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.