DISCUSSION
Dolichol is a vital component of eukaryotic cell synthesis and accumulation of which is tightly regulated in response to physiological requirements and environmental stimuli. The identification of a long searched for CPT3 makes the biosynthetic route of Dol in plants complete and implicates possible integration of this pathway into Dol-producing biotechnological platforms. Association of a putative alpha-beta hydrolase, encoded by AT1G52460, with Dol accumulation in Arabidopsis provides novel insight into possible determinants of Dol level in all eukaryotes (Figure 6). Understanding of the cellular mechanisms underlying this connection appeals for clarification still it would be difficult to get unrevealed without genetic association approach applied in this study.
It is intriguing that we could detect QTLs for four different compounds: Prens, Dols, chlorophylls, and carotenoids, while we found significant GWAS associations for three: phytosterols, plastoquinone, and Dols. Consequently, Dols are the only compounds where both approaches detected associations. Still, the reported QTL on chromosome 2 does not overlap with the GWAS results, which are located on chromosomes 1 and 3, respectively (summarized in Table S7). While, at a first glimpse, this lack of accordance might be disturbing, there could be many good reasons for it. It is well known that both methods have different power to detect associations (see Figure 4 in Weigel and Nordborg, 2015). For example, on chromosome 1, we identified a significant GWAS association for three different compounds, but we detected no corresponding QTL in the mapping population even though the associated polymorphism segregates in the AI-RIL population. The three traits for which this association is detected (the content of phytosterols, plastoquinone, and Dols) show a strong genetic correlation, so one would expect to find shared genetic factors that regulate all three traits, despite a slightly lesser phenotypic correlation of the traits. The associated sequence variant is located in the gene AT1G52450, which is thus an excellent candidate to modulate all three traits and would not have been found using QTL mapping alone. AT1G52450 is annotated to encode a ubiquitin carboxyl-terminal hydrolase (UCH)-related protein, while the neighboring gene AT1G52460 encodes an alpha-beta hydrolase, ABH (PubMed Gene database). Neither of these proteins has been characterized yet. Eukaryotic cells usually possess a family of UCHs (e.g., three in Arabidopsis) (Isono and Nagel, 2014) responsible for releasing ubiquitin (Ub) from ubiquitinated proteins. A tight balance between ubiquitination and deubiquitination is required for cellular survival since ubiquitin controls numerous bioactivities, such as protein degradation by the 26S proteasome, cell cycle regulation, signal transduction, or membrane trafficking. In turn, the ABH superfamily proteins are found across all domains of life. They are implicated in primary and secondary metabolism by serving highly diverse enzymatic activities, e.g., as esterases, thioesterases, lipases, proteases. Additionally, proteins with the α/β hydrolase fold function as receptors in the strigolactone, gibberellin and karrikin-smoke response pathways (Mindrebo, Nartey, Seto, Burkart, & Noel, 2016 and references therein). In Arabidopsis, more than 600 proteins with ABH folds have been predicted by the InterPro database (Mitchell et al., 2019) with the majority remaining uncharacterized.
Taken together, hydrolytic enzymes, as ABH, encoded by AT1G52460, and/or UCH, encoded by AT1G52450, might control isoprenoid biosynthesis in eukaryotic cells. Interestingly, both ABH and UCH show a high dN/dS ratio (ratio of nonsynonymous to synonymous divergence) in the Arabidopsis population, arguing for strong selection on these genes (see Table S8). Further studies are needed to identify the cellular target(s) of AT1G52460 and the mechanisms underlying its involvement in the metabolism of Dol, phytosterol, and plastoquinone.
It is worth noting that in previous reports, the AT1G52460 gene was identified as one of the maternally expressed imprinted genes (MEGs) that was shown to be predominantly expressed from maternal alleles in reciprocal crosses (Wolff et al., 2011). Notably, the AT1G52460 was among the MEGs (∼30% of all the MEGs tested in that study) for which authors reported a dN/dS value greater than one (Wolff et al., 2011). The dN/dS value can be used to measure the rate of molecular evolution of genes (Warren et al., 2010); therefore, the results of Wolff et al. (2011) provide particularly strong evidence for the fast evolution of AT1G52460. Taking into account that we detected only heterozygotic lines for the AT1G52460 gene, we consider that a loss-of-function allele may lead to a lethal phenotype. A 2:1 ratio (the frequency of heterozygous:WT plants in F2) fitted the data (χ²=2.6 and χ²=0.2 for GK_823G12 and SALK_066806 lines, respectively, at the value of p>0.05). This finding could be particularly important, and it deserves further investigation since very few imprinted genes have been confirmed in plants and even fewer of them have been functionally investigated (He et al., 2017).
