accumulation of (−)-lariciresinol
ing comparable Kcat/Km values for both reduction steps in as intermediate, indicating a higher catalytic efficiency
the range of 0.9–1.6 µM−1 min−1 although no experiments for the conversion of lariciresinol over pinoresinol. In the
with respect to the enantiospecificity of this enzyme were reaction of LuPLR1, the conversion of (+)-pinoresinol to
performed (Xiao et al. 2015). (+)-lariciresinol seems to be more effective than the further
The PLRs 1 and 2 from Thuja plicata have an opposite conversion of the latter to (−)-SECO. In contrast, SECO
enantiospecificity: TpPLR1 prefers the (−)-enantiomers 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
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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
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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- Arabidopsis and  Isatis  Arabidopsis thaliana root MeOH
sion of (−)-pinoresinol to (−)-lariciresinol and (−)-laricires- extracts after β-glucosidase treatment show small amounts
inol to (+)-SECO. However, unlike FiPLR1, TpPLRs are of (−)-lariciresinol in 88% e.e. (enantiomeric excess) that
not completely enantiospecific, and, based on the enanti- probably exists only as a glucoside because no lariciresinol
omers available, exhibit both pinoresinol–lariciresinol and is detected without a β-glucosidase treatment. Pinoresinol,
pinoresinol activity. If the reaction with racemic pinoresinol SECO and matairesinol are absent in the roots and none
is left to run its course, both pinoresinol enantiomers will be of the lignans are detected in aerial parts (Nakatsubo et al.
converted albeit formed (+)-lariciresinol and (−)-laricires- 2008). While most PLRs show the ability to reduce both
inol will not be converted further by TpPLR1 and TpPLR2, pinoresinol and lariciresinol, the same cannot be said for
respectively, meaning that T. plicata PLRs are strictly spe- pinoresinol reductases of A. thaliana. The two character-
cific only on the level of lariciresinol conversion (Fujita ized AtPrR1 (AY065214.1) and AtPrR2 (BT002882.1)
et al. 1999). Recently, new PLRs were described from fam- are named pinoresinol reductases since they show no or
ily Cupressaceae expressed in the wood of Taiwania cryp- a very weak activity towards the reduction of laricires-
tomerioides. Here TcPLR2.2 and TcPLR3 showed pinores- inol (Nakatsubo et  al. 2008). Microarray expression data
inol–lariciresinol activity converting (+)-pinoresinol to show that AtPrR1 is expressed in most tissues with highest
(+)-lariciresinol and then to (−)-lariciresinol while TcPLR1 expression in stem (lignified 2nd internode) while AtPrR2
showed pinoresinol activity converting (+)-pinoresinol has highest expression in roots (Zhao et al. 2015). AtPrR1
only to (+)-lariciresinol (note activity of TcPLRs has been and AtPrR2 are expressed in stems and even more in roots
tested using only (+)-pinoresinol and no data about conver- (Nakatsubo et al. 2008). When supplied with both pinores-
sion of (−)-pinoresinol are available) (Chiang et al. 2018). inol enantiomers AtPrR2 reduces only (−)-pinoresinol to
Two other gymnosperm PLRs, ThPLR1 (AF242501_1) and (−)-lariciresinol with 96% e.e while AtPrR1 reduces both
ThPLR2 (AAF64185.1), were identified in the young stem pinoresinol enantiomers and forms both lariciresinol enan-
of Tsuga heterophylla (western hemlock) but no investiga- tiomers with comparable kinetic values [6% e.e. in favor of
tion of their specificities has been performed so far (Gang the (+)-lariciresinol]. Better understanding of lariciresinol
et al. 1999). production was obtained from analyses of single and double

