2 abc1k3 regulates vitamin e metabolism 3 corresponding ... · 1/23 1 running title 2 abc1k3...

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Running Title 1

ABC1K3 regulates Vitamin E metabolism 2

3

Corresponding author 4

Felix Kessler 5

Laboratory of Plant Physiology, University of Neuchâtel, 2000 Neuchâtel, Switzerland 6

Tel: +41 32 718 2292 7

Email: [email protected] 8

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Journal research area 10

Environmental Stress and Adaptation 11

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Estimate of length (from Plant Physiology page calculator): 10.2 13

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Plant Physiology Preview. Published on April 30, 2013, as DOI:10.1104/pp.113.218644

Copyright 2013 by the American Society of Plant Biologists

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Title 1

A chloroplast ABC1-like kinase regulates Vitamin E metabolism in Arabidopsis thaliana 2

3

Jacopo Martinis1, Gaétan Glauser2, Sergiu Valimareanu1 and Felix Kessler1* 4 1Laboratory of Plant Physiology, University of Neuchâtel, 2000 Neuchâtel, Switzerland 5 2Chemical Analytical Service of the Swiss Plant Science Web, University of Neuchâtel, 2000 6

Neuchâtel, Switzerland 7

8

Email addresses: 9

JM: [email protected] 10

GG: [email protected] 11

SV: [email protected] 12

FK: [email protected] 13

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Financial sources 1

GG acknowledges support from the Swiss Plant Science Web. FK was supported by UniNE, SystemsX 2

PGCE, NCCR Plant Survival and SNF 31003A_127380. SV was supported by Sciex-12.095. 3

4

5 *Corresponding author: Felix Kessler, [email protected] 6

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Abstract 1

In bacteria and mitochondria, ABC1-like kinases regulate ubiquinone synthesis, mutations causing 2

severe respiration defects, including neurological disorders in humans. Little is known about plant 3

ABC1-like kinases: in Arabidopsis thaliana five are predicted in mitochondria, but surprisingly six are 4

located at lipid droplets in chloroplasts. These are a known site of prenylquinone (including tocopherol 5

(Vitamin E), phylloquinone (Vitamin K) and plastoquinone) metabolism and contain a large proportion 6

of the tocopherol cyclase (VTE1) required for Vitamin E synthesis and recycling. Therefore, ABC1-7

like kinases may be involved in the regulation of chloroplast prenylquinone metabolism. 8

Using a non-targeted lipidomics approach we demonstrate that plants lacking the plastoglobule ABC1-9

like kinase ABC1K3, are defective both for the production of plastochromanol-8 (a plastoquinone-10

derived lipid antioxidant) and the Redox recycling of α-tocopherol, while tocopherol production is not 11

affected. All of these pathways require tocopherol cyclase (VTE1) activity. However, in the abc1k3 12

mutant VTE1 levels are strongly reduced post-transcriptionally. 13

Evidence is provided that the ABC1-like kinase ABC1K3 phosphorylates VTE1 possibly stabilizing it 14

at plastoglobules. But ABC1K3 may also have other targets and be involved in a wider chloroplast 15

regulatory network. 16

17



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Introduction 1

Prenylquinones make up a class of lipophilic molecules which include essential electron carriers in 2

bacterial respiration, mitochondria and chloroplasts (ubiquinone, plastoquinone, phylloquinone) and 3

compounds with physiological antioxidant activity (tocopherols, tocotrienols, plastochromanol), some 4

of which (tocopherol/Vitamin E and phylloquinone/Vitamin K) are required as Vitamins in human 5

nutrition (Bouvier et al., 2005; Mene-Saffrane and DellaPenna, 2010). The key enzymes involved in 6

prenylquinone biosynthesis are mostly known, but the regulation of the pathways is still poorly 7

understood. Members of the ABC1/ADCK/UbiB family of atypical kinases are candidates for such 8

regulators. The ABC1/ADCK/UbiB family consists of putative kinases identified by sequence 9

alignment methods (Psi-Blast and HMMs) and which have a domain that is similar to the eukaryotic 10

protein kinase (ePK) domain. This includes the most conserved metal binding residues and catalytic 11

motifs. Members of the ABC1 family were found both in bacteria and eukaryotes. These findings 12

suggest that the family evolved in bacteria and entered early eukaryotes by horizontal transfer (Leonard 13

et al., 1998). The prototypical family member, S. cerevisiae ABC1 (Activity of bc1 complex), is 14

defective in aerobic respiration because of a reduction in the activity of the mitochondrial bc1 complex. 15

The defect can be overcome by exogenously supplied decylubiquinol (Bousquet et al., 1991; Brasseur 16

et al., 1997). Similarly, mutations of the human ABC1 homolog (CABC1 or ADCK3) cause 17

ubiquinone deficiency and defects in the respiratory chain, resulting in a progressive neurological 18

disorder with cerebellar atrophy, developmental delay, and hyperlactatemia (Mollet et al., 2008). 19