The confidence intervals of the detected QTLs include hundreds of different genes. This is within the typical mapping resolution of QTL studies but leads to the problem of prioritizing candidate genes. The most promising gene identified in the QTL analysis, AT2G17570 (CPT3 ), is a long-searched enzyme responsible for backbone synthesis for the major family of dolichols in Arabidopsis, with Dol-15 and Dol-16 dominating. Interestingly, the different product specificity of the Arabidopsis enzymes CPT3, CPT6 (which produces in planta a single Dol-7 (Surmacz, Plochocka, Kania, Danikiewicz, & Swiezewska, 2014)) and the recently characterized CPT1 (producing a family of Dols with Dol-21 dominating (Surowiecki, Onysk, Manko, Swiezewska, & Surmacz, 2019)) suggests that the particular AtCPTs play dedicated, non-redundant roles in isoprenoid synthesis in Arabidopsis tissues. For further comments regarding CPT3 see also Table S8.
Even though no overlapping associations have been found for the GWAS and QTL results, one can try, using the GWAS results, to prioritize candidate genes in the QTL interval. In the confidence interval of the detected QTL for Dol on chromosome 2, we could analyze 6,668 independent segregating polymorphisms with a minor allele frequency greater than 5%. None of these reached the genome-wide significance threshold; the most significant polymorphism had a p-value of 4.88*10^-6 and was located in the proximity of AT2G17570, which encodes CPT3. Although this score is marginal, it is locally significant, if we restrict our analysis to sequence variants within the QTL region. So, the combined results of GWAS and QTL strongly indicate that CPT3 is the gene underlying the detected QTL for Dol, despite the plethora of other tempting candidate genes. Detailed SNP analyses of CPT3 revealed that this gene shows a high amount of variation with a total number of 30 non-synonymous substitutions and 5 alternative starts and 1 premature stop codon in the Arabidopsis population (Table S8).
It is worth underlying that both genetic- and metabolic-based analysis revealed correlations of the analyzed traits indicating genetic co-regulation of the biosynthesis of specific isoprenoids.
In summary, several candidate genes for potential new factors that might affect polyisoprenoid accumulation have been identified in this study. The regulation of isoprenoid pathways is complex but using the combination of both GWAS and QTL it is possible to prioritize the underlying genes. Genetic and biochemical evidences described in this report document the role of CPT3 and ABH in Dol pathway (Figure 6) still further studies are needed to prove their causal role in the natural variation of this trait.
Last but least, it should be kept in mind that this study is based on terpene levels at the seedling stage and might not be representative for later growth stages. Anyhow, obtained results clearly suggest the role of CPT3 and ABH in Dol accumulation.
Understanding of the mechanisms of Dol synthesis/accumulation in eukaryotes is important since the shortage of dolichol/dolichyl phosphate results in serious defects in all studied organisms, most probably caused by defective protein glycosylation. In plants, it is lethal due to male sterility (Jozwiak et al., 2015; Lindner et al., 2015) while in humans mutations in genes encoding enzymes involved in Dol/DolP synthesis lead to rare genetic disorders collectively called Congenital Disorders of Glycosylation (CDG type I); supplementation of the diet with plant tissues that can be utilized as a source of dolichol/dolichyl phosphate has been suggested (summarized in Buczkowska, Swiezewska, & Lefeber, 2015). The identification of genes involved in the synthesis/accumulation of Dols – such as the here detected CPT3 and ABH – opens a perspective for the manipulation of Dol content in plants and consequently makes it feasible to think of constructing plants with increased Dol content. Moreover, involvement of ABH in Dol synthesis in Arabidopsis might also suggest analogous role of ABH in mammalian cells indicating new potential therapeutic strategy for CDG patients.