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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- (PhyML v3.0 at http://www.phylo​geny.fr). The protein list is in Sup-
tases from different plant species created from complete protein plementary material Table S1. +L (+)-Lariciresinol, −L (−)-laricires-
sequence alignment constructed with MUSCLE 3.8.31 using the inol, +S (+)-secoisolariciresinol, −S (−)-secoisolariciresinol, A algae
maximum likelihood method and bootstrap analysis 500 replicates group, G gymnosperm group, B1 and B2 Brassicaceae groups

AtPrR1 and AtPrR2 mutants. While the lariciresinol con- (±)-pinoresinol and (±)-lariciresinol. IiPLR1 is expressed
tent does not change significantly in single mutants there in stems, leaves, flowers and has the highest expression in
is a change in the enantiomeric composition. A knockout roots.
of AtPrR1 brings (−)-lariciresinol to 94–96% e.e. while
knockout plants for AtPrR2 show only 82% e.e. in favor Linum  The genus Linum of the Linaceae family consists of
of (−)-lariciresinol. Contrarily, double mutants contain no more than 200 species that are great lignan producers (Gar-
lariciresinol but accumulate pinoresinol [74% e.e. in favor ros et  al. 2018) many of them with importance in human
of the (−)-pinoresinol] (Nakatsubo et al. 2008). The authors health (podophyllotoxin, SDG). PLRs with different enan-
postulate that enantiomeric specificity arises from the action tiospecificity were isolated from four Linum species (L.
of pinoresinol reductases along with DIR. usitatissimum, L. album, L. perenne and L. corymbulosum)
PLRs have also been identified in another member of and share 63–89% identity among each other and 60–76%
the Brassicaceae family, the tinctorial plant Isatis indig- identity with FiPLR1 on amino acid level.
otica which expresses IiPLR1 (AEA42007.1) responsible Linum usitatissimum, the commonly cultivated flax,
for the accumulation of (±)-lariciresinol from (±)-pinores- expresses at least two PLRs with opposite enantiospeci-
inol (specificity not determined) (Xiao et al. 2015). Gene ficity. First flax PLR was isolated by Lewis et al. (1999).
silencing of IiPLR1 by RNAi leads to decrease in laricires- Later, LuPLR1 (AJ849359.1) was isolated from a cell sus-
inol quantity while overexpression causes its increase, pension culture and characterized by von Heimendahl et al.
which indicates that, as it is the case in A. thaliana PrR (2005). LuPLR1 converts (−)-pinoresinol to (+)-SECO via
with whom it shares highest identity (82–85%), IiPLR1 (−)-lariciresinol. A L. usitatissimum cell culture contains
in vivo acts mainly as a PrR. Nevertheless, enzymatic assay both pinoresinol enantiomers and pure (+)-SDG formed
showed comparable Kcat/Km values for reduction of both from (+)-SECO (von Heimendahl et al. 2005) while the