Studies performed on bacteria showed that YigR, the E. coli homologue of ABC1, corresponds to UbiB, 20

a gene required for the first monooxygenase step in ubiquinone biosynthesis (Poon et al., 2000). Prior 21

experiments identified a pool of octaprenylphenol that remained bound to a protein complex until it 22

was activated in the presence of oxygen to continue the ring modifications required in ubiquinone 23

biosynthesis (Knoell, 1981). The rapid conversion of octaprenylphenol to UQ-8 upon transfer from 24

anaerobic to aerobic growth conditions is consistent with a mechanism that may require kinase 25

regulation. Moreover, ABC1 has been shown to be able to complement the yeast mutant coq8-1, 26

defective in ubiquinone synthesis (Do et al., 2001). Despite its widely accepted role in ubiquinone 27

synthesis, little is known about the exact function and the in vivo targets of ABC1. In yeast, it has been 28

demonstrated that Coq3, Coq5, and Coq7 polypeptides are phosphorylated in a ABC1/Coq8-dependent 29

manner and that the expression of the human homolog ADCK3/CABC1 rescued the growth of yeast 30

coq8 mutants as well as the phosphorylation state of several of the Coq polypeptides, suggesting the 31



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existence of multiple enzyme targets of the kinase in the ubiquinone biosynthesis pathway (Xie et al., 1

2011). 2

Until recently, ABC1-like kinases have been mainly studied in mitochondria and bacteria, while little 3

evidence was obtained on the role of their chloroplast homologs. One of these putative kinases, 4

AtOSA1 (A. thaliana Oxidative Stress-related ABC1-like protein) was identified in the inner envelope 5

membrane of A. thaliana chloroplasts (Jasinski et al., 2008). The analysis of gene expression showed 6

that AtOSA1 transcription is induced by Cd2+ treatment and oxidative stress conditions, and knock-out 7

plants for this gene permanently suffer from oxidative stress. However, AtOSA1 was unable to 8

complement yeast strains lacking the endogenous ABC1 gene, and this suggests a different function for 9

this chloroplast kinase when compared to mitochondrial ABC1. Another chloroplast ABC1-like kinase, 10

AtACDO1 (ABC1-like kinase related to chlorophyll degradation and oxidative stress), has been 11

recently associated with defects in chlorophyll degradation and oxidative stress response under high 12

light conditions (Yang et al., 2012). The Chlamydomonas reinhardtii serine/threonine protein kinase 13

EYE3, belonging to the ABC1 family, has been recently demonstrated to localize to the pigment 14

granule arrays in the eyespot apparatus and to be required for their assembly (Boyd et al., 2011). These 15

carotenoid-filled globules are necessary for the correct assembly of the eyespot complex and are related 16

to chloroplast lipid droplets (plastoglobules) (Kreimer, 2009). Plastoglobules are associated with 17

thylakoid membranes (Brehelin and Kessler, 2008) and known to accumulate carotenoids and 18

prenylquinones (Steinmuller and Tevini, 1985; Deruere et al., 1994). Interestingly, six out of the eight 19

ABC1-like kinases currently identified in A. thaliana chloroplasts were identified in the proteome of 20

highly purified plastoglobules in several independent studies (Vidi et al., 2006; Ytterberg et al., 2006; 21

Lundquist et al., 2012). Considering the coincident presence of several enzymes involved in 22

prenylquinone metabolism in plastoglobules, it appears possible that the ABC1-like kinases are 23

involved in the regulation of prenylquinone metabolism comparable to ABC1 in the bacterial and 24

mitochondrial ubiquinone pathways. 25

In this paper we focused on the ABC1-like kinase family member ABC1K3 and on its influence on 26

chloroplast prenylquinone composition. ABC1K3 is known to be highly enriched in chloroplast 27

plastoglobules (Vidi et al., 2006; Ytterberg et al., 2006; Lundquist et al., 2012). Moreover, gene 28

coexpression analysis has recently demonstrated that this kinase clusters together with plastoglobule 29

proteins predicted to be involved in carotenoid metabolism and plastid proteolysis, thus suggesting that 30

ABC1K3 is functionally linked to genes involved in diverse aspects of chloroplast and plastoglobule 31

metabolism (Lundquist et al., 2012). 32

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1

Results 2

Isolation of ABC1K3 mutants 3

Two independent T-DNA insertion mutant lines (SALK_128696 and SAIL_918E10) were identified 4

for ABC1K3 in the SALK database (Alonso et al., 2003), and homozygous plants were selected by PCR 5

genotyping (Figure S1A-C). The visual phenotype and protein patterns, as well as photosynthetic 6

parameters and lipid composition of the two mutants were identical (Figure S2A-C). For these reasons, 7

only the abc1k3 mutant line SALK_128696 was used for further experiments. 8

Moreover, a quantitative RT-PCR analysis was carried out on total RNA extracted from fresh wild-type 9

