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.