INTRODUCTION
Isoprenoids (also known as terpenes) are a large and diverse group of
compounds comprised of more than 40,000 chemical structures (Bohlmann &
Keeling, 2008). Linear polymers containing from 5 to more than 100
isoprene units are called polyisoprenoids (Swiezewska & Danikiewicz,
2005). Due to the hydrogenation
status of their OH-terminal, (α-) isoprene unit, polyisoprenoids are
subdivided into α-unsaturated polyprenols (hereafter named Prens) and
α-saturated dolichols (hereafter named Dols) (Figure 1). Prens are
common for bacteria, green parts of plants, wood, seeds, and flowers,
while Dols are constituents of plant roots as well as animal and fungal
cells (Rezanka & Votruba, 2001). In eukaryotic cells, the dominating
polyisoprenoid components are accompanied by traces of their
counterparts, e.g., Prens are accompanied by Dols in photosynthetic
tissues (Skorupinska-Tudek et al., 2003).
All isoprenoids are synthesized from isopentenyl and dimethylallyl
diphosphate (IPP and DMAPP) molecules, which in plants are derived from
the cytoplasmic mevalonate (MVA) and plastidial methylerythritol
phosphate (MEP) pathways (Hemmerlin, Harwood, & Bach, 2012; Lipko &
Swiezewska, 2016). Formation of the polyisoprenoid chains of both Pren
and Dol from IPP is executed by enzymes calledcis -prenyltransferases (CPTs), which are responsible for
elongation of an all-trans initiator molecule, most commonly
farnesyl or geranylgeranyl diphosphate. This reaction generates a
mixture of polyprenyl diphosphates (PolyprenylPP) of similar,
CPT-specific, lengths. In Arabidopsis thaliana (hereafter named
Arabidopsis), only three (Oh, Han, Ryu, & Kang, 2000; Cunillera, Arrὀ,
Forẻs, Manzano, & Ferrer, 2000; Surowiecki, Onysk, Manko, Swiezewska,
& Surmacz, 2019; Kera, Takahashi, Sutoh, Koyama, & Nakayama, 2012;
Surmacz, Plochocka, Kania, Danikiewicz, & Swiezewska, 2014; Akhtar et
al., 2017) out of nine putative CPTs (Surmacz & Swiezewska, 2011) have
been characterized at the molecular level. Interestingly, none of these
well-characterized CPTs (CPT1, -6 or -7) is responsible for the
synthesis of the major ‘family’ of Dols (Dol-16 dominating) accumulated
in Arabidopsis tissues. The polyprenyl diphosphates resulting from CPT
activity undergo then either dephosphorylation to Prens and/or reduction
to Dols. The reduction reaction is catalyzed by polyprenol reductases,
two of which have been recently described in Arabidopsis (Jozwiak et
al., 2015). Although this biosynthetic scheme is generally accepted some
steps of Pren and Dol biosynthesis pathways remain unknown.
Isoprenoids are implicated in vital processes in plants, e.g. in
photosynthesis and stress response (chlorophylls, carotenoids,
plastoquinone, and tocopherols), or in the synthesis of plant hormones
(carotenoids, sterols), or they function as structural components of
membranes (sterols) (Tholl, 2015). Polyisoprenoids are modulators of the
physico-chemical properties of membranes, but they are also involved in
other specific processes. Dolichyl phosphate (DolP) serves as an
obligate cofactor for protein glycosylation and for the formation of
glycosylphosphatidylinositol (GPI) anchors, while Prens, in turn, have
been shown to play a role in plant photosynthetic performance (Akhtar et
al., 2017). Importantly, an increased content of Prens improves the
environmental fitness of plants (Hallahan & Keiper-Hrynko, 2006).
Additionally, it has also been suggested that in plants Prens and Dols
might participate in cell response to stress since their content is
modulated by the availability of nutrients (Jozwiak et al., 2013) and by
other environmental factors (xenobiotics, pathogens, and light
intensity) (summarized in Surmacz & Swiezewska, 2011). Moreover, the
cellular concentration of Prens and Dols is also considerably increased
upon senescence (summarized in Swiezewska & Danikiewicz, 2005). These
observations suggest that eukaryotes might possess, so far elusive,
regulatory mechanisms allowing them to control polyisoprenoid synthesis
and/or degradation.
Most traits important in
agriculture, medicine, ecology, and evolution, including variation in
chemical compound production, are of a quantitative nature and are
usually due to multiple segregating loci (Mackay 2001).