and abc1k3 leaves. No ABC1K3 transcript was detected in abc1k3, demonstrating that it corresponds to 10

a null mutant (Figure S1D). 11

12

NPQ is moderately affected in the abc1k3 mutant under HL treatment 13

Chlorophyll fluorescence measurements using a Mini-PAM device indicate photosynthetic functions 14

and were carried out on abc1k3 and tocopherol cyclase (vte1) mutants using wild-type plants (Col-0) as 15

the control. The fluorimetric determination of the photosynthetic parameters was performed at intervals 16

of 24 h on leaves (n = 5) from rosettes grown either under normal light conditions (150 μE⋅m-2⋅s-1) or 17

exposed to continuous high light intensity (HL, 500 μE⋅m-2⋅s-1) for a total duration of 7 days. No 18

significant differences in PSII quantum efficiency (Fv/Fm) or total electron transport rate (ETR, not 19

shown) were detected between the three plants lines either under normal light intensity or after short 20

HL treatment. A significant reduction in PSII efficiency was observed in the vte1 mutant starting from 21

4 days of continuous HL exposure (Figure 1A). Non-photochemical quenching (NPQ) in abc1k3 was 22

slightly lower than in the wild-type. In vte1, NPQ levels were higher than in wild-type plants after a 23

short exposure (1-3 days) to continuous HL but they significantly decreased under prolonged HL 24

treatment (Figure 1B). 25

26

abc1k3 mutation affects chloroplast ultrastructure 27

Transmission electron microscopy was used to analyze the chloroplast ultrastructure in abc1k3 plants 28

and compare it to both the wild-type and the vte1 mutant (Figure 2A-C). Leaf sections from plants 29

grown under different light intensities were analyzed. Under normal light conditions, abc1k3 30

chloroplasts appeared to be morphologically similar to wild-type but the thylakoid grana appeared 31

slightly scattered and there were very few or no visible starch granules. However, under HL treatment, 32



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abc1k3 chloroplasts significantly differed from the wild-type, presenting large scattered grana, 1

extensive vacuolation as well as an increase in plastoglobule size and number of plastoglobule clusters. 2

Moreover, under HL no starch granules were observed in abc1k3 chloroplasts (Figure 2B). 3

In contrast, vte1 chloroplasts did not significantly differ from the wild-type under normal light 4

conditions. But a slight reduction of the number of starch granules and a reduction in the number and 5

size of plastoglobules in vte1 after HL treatment was observed when compared to the wild-type (Figure 6

2C), as previously reported (Zbierzak et al., 2010). 7

8

9

ABC1K3 has a major effect on membrane prenylquinone composition 10

Ultra-high pressure liquid chromatography coupled to atmospheric pressure chemical ionization-11

quadrupole time of flight mass spectrometry (UHPLC-APCI-QTOFMS), a powerful analytical 12

technique for rapid and reliable lipid profiling of plant samples, was used to test whether the abc1k3 13

mutant is affected in prenylquinone and/or carotenoid composition. 14

Total leaf extracts from 2-month-old A. thaliana rosettes grown under normal light conditions or 15

exposed to continuous HL stress for an increasing period of time (1, 2 and 7 days, respectively) were 16

subjected to analysis, yielding more than 400 peaks in negative APCI mode. The identified 17

prenylquinones and carotenoids are presented in Table S1. 18

Principal component analysis (PCA) was applied to highlight differences in the lipid composition of 19

wild-type and abc1k3 plants (Figure 3A-B). Both under normal light conditions and after HL exposure, 20

wild-type and abc1k3 samples clustered separately and differed significantly from vte1. Interestingly, 21

the abc1k3 cluster co-localized with the α-T oxidative-derivate α-TQ but not with the other 22

tocopherols, which were typical of wild-type samples. On the contrary, vte1 samples co-localized with 23

the xanthophyll lutein and the tocopherol precursor DMPBQ. 24

A quantitative analysis of these prenylquinones and carotenoids was also performed to determine the 25

variations in their content between wild-type, abc1k3 and vte1 in response to HL exposure (Figure 4A-26

F). Similar α-, γ- and δ-T levels were detected in wild-type and abc1k3, although a slightly slower 27

accumulation rate was observed in abc1k3 after prolonged HL exposure. The tocopherols were absent 28

from vte1 as expected (Figure 4A, 4C and 4D). However, under HL treatment, abc1k3 accumulated 29

significantly higher amounts of α-TQ, a tocopherol Redox cycle intermediate generated in thylakoid 30

membranes by spontaneous oxidation that is recycled in a VTE1-dependent manner (Figure 4B). 31

Strikingly, the levels of another VTE1 product, plastochromanol-8 (PC-8), were about 4-fold lower in 32

abc1k3 than in the wild-type and did not significantly change after HL exposure (Figure 4E). The 33