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seeds contain almost pure (+)-SDG (99% e.e.) (Ford et al. Two other members of the genus Linum, Linum album
2001; Sicilia et al. 2003). Transgenic plants with LuPLR1 and Linum corymbulosum express, respectively LaPLR1
knocked down produced seeds devoid of lignan which con- (AJ849358.1) and LcPLR1 (EU107358.1) (both cloned from
firmed the involvement of LuPLR1 in the lignan synthesis cell suspensions), which convert (+)-pinoresinol to (−)-
occurring in the flax seedcoat (Renouard et al. 2014). The SECO using (+)-lariciresinol (von Heimendahl et al. 2005;
down-regulation of the LuPLR1 gene expression does not Bayindir et al. 2008). Formation of the (−)-SECO enanti-
result only in pinoresinol accumulation but the synthesis omer corresponds to the accumulation of (−)-hinokinin in
of the SECO is partly replaced by the synthesis of the L. corymbulosum (Bayindir et al. 2008) and (−)-podophyl-
neolignans with 8-O-4′ or 8-5′ linked monolignols (Ren- lotoxin accumulation in L. album (Smollny et al. 1998).
ouard et al. 2014). Aerial parts of L. usitatissimum contain Silencing of the LcPLR1 shows its role in (−)-hinokinin
levorotatory non-polar dibenzylbutyrolactone (DBL) lig- synthesis that goes through (−)-matairesinol via either
nans with absolute stereochemistry 8R, 8′R derived from (−)-haplomyrfolin or (−)-pluviatolide (Bayindir et al. 2008).
8R, 8′R-SECO and 8R, 8′R-matairesinol (Schmidt et al. In addition to its PLR activity, LcPLR1of Linum corymbulo-
2006, 2010). With regard to the presence of 8R, 8′R-lig- sum can also act as a PCBER (Bayindir et al. 2008). Enan-
nans in aerial parts, a second PLR was isolated from flax tiospecificity assays show no conversion of (−)-pinoresinol.
leaves. LuPLR2 (EU029951.1) forms 8R, 8′R-(−)-SECO Characteristic of LaPLR1 action is the formation of (−)-
by converting (+)-pinoresinol and (+)-lariciresinol (Hem- SECO only when all the (+)-pinoresinol is converted to
mati et al. 2010). Both PLRs are exclusive in which enan- lariciresinol (von Heimendahl et al. 2005). A similar situa-
tiomers they use and produce. tion is found with LpPLR1 (see above) where (±)-pinores-
Analysis of the lignan content in different plant parts inol is completely used up before lariciresinol is converted
and monitoring of the LuPLRs gene expression shows cor- to SECO (Hemmati et al. 2007). LaPLR1 is specific in the
relation between lignan synthesis and gene expression. step of conversion of pinoresinol to lariciresinol converting
LuPLR2 gene expression was detected in stem, leaves, only the (+)-pinoresinol. In contrast, FiPLR1 shows high
roots and seed while LuPLR1 expression was detected preference to (+)-pinoresinol (> 99:1) while two PLRs of T.
only in roots and seeds (Hemmati et al. 2010). Expression plicata are only strictly specific in the lariciresinol to SECO
of LuPLR1 and LuPLR2 in roots is comparable to ones in conversion (Katayama et al. 1993; Fujita et al. 1999; Ford
the seeds and leaves even though only small amounts of et al. 2001; von Heimendahl et al. 2005). The down-regula-
lignans are detected [traces of 8R, 8′R-(−)-SDG]. Interest- tion of the LaPLR1 gene expression in hairy roots of Linum
ingly, coniferin (a glycoside of coniferyl alcohol) seems album leads to pinoresinol and to less extent lariciresinol
to be a major constituent of roots (Hemmati et al. 2010) accumulation and reduces levels of podophyllotoxin and
and could perhaps serve as an alternative precursor in lig- 6-methoxy podophyllotoxin (Tashackori et al. 2019).
nan production (Smollny et al. 1998). Aerial parts of L. An interesting situation is found in Linum perenne which
usitatissimum contain non-polar DBL lignans with 8R, accumulates the achiral lignan justicidin B (AN) (Schmidt
8′R-configuration but their precursor matairesinol is not et al. 2007). LpPLR1 (EF050530.1) was cloned from a cell
detected. 8R, 8′R-configuration corresponds to transcrip- suspension culture of L. perenne Himmelszelt (Hemmati
tional activity of LuPLR2 in stem and leaves (Hemmati et al. 2007). Hairy roots lines with suppressed LpPLR1 gene
et al. 2010). Transcription levels of LuPLR1 and LuPLR2 expression show a decrease in justicidin B quantity impli-
in seeds are similar even though (+)-SDG is the predomi- cating LpPLR1 in justicidin B production (Hemmati et al.
nant form in the seeds. Hemmati et al. 2010 speculate that 2007). The LpPLR1 converts both pinoresinol and laricires-
(+)-SECO is the main substrate for glycosylation whereas inol enantiomers but exhibits a preference for (+)-pinores-
(−)-SECO is further converted. In case of L. usitatissi- inol and (−)-lariciresinol (Hemmati et al. 2007). Unlike
mum possible gene duplication enabled development of PLRs in F. intermedia, T. plicata, L. album, L. usitatissi-
two parallel biosynthetic pathways. It is suggested that mum and L. corymbulosum, LpPLR1 is a first PLR to show
gene duplication occurred in Linum bienne, the progenitor opposite enantiospecificity towards pinoresinol and laricires-
of L. usitatissimum (Hemmati et al. 2010). A search for inol, i.e., LpPLR1 preferentially converts R,R-pinoresinol
LuPLR genes in the flax genome revealed the presence of and S,S-lariciresinol.
at least five LuPLRs. Another case of gene duplication are
pinoresinol reductases of A. thaliana AtPrR1 and AtPrR2 Other genera  Two PLRs have been cloned from Podophyl-
where the authors suggest that enantiomeric specificity is lum species known for the production of podophyllotoxin. A
controlled by the actions of both PrR and DIR (Nakatsubo putative PLR (PhPLR, EU855792) was identified in a Podo-
et al. 2008). Another example where PLRs from same spe- phyllum hexandrum (Wankhede et  al. 2013), the current
cies catalyze the synthesis of opposite SECO enantiomers ­ toposide®,
source of the podophyllotoxin used to produce E
are T. plicata TpPLR1 and TpPLR2. the anticancer drug. PhPLR is strongly expressed in leaf

