The role of CPT3 in Dol synthesis in Arabidopsis – genetic and
biochemical studies
Remarkably, the CPT responsible for the formation of the hydrocarbon
backbone of the major Dols (Dol-15 to Dol-17) accumulated in Arabidopsis
has not been identified yet. The AT2G17570 gene encoding CPT3 (sometimes
named CPT1 (Kera, Takahashi, Sutoh, Koyama, & Nakayama, 2012)) is
ubiquitously expressed in Arabidopsis organs and, among all nine AtCPTs,
it is by sequence homology the closest counterpart of the yeast CPTs
that synthesize Dols (Surmacz & Swiezewska, 2011). Preliminary studies
revealed that CPT3, when co-expressed with LEW1 , was
capable of rescuing the growth defect of a yeast strain devoid of both
yeast CPTs: rer2Δ srt1Δ , and a microsomal fraction of thus
obtained yeast transformant was able to incorporate in vitro a
radioactive precursor into polyisoprenoids, although their profile had
not been presented (Kwon, Kwon, & Ro, 2016).
At the time, no T-DNA insertion mutant in the CPT3 gene was
available from the NASC collection. For this reason, to analyze in
planta the involvement of CPT3 in Dol formation, four independent RNAi
lines targeting CPT3 for mRNA knockdown (RNAi-1, -12, -14 and -23) and a
transgenic line overexpressing CPT3 (OE-7) were generated. The
expression level of CPT3 and the polyisoprenoid content were
examined in 4-week-old leaves of these mutants. qRT-PCR analyses
revealed that the CPT3 transcript is significantly reduced (by
40-50%) in the four RNAi lines, and it is nearly 5-fold elevated in the
OE line, in comparison to wild-type plants (Figure 2A). No visible
phenotypic changes were observed between wild type plants and the
studied mutant lines under standard growth conditions (data not shown).
In contrast, HPLC/UV analysis of total polyisoprenoids revealed a
significant decrease in dolichol (Dol-15 – Dol-17, dominating Dol-16)
accumulation in CPT3 RNAi lines – to approx. 50% of the WT for
three lines (RNAi-1, -12, and -23) and to approx. 80% for RNAi-14. Not
surprisingly, CPT3-OE plants accumulated significantly higher
amounts of dolichols, reaching 300% of the WT levels (Figure 2B). These
results clearly suggest that CPT3 is involved in the biosynthesis of the
major family of Dols in Arabidopsis. In line with this, we observed a
positive correlation between the level of CPT3 transcript and the
content of Dol during plant development for three of the selected
accessions (Figure 2C). This further supports the role of CPT3 in Dol
formation; interestingly, no such correlation was noted for Prens
(Figure 2C).
CPT3, similarly to numerous other eukaryotic CPTs engaged in Dol
biosynthesis (Grabińska, Park, & Sessa, 2016), is located in the
endoplasmic reticulum (ER), as documented by confocal laser microscopy
– in transiently transformed N. benthamiana leaves the
fluorescence signal of CPT3-GFP fully overlapped with that of the ER
marker ER-CFP (Figure 2D). Moreover, the physical interaction of CPT3
with Lew1 (Arabidopsis homologue of mammalian NgBR and yeast Nus1, an
accessory protein required for activity of some eukaryotic CPTs,
Grabińska, Park, & Sessa, 2016) was confirmed in planta using a
BiFC assay (nEYFP-C1/CPT3 was transiently co-expressed with
cEYFP-N1/Lew1 in N. benthamiana leaves, Figure 2E) and Y2H system
(Figure 2F).
Finally, functional complementation of the yeast mutant rer2 Δ by
Arabidopsis CPT3 followed by an analysis of the polyisoprenoid
profile of transformants (Figure 2G) revealed that solely co-expression
of CPT3 and LEW1 resulted in the synthesis of the major
family of Dols (Dol-14 to Dol-16, Dol-15 dominating, Figure 2G).
Moreover, in line with the cellular function of Dol as an obligate
cofactor of protein N -glycosylation, only simultaneous expression
of CPT3 and Lew1 fully rescued the defective glycosylation
of the marker protein CPY in rer2 Δ mutant cells (Figure 2H).
Taken together, the genetic and biochemical data presented here clearly
show that Arabidopsis CPT3 is a functional ortholog of yRer2 and this
verifies the QTL mapping by demonstrating that CPT3 is responsible for
Dol synthesis in Arabidopsis. It should be kept in mind however, that
further experiments are needed to document that CPT3 is causal of
the natural variation between Col-0 and Est.
Genetic analyses of the
variations in metabolite levels in natural accessions - GWAS
As a following step, we used a multi-trait mixed model (Korte et al.,
2012) to calculate the genetic correlations between the different traits
studied (see Table S4). Here, we found a strong correlation for the four
traits – Prens, phytosterols, plastoquinone, and Dols, which argues for
a common genetic correlation of these four traits, and at the same time
it shows that they have a negative genetic correlation with the
remaining three traits, namely tocopherols, chlorophylls, and
carotenoids.