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abc1k3 phenotype is not a phenocopy of vte1, because vte1 completely lacks PC-8 (in addition to the 1

absence of all tocopherols) but accumulates the precursor DMPBQ instead. DMPBQ was not detectable 2

in abc1k3 samples (data not shown) which contained all the tocopherols. Moreover, an accumulation of 3

the xanthophyll lutein was observed in vte1 both under normal growth conditions or after prolonged 4

HL exposure, while its content in abc1k3 was slightly lower than in the wild-type under normal light, 5

but increased after HL treatment (Figure 4F). As previously observed by PCA of total lipidomics data, 6

other molecules could be affected by the abc1k3 mutation, yet no unambiguous identification could be 7

achieved based on their m/z ratio. A complete list of all the detected molecules, as well as their relative 8

abundance in the three tested plant lines and under different light conditions, is provided in Table S2. 9

10

abc1k3 mutants have a low VTE1 content 11

Considering that the most relevant differences in prenylquinone composition of abc1k3 plants 12

depended on the enzymatic products of the tocopherol cyclase, a possible variation in VTE1 content or 13

sub-cellular localization was investigated by Western Blot analysis. Besides, in order to investigate a 14

potential effect of ABC1K3 on other plastoglobule proteins, antibodies raised against the two most 15

abundant plastoglobule fibrillins FBN1a and FBN2 were used as well (Lundquist et al., 2012). The 16

thylakoid marker LHCb2 was used as control. Equal protein amounts of total chloroplast membrane 17

fractions as well as purified chloroplast membrane sub-fractions corresponding to plastoglobules and 18

thylakoids were extracted from plants grown under normal light conditions, then separated by SDS-19

PAGE, transferred to nitrocellulose and probed using αVTE1, αFBN1a/b, αFBN2 and αLHCb2 20

antibodies (Figure 5A-C). VTE1 was detected in wild-type plastoglobules but was below the detection 21

limit in abc1k3. As expected, VTE1 was not present in the knock-out line vte1. Interestingly, the 22

fibrillin FBN2 appeared to be much less abundant in abc1k3 plastoglobules than in wild-type, while its 23

content in vte1 samples was only moderately lower than in wild-type. The use of αFBN1a antibodies 24

for immunoblotting resulted in the detection of two distinct proteins with a predicted molecular mass 25

slightly higher than 30 kDa and corresponding to the two fibrillins FBN1a and FBN1b, as previously 26

observed (Giacomelli et al., 2006). In abc1k3 plastoglobules, both proteins were less abundant than in 27

wild-type, while vte1 plastoglobules showed a similar intensity in the upper band (FBN1a), whereas 28

that of the lower one (FBN1b) was reduced. With regard to the controls, no significant differences 29

between wild-type, abc1k3 and vte1 were observed for the thylakoid marker LHCb2. 30

31

VTE1 and FBN1a are overexpressed in abc1k3 plants 32



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Both VTE1 and FBN1a protein levels were reduced in abc1k3. In order to determine whether the 1

expression of the corresponding genes is affected or whether the reduction occurs at a post-2

transcriptional level, quantitative RT-PCR experiments were carried out for the two genes VTE1 and 3

FBN1a on total RNA extracted from fresh wild-type, abc1k3 and vte1 leaves. A significant increase 4

(2.75-fold) in VTE1 transcript levels was observed in abc1k3 compared to wild-type plants, while only 5

a weak, presumably background signal was detected in vte1 (Figure 6). At the same time, a significant 6

increase in the expression of FBN1a was detected in both abc1k3 and vte1 mutants (1.5 and 2.2-fold 7

more, respectively) when compared to the wild-type (Figure 6). Gene (co-) expression was also 8

investigated in silico using the ATTED-II database. ABC1K3 and VTE1 clustered together, and a strong 9

correlation between the expression of the two genes was predicted under stress conditions, suggesting a 10

functional interaction (Figure S3). 11

12

ABC1K3 phosphorylates VTE1 13

ABC1K3 is a predicted kinase located in plastoglobules. However, its targets are currently unknown 14

but our results suggest VTE1 is one of them. In order to test this, the mature form of the kinase was 15

synthesized in a cell-free reticulocyte lysate (IVT mix) and used in phosphorylation reactions in the 16

absence or presence of purified, recombinant tocopherol cyclase and [γ-33P]ATP. The reticulocyte 17

lysate alone was used as negative control. The lysate contains all kinds of cellular proteins including 18

kinases as well as their substrates and therefore a number of faint background bands can be observed 19

(Figure 7A). 33P incorporation above the background levels (Figure 7C) was detected in a protein band 20

with an apparent molecular mass of about 45 kDa in samples containing both the ABC1K3 in vitro 21

translation product and the purified VTE1, but it was absent both in the reticulocyte lysate alone and in 22

the samples containing the lysate supplemented with ABC1K3 or VTE1 alone. The phosphoprotein had 23

a predicted molecular mass which was consistent with that of VTE1 (43.5 kDa) (Figure 7B). The 24

absence of additional bands phosphorylated by ABC1K3 points to the specificity of the kinase reaction. 25

