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.