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and at a lower level in rhizome and root. PhPLR expres- Another mostly uninvestigated area is the area of struc-
sion increases in response to wounding and MeJa treat- ture of PLR along with the knowledge of the way PLRs
ment as well as response to UV treatment of leaves which cope with two different substrates. Another question is do
can be linked to the fact that synthesis of podophyllotoxin the PLRs form a complex with other proteins such as DIR
was shown to be MeJa responsive (Wankhede et al. 2013). or proteins found downstream in lignan production that
There is no extensive biochemical characterization of could allow channeling at least from coniferyl alcohol to
PhPLR. However, during investigation of podophyllotoxin SECO? Such a hypothesis can be raised from the data of
synthesis pathway it was shown that PhPLR is able to con- co-expression and molecular modeling in silico but further
vert (+)-pinoresinol to (−)-SECO (no data about laricires- experiments using recombinant proteins will help shade light
inol production and conversion) (Lau and Sattely 2015a). on that.
Podophyllotoxin might play a role in the plant response to Expanding the knowledge base in the field of PLRs will
wounding in leaves since an increase in PhDPO, PhPLR also be of interest for the production of lignans used in can-
and PhSDH is observed in this case (Oliva et  al. 2002). cer treatments by biotechnological means (Malik et al. 2014)
PpPLR1, identified from Podophyllum pleianthum, shows or by enzyme-assisted chemistry (Ricklefs et al. 2016).
enantiospecificity towards conversion of (+)-pinoresinol
to (+)-lariciresinol and then to (−)-lariciresinol (Kuo et al. Author contribution statement  LM, CH, EL and EF designed
2014). the outline of the article, composed the manuscript and fig-
PLR activity was also detected in other plants such as ures. CC, DA, LG, SD and SR provided critical comments
Arctium lappa (Suzuki et al. 2002), Wikstroemia sikokiana and revised the content. All authors read and approved the
(Umezawa et al. 1998) and two Daphne species (Okunishi manuscript.
et al. 2001) but has not been investigated in more detail.

Future prospects Acknowledgements  Lucija Markulin received a grant from the French
Ministry of Research and Higher Education

Despite the amount of data provided by researchers dur-

ing the last decade, numerous points remain unclear about
Compliance with ethical standard 
the PLRs and their activity. Moreover, the whole diversity Conflict of interest  The authors declare that they have no competing
of PLRs is still to be uncovered as we lack knowledge on interest.
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 References
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