In parallel, protein identification was performed by Western Blotting using antibodies raised against 26

VTE1 or a His6 tag on the recombinant kinase, which confirmed both ABC1K3 and VTE1 distribution 27

in the samples and strongly support VTE1 as the likely phosphoprotein (Figure 7D). 28

29

30

Discussion 31

ABC1K3 affects membrane lipid composition 32



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To analyze the role of ABC1K3 in lipid metabolism we analyzed total leaf lipid extracts of wild-type, 1

abc1k3 and vte1 mutant plants grown both under standard conditions and high light stress using 2

UHPLC-APCI-QTOFMS. Principal component analysis highlighted differences in prenylquinone and 3

carotenoid composition between the wild-type and the mutant plant lines. Most strikingly, lower PC-8 4

and elevated α-TQ levels were observed in abc1k3 already under normal growth conditions, while α-, 5

γ- and δ-T contents remained similar to the wild-type. PC-8 is a chromanol-derivative of plastoquinone 6

and α-TQ is an intermediate of the recently discovered tocopherol Redox cycle. Both PC-8 synthesis 7

and α-TQ recycling require the tocopherol cyclase (VTE1) activity (Kobayashi and DellaPenna, 2008; 8

Szymanska and Kruk, 2010) and were absent from the vte1 mutant. Interestingly though and in contrast 9

to vte1, no significant variations in the levels of DMPBQ were observed in the abc1k3 mutant when 10

compared to the wild-type. These results demonstrate a very specific role of ABC1K3 in prenylquinone 11

metabolism in regulating branches of VTE1 activity that may be located at the plastoglobule lipid 12

droplets. 13

These results are consistent with the finding that VTE1 activity can be a limiting factor for PC-8 but 14

not for tocopherol synthesis (Raclaru et al., 2006; Zbierzak et al., 2010). However, a comparative 15

lipidomics analysis performed using the vte1 mutant as a reference, lacking both PC-8 and tocopherols, 16

showed that the abc1k3 mutant does not simply correspond to an intermediate phenotype between wild-17

type and vte1 plants but clusters separately, suggesting that molecules other than these prenylquinones 18

may be affected as well. 19

20

ABC1K3 affects VTE1 activity and plastoglobule proteins 21

VTE1 levels were strongly reduced or absent in the total chloroplast membranes and sub-fractions 22

derived from abc1k3 and vte1 mutants, respectively. However, the VTE1 gene was transcribed at higher 23

levels in abc1k3 than in the wild-type. These findings indicate that VTE1 activity is post-24

transcriptionally downregulated in abc1k3 mutants. Moreover, evidence is provided that the mature 25

form of ABC1K3 phosphorylates VTE1 in vitro, suggesting that it is a target of the kinase. We 26

hypothesize that phosphorylation by ABC1K3 stabilizes VTE1 at plastoglobules. It is conceivable that 27

in the absence of ABC1K3-dependent phosphorylation VTE1 is degraded, explaining its lower content 28

in abc1k3 plastoglobules and the effect on the cellular levels of some of its metabolic products. 29

30

ABC1K3 deletion affects NPQ and chloroplast ultrastructure 31

When photosynthetic parameters and the response to HL intensity were measured, abc1k3 did not 32

significantly differ from the wild-type, although it showed slightly lower NPQ levels under HL stress, 33



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possibly because of a slightly lower xanthophyll (lutein and zeaxanthin) content. Conversely, vte1 had 1

higher NPQ levels than the wild-type after a short HL exposure because of the higher xanthophyll 2

content, as previously reported (Havaux et al., 2005). However, after prolonged HL treatment, a 3

reduction in both ETR and NPQ levels was observed in vte1 plants, possibly because of photo-4

oxidative damage caused by the lack of PC-8 and tocopherols, which were instead present in abc1k3, 5

although PC-8 at much lower concentration than in the wild-type. 6

Moreover, the comparison of transmission electron micrographs of wild-type and abc1k3 plants 7

indicates a possible effect of the mutation on chloroplast ultrastructure under HL conditions, which was 8

not observed in plants lacking VTE1. In particular, abc1k3 chloroplasts were characterized by large 9

plastoglobule clusters, extensive vacuolation and the absence of starch granules. These findings suggest 10

that ABC1K3 may have targets other than VTE1 that are still unknown. 11

12

13

Conclusion 14

We here demonstrate that ABC1K3 affects the accumulation of VTE1-dependent metabolites, α-15

tocopherol quinone and plastochromanol, most likely via phosphorylation of VTE1. Moreover, the 16

evidence presented suggests that ABC1K3 acts on other targets that we have not yet identified. In 17

conclusion, ABC1K3 may be involved in a far-ranging regulatory network not only regulating the 18

synthesis of VTE1-derived prenylquinone products but also plastoglobule morphology, thylakoid 19

integrity and starch accumulation. 20

21

22

Materials and Methods 23

Chemicals: 24

The solvents used for plant lipid profiling were tetrahydrofuran (THF, analytical grade, Normapur) 25

from VWR (Leuven, Belgium) and ethylacetate (EtAc, analytical grade) from Acros Organics (Geel, 26