Next, we used the mean phenotypic values of the 116 natural Arabidopsis
accessions per trait to perform GWAS. Eighty-six of these lines have
been recently sequenced as part of the 1,001 genomes project and full
sequence information is readily available (1001 Genomes Consortium,
2016). For the remaining accessions, high-density SNP data have been
published earlier (Horton et al., 2012). We used an imputed SNP dataset
that combined both sets and has been published earlier (Togninalli et
al., 2008). This data set contains ~ 4 million
polymorphisms that segregate in the analyzed accessions. Two million
polymorphisms, which had a minor allele count of at least 5, were
included in the analysis. At a 5% Bonferroni corrected significance
threshold of 2.4 *10^-8, significant associations have been found
only for three of the seven different compounds analyzed (Dols,
plastoquinone, and phytosterols), while no significant associations have
been found for the other four compounds (chlorophylls, carotenoids,
Prens, and tocopherols). In summary, 2, 7, and 5 distinct genetic
regions were significantly associated with Dols, plastoquinone, and
phytosterols, respectively. One region on chromosome 1 is found for all
three traits. The respective Manhattan plots are shown in Figure 3 and
Figure S5.
Summarizing, we found 4 SNPs, representing two different regions, that
were associated with Dol content. The first of the associated
polymorphisms is at position 19,545,459 on chromosome 1 and it codes for
a non-synonymous AA-exchange (Q270K) in the first exon of AT1G52450, a
gene involved in ubiquitin-dependent catabolic processes. The second
polymorphism is located at position 19,540,865: it is upstream of
AT1G52450 and in the 3′ UTR of the neighboring gene AT1G52440, which
encodes a putative alpha-beta hydrolase (ABH). A second putative ABH
(AT1G52460) is also within 10 kb of these associations. The remaining
two significant associations are on chromosome 3 (positions 18,558,714
and 18,558,716, respectively) and they code for one non-synonymous
(V113G) and one synonymous substitution in an exon of the gene
AT3G50050, which encodes the auxin-related transcription factor Myb77
(Shin et al., 2007).
Moreover, several SNPs were detected for plastoquinone (26) and
phytosterols (10) – for details see Figure S5.
The identification of AT1G52450 and two neighboring genes as putative
effectors of the accumulation of Dols, plastoquinone, and phytosterols
prompted us to analyze the phenotypes of the respective Arabidopsis
T-DNA insertion mutants (Figure 4). Interestingly, a significant
increase in the content of Dols (approx. 2-fold, comparing to control WT
plants) was noted for two analyzed heterozygous AT1G52460-deficient
lines: SALK_066806 and GK_823G12. Moreover, in the SALK_066806 line,
phytosterol content was also increased (167.8 ± 20.3 vs. 117.4 ± 23.2
μg/g of fresh weight) and plastoquinone content was considerably
decreased (27.3 ± 2.0 vs 56.7 ± 5.2 μg/g of fresh weight). It is worth
noting that mutations in the AT1G52460 gene did not affect the content
of Prens – this gene has not come up as that putatively affecting Pren
accumulation (Figure 4). Additionally, these mutant plants developed
deformed, curled leaves (Figure S6). Expression analysis of genes of
interest in the genetic backgrounds of heterozygous AT1G52460-deficient
lines (both SALK_066806 and GK_823G12) revealed that in comparison to
WT (Columbia-0) plants, the level of AT1G52460 mRNA was considerably
decreased while that of AT1G52440 and AT1G52450 remained unchanged
(Figure 4B).
To establish the reason for the inability to obtain homozygous
AT1G52460-deficient mutant plants, we analyzed and genotyped the
progenies of heterozygous plants originating from the SALK_066806
(n=61) and GK_823G12 (n=151) lines. The lack of AT1G52460-deficient
homozygotes among analyzed plants of each mutant line suggested that
disruption of this gene was lethal (Table S5). Since the fraction of
aborted seeds per silique was higher for both mutants (approx. 17.9%
and 25.5% for GK_823G12 and SALK_066806, respectively) than for WT
line (2.6%), the seeds produced by mutants showed a reduced germination
rate comparing to WT plants (Table S5). It suggests that homozygous
mutation in AT1G52460 most probably results in embryolethality. Other
analyzed homozygotic mutants (carrying insertions in the genes AT1G52440
and AT1G52450) did not show significant differences neither in
isoprenoid content nor in macroscopical appearance (data not shown).
Taken together, identification of the involvement of putative ABH,
encoded by AT1G52460, in Dol biosynthesis sheds new light on metabolic
pathway in eukaryotes, although the cellular mechanism underlying this
process as well as the causative role of ABH variants in the natural
variation of Dol accumulation awaits clarifications.