Arabidopsis is an excellent model
for studying natural variation due to its genetic adaptation to
different natural habitats and its extensive variation in morphology,
metabolism, and growth (Alonso-Blanco et al., 2009; Fusari et al.,
2017). Natural variation for many traits has been reported in
Arabidopsis, including primary and secondary metabolism (Mitchell-Olds
& Pedersen, 1998; Kliebenstein, Gershenzon, & Mitchell-Olds, 2001;
Sergeeva et al., 2004; Tholl, Chen, Petri, Gershenzon, & Pichersky,
2005; Keurentjes et al., 2006; Meyer et al., 2007; Lisec et al., 2008;
Rowe, Hansen, Halkier, & Kliebenstein, 2008; Siwinska et al., 2015).
Until now, no systematic analysis of the natural variation of
polyisoprenoids has been performed for any plant species.
Therefore, in this study, we decided to use the model plant Arabidopsis
to explore the natural variation of Prens and Dols. Importantly,
Arabidopsis provides the largest and best-described body of data on the
natural variation of genomic features of any plant species (Kawakatsu et
al., 2016; The 1001 Genomes Consortium, 2016). Over 6,000 different
Arabidopsis accessions that can acclimate to enormously different
environments (Kramer, 2015) have been described so far (Weigel & Mott,
2009).
In order to identify genes that are responsible for modulation of
polyisoprenoid content, we used both a quantitative trait loci (QTL)
mapping approach and genome-wide association studies (GWAS). So far,
neither QTL nor GWAS has been used for the analysis of Prens and Dols.
Traditional linkage mapping usually results in detection of several QTLs
with a high statistical power, making it a powerful method in the
identification of genomic regions that co-segregate with a given trait
in mapping populations (Koornneef,
Alonso-Blanco, & Vreugdenhil, 2004; Korte & Farlow, 2013). But the
whole procedure including the identification of underlying genes is
usually time-consuming and laborious. Moreover, the mapped QTL regions
can be quite large, making it sometimes impossible to identify the
causative genes. Another issue is that the full range of natural
variation is not analyzed in QTL studies using bi-parental populations,
because they are highly dependent on the genetic diversity of the two
parents and may reflect rare alleles. GWAS studies profit from a wide
allelic diversity, high resolution and may lead to the identification of
more evolutionarily relevant variation (Kooke et al.,
2016).Therefore, it is possible to
overcome some limitations of QTL analyses by using the GWAS approach,
which can be used to narrow down the candidate regions (Korte & Farlow,
2013; Han et al., 2018). But it
should be kept in mind that GWAS also has its limitations, such as
dependence on the population structure, the reliance on SNPs rather than
gene structural variants or the potential for false‐positive as well as
false-negative errors (Zhu, Gore, Buckler, & Yu, 2008; Korte & Farlow,
2013). We have applied here both
QTL mapping and GWAS analyses because it has been shown that the
combination of these two methods can alleviate their respective
limitations (Zhao et al., 2007; Brachi et al., 2010).
The described here application of QTL and GWAS led to identification of
several candidate genes underlying the accumulation of polyisoprenoids.
Additionally, to get insight into the biosynthetic pathways of Dols and
Prens in a broader cellular context, a set of seven isoprenoid compounds
was analyzed and subsequently candidate genes were selected. The most
interesting of the identified genes were cis-prenyltransferase 3(CPT3 , AT2G17570, identified through QTL) and alpha-beta
hydrolase (ABH , AT1G52460, identified through GWAS). CPT3,
although biochemically not characterized, has been demonstrated to
efficiently incorporate in vitro IPP intocis -polyisoprenoid of an undefined chain-length thus to possess a
CPT-like activity; moreover, its expression complemented the yCTP
deficiency (Kwon, Kwon, & Ro, 2016), whereas alpha-beta hydrolase has
not been previously connected with polyisoprenoid biosynthesis. In this
work, their involvement in Dol biosynthesis/accumulation is
experimentally confirmed using mutant approach, metabolite profiling,
yeast transformation, transient expression in Nicotiana
benthamiana leaves, bimolecular fluorescence complementation (BiFC) and
yeast two-hybrid (Y2H) assays. Although obtained results clearly suggest
the role of CPT3 and ABH in Dol accumulation one should remember that in
this report analysis of the level of terpene was limited to the seedling
stage and might differ for mature plants. Moreover, it should be kept in
mind, however, that although CPT3 and ABH, together with other genes
depicted in this report, are strong candidates for being causal for the
observed natural variation more studies are required to prove such role.
Importantly, identification of CPT3 fills the gap in the Dol
biosynthetic route in Arabidopsis and, together with newly depicted ABH,
makes the manipulation of Dol content in plants feasible. Consequently,
an option for the generation of plant tissues with increased Dol content
as dietary supplements for individuals suffering from Dol-deficiency is
emerging. Moreover, presuming conserved role of ABH in Dol pathway in
eukaryotes a design of a new therapeutic strategy ameliorating Dol
deficiency via manipulation of the activity of respective human ABH
seems plausible.