Belgium). UPLC/MS grade MeOH and water from Biosolve (Valkenswaard, The Netherlands) were 27

used for the UHPLC-APCI-QTOFMS analyses. Unless stated otherwise, all the other chemicals were 28

purchased from Sigma-Aldrich (Steinheim, Germany). 29

30

Purified standards for Lipidomics analysis: 31



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α- and γ-tocopherol (α-T and γ-T, respectively) and phylloquinone (K) standards of HPLC grade (≥ 1

99.5 %) were purchased from Sigma-Aldrich. δ-tocopherol (δ-T) standard was purchased from Supelco 2

(Bellefonte, PA, USA). α-tocopherol quinone (α-TQ), plastoquinone-9 (PQ-9) and plastochromanol-8 3

(PC-8) standards were kindly provided by Dr. Jerzy Kruk (Jagiellonian University, Kraków, Poland). 4

The oxidized and reduced PQ-9 standards were obtained as described in (Suhara et al., 2005) with 5

slight changes (Martinis et al., 2011). Lutein (Lut) and β-Carotene (β-Car) standards were purchased 6

from Extrasynthese (Lyon, France). 7

8

Plant material and treatments: 9

Wild-type Arabidopsis thaliana plant always refers to Arabidopsis thaliana (L.) Heynh. var. Columbia 10

0. ABC1K3 disruption was tested using two T-DNA insertion lines, SALK_128696 and 11

SAIL_918_E10, both obtained from the Nottingham Arabidopsis Stock Centre (NASC, 12

http://arabidopsis.info) (Alonso et al., 2003). However, no phenotypical differences were observed 13

between the two lines. For this reason, in this paper abc1k3 always refers to SALK_128696. The 14

mutant line for VTE1 was a gift from Dr. P. Dörmann (Max Planck Institute, Golm, Germany) and was 15

obtained by ethyl methanesulfonate mutagenesis as described in (Porfirova et al., 2002). Plants were 16

grown on soil under standard growth conditions (150 μE⋅m-2⋅s-1, 8/16 h light/dark period, 21/18 °C, 17

55% relative air humidity) according to the protocol described in (Hiltbrunner et al., 2001) with slight 18

modifications. 19

High light treatments were performed on 1 or 2-month-old rosettes by exposure to continuous high 20

light conditions (500 μE⋅m-2⋅s-1, 21 °C, 55% relative air humidity); light was provided by 400 W metal 21

halide lamps (Philips, USA). 22

23

Mutant genotyping 24

Rapid genomic DNA (gDNA) extraction from A. thaliana leaves was performed as described in 25

(Edwards et al., 1991). The predicted location of the T-DNA insertion in the two mutant lines 26

SAIL_918E10 and SALK_128696 was at the 5’ of the second exond and inside the third exon of 27

ABC1K3, respectively. In order to select homozygous lines for T-DNA insertions, a three-primer PCR 28

analysis was employed. The primer sets included a Left and Right genomic Primer designed on the 29

regions flanking the predicted T-DNA insertion site (LP and RP, at the 5’ and 3’ of the T-DNA, 30

respectively) and the left T-DNA Border Primer (BP), designed on the inserted sequence. For SALK 31

lines LBa1 of pBIN-pROK2 for was used, while LB1 of pCSA110-pDAP101 was selected for SAIL 32



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lines. LB and RP were designed using the T-DNA Primer Design tool from the Salk Institute Genomic 1

Analysis Laboratory (La Jolla, CA, USA). The T-DNA insertion line SAIL_918_E10 was genotyped 2

using the primer couples LPa (5’-GGGAGGAGGTAGTGACAAAGG-3’) and RPa (5’-3

AAGGTAATCGGGTGGACAGAG-3’), while for the SALK_128696 line the primers LPb (5’-4

TGTTGCTGTCAAAGTTCAACG-3’) and RPb (5’-CAAGCGTACTTTGAAGTTCCG-3’) were used. 5

DNA extracted from wild type A. thaliana (Columbia-0) plants was used as reference. PCR reactions 6

were then analyzed by agarose gel electrophoresis according to standard protocols (Sambrook and 7

Russell, 20012001). 8

9

Determination of the photosynthetic parameters: 10

Maximum quantum efficiency of photosystem II (Fv/Fm), electron transport rate (ETR) and non-11

photochemical quenching (NPQ) were fluorometrically determined using a MINI-PAM Photosynthesis 12

Yield Analyzer (Walz, Effeltrich, Germany). 13

14

Arabidopsis chloroplast ultrastructural analysis: 15

Leaves from 4- to 5-week-old A. thaliana rosettes were fixed overnight in 0.1 M phosphate buffer (pH 16

6.8) containing 4% (w/v) formaldehyde and 5% (w/v) glutaraldehyde, washed three times in 0.1 M 17

phosphate buffer (pH 6.8) for 20 min and post-fixed for 2 h with 1% (w/v) osmium tetraoxide (OsO4) 18

at 20°C. Samples were dehydrated in ethanol and acetone series and infiltrated overnight with a low 19

viscosity epoxy resin (Spurr; Polyscience, Eppelheim, Germany). Leaf fragments were then placed in 20

an appropriate mold, included in Spurr and heated for 4 h at 60°C to allow the resin to solidify. 21

Ultrathin sections of 90 nm were prepared using an Ultracut-E microtome (Reichert-Jung) equipped 22

with a diamond knife (Diatome, Biel, Switzerland), mounted on copper grids and contrasted with a 23

saturated uranyl acetate solution in 50% ethanol and with Reynolds' lead citrate according to 24

(Reynolds, 1981). Ultrathin sections were observed with a Philips CM-100 electron microscope 25

operating at 60 kV. 26

27

Arabidopsis membrane prenylquinone profiling: 28

Liquid chromatography-mass spectrometry analysis of leaf samples was performed as described in 29

(Martinis et al., 2011) with the following modifications to allow the simultaneous separation and 30

detection of prenylquinones and carotenoids: the initial MeOH percentage used for the 31

chromatographic gradient was set to 80%, increased to 100% in 3 min, and maintained at 100% for 2 32

min. α-T, α-TQ, γ-T, δ-T, K, PQ-9, PC-8, lutein and β-carotene were quantified based on calibration 33



15/23

curves built from standard compounds. Data were processed using Masslynx v4.1 (Waters) and 1

multivariate analysis was carried out using MarkerLynx XS™ (Waters). Variables were Pareto-scaled 2

before applying principal component analysis (PCA). 3

4

Chloroplast fractionation and Western Blot analysis: 5

Chloroplast membranes were extracted and fractionated as described in (Vidi et al., 2006) with slight 6

modifications. To prevent proteolytic degradation and spontaneous dephosphorylation, 0.5% (v/v) 7

protease inhibitor cocktail for plant cell extracts (P9599, Sigma) and 1% (v/v) phosphatase inhibitor 8

cocktail 2 (P5726, Sigma) was added. Total proteins were extracted by chloroform-methanol 9

precipitation (Wessel and Flugge, 1984), resuspended in an appropriate volume of sample buffer [50 10

mM Tris/HCl pH 6.8, 0.1 M DTT, 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol] 11

and separated by SDS-PAGE. Blots were then probed with sera raised against the light-harvesting 12

antenna type II chlorophyll a/b-binding protein (LHCb2, kindly provided by Dr. K. Apel, ETH Zurich, 13

Switzerland), the tocopherol cyclase (VTE1, (Kanwischer et al., 2005)) and the two fibrillins FBN1a 14

and FBN2 (Vidi et al., 2006) 15

16

Phosphorylation assays: 17

In order to test VTE1 as a possible target of ABC1K3, the coding sequence for the kinase without the 18

transit peptide (in silico prediction performed using the TargetP 1.1 server (Emanuelsson et al., 2007)) 19

and including a C-terminal His6 tag was cloned in the p0GWA expression vector, a derivate of pET22d 20

(Merck, Darmstadt, Germany) described in (Busso et al., 2005). The protein was then synthesized in 21

vitro using the TNT® Quick Coupled Transcription/Translation System (Promega) according to the 22

manufacturer’s recommendations. The expression vector VTE1-pQE31, encoding for the mature part 23

of the tocopherol cyclase without the predicted signal peptide, was kindly provided by Dr. P. Dörmann 24

(University of Bonn, Germany) and was used for protein expression, as previously described (Porfirova 25

et al., 2002). VTE1 was then purified by affinity chromatography (Qiagen, Hilden, Germany), mixed 26

with the ABC1K3 in vitro translation product in different combinations and used in 100 μL in vitro 27

phosphorylation reactions in a suitable phosphorylation buffer [50 mM HEPES/KOH pH 7.5, 5 mM 28

MgCl2, 0.5 mM MnCl2, 50 mM NaCl, 0.1 mM DTT] supplemented with 50 μM ATP and 2 μCi [γ-29 33P]ATP. Reactions were incubated for 30 min at 25 °C and under gentle shaking (300 rpm), then 30

proteins were swiftly precipitated in chloroform-methanol (Wessel and Flugge, 1984) and resuspended 31

in an appropriate volume of the previously described sample buffer. Proteins were separated by SDS-32

PAGE followed by Coomassie Blue staining and dried on 2 layers of Whatman filter paper. 33P 33



16/23

incorporation was detected after three or more days using a Personal FX Phosphorimager and analyzed 1

using the QuantityOne software (both from Bio-Rad Laboratories). The identity of the radioactive band 2

was confirmed by parallel Western Blot analysis using antibodies raised against VTE1 (see previous 3

section). 4

5

6

Gene expression: 7

Total RNAs were purified from A. thaliana leaves using the RNeasy Plant Mini Kit (Qiagen), 8

according to the supplier’s recommendations, and DNase treatment was performed on column using the 9

RNase-free DNase set (Qiagen). cDNAs were then prepared using the GoScript™ Reverse 10

Transcriptase kit (Promega) in the presence of RNasin RNase inhibitors (Promega), and stored at -11

20°C. For quantitative PCR, the housekeeping gene ACTIN2 was used as reference and amplified using 12

the primer couple act2-S (5’-TGGAATCCACGAGACAACCTA-3’) and act2-AS (5’-13

TTCTGTGAACGATTCCTGGAC-3’). For the analysis of ABC1K3 expression level in the mutant, the 14

primer couple ABC1K3_q_F (5’- GCTTTGATCCTGCTATTTTG-3’) and ABC1K3_q_R (5’- 15

GTGGCTCGAAGAAGTTGTTG-3’) were used. For the analysis of VTE1 and FBN1a expression, the 16

following primers were designed: VTE1_q_F (5’-GTGCTCCTACCACAGAAGTT-3’), VTE1_q_R 17

(5’-TATCTCCACTGCTGCCATTG-3’), FBN1a_q_F (5’-CAAACCATTGATTCCGATAG-3’) and 18

FBN1a_q_R (5’-AGTCCCTATAACACCTTGCT-3’). Real-Time PCR reactions (n = 3) were 19

assembled in a final volume of 20 μL using the ABsolute™ qPCR SYBR® Green mix (ABgene, 20

Epsom, UK), 10 µM of both 5' and 3' primers, and adjusted amounts of cDNA diluted 40 times in 21

ddH2O. The PCR program was as follows: 15 min, 95°C (thermo-start); 45x (20 s, 95°C; 30 s, 55°C; 45 22

s, 72°C); 1 min, 55°C, and was followed by an 80-step dissociation (55 to 95°C). SYBR® Green 23

emission was detected in real-time in an iQ5 Optical System (Bio-Rad Laboratories, Reinach, 24

Switzerland) and data were treated using the included software. A one-way analysis of variance 25

(ANOVA) was conducted on the obtained data. When a significant difference was detected, a Tukey 26

HSD test was applied at a significance level of P < 0.05 to separate the means. 27

Gene coexpression was determined in silico using the ATTED-II 6.0 database (Obayashi et al., 2011). 28

29

Accession numbers: 30

Sequence data from this article can be found in the EMBL/GenBank data libraries under accession 31

numbers AEE36271.1 (ABC1K3, At1g79600.1), AEE86116.1 (VTE1, At4g32770.1), AEE82363.1 32



17/23

(FBN1a, At4g04020.1), AEC09113.1 (FBN2, At2g35490.1), AEC05893.1 (LHCB2, At2g05070.1) and 1

AEE76147.1 (ACTIN2, At3g18780.1). 2

3

4

Acknowledgments 5

We thank Jerzy Kruk from Jagiellonian University for kindly providing purified α-tocopherol quinone, 6

plastoquinone-9 and plastochromanol-8 standards, and Claus Wedekind from University of Lausanne 7

for providing carotenoid standards. 8

9

10

Author Contributions 11

JM performed the research and analyzed the data. GG conducted the UHPLC-APCI-QTOFMS 12

measurements. SV carried out the purification of the tocopherol cyclase used in the phosphorylation 13

assays. JM and FK conceived the study and wrote the manuscript. All authors read, commented and 14

approved the final manuscript. 15

16

17

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33



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1

Figure legends 2

Figure 1: Effect of continuous HL exposure on plant photosynthetic parameters. A. PSII activity 3

(Fv/Fm). B. Non-photochemical quenching (NPQ). Leaves (n=5) from plants exposed to normal (150 4

μE × m-2 × s-1) or high (500 μE × m-2 × s-1) light intensity were analyzed at intervals of 24 h. 5

6

Figure 2: Changes in chloroplast ultrastructure observed under HL treatment. Transmission 7

electron micrographs of leaves from A. wild-type, B. abc1k3 and C. vte1 plants exposed to normal light 8

intensity (left panels, 150 μE × m-2 × s-1) or to continuous high light (right panels, 500 μE × m-2 × s-9

1) for 4 days. Black bars correspond to a length of 1 μm. 10

11

Figure 3: Bi-plots derived from an untargeted principal component analysis (PCA) showing 12

differences between lipid profiles of different plant lines and after HL treatment. A. Plants grown 13

under normal light conditions (150 μE × m-2 × s-1). B. Plants exposed to continuous high light (500 μE 14

× m-2 × s-1) for 7 days. Inset on the right: Bi-plots from a PCA performed exclusively on wild-type 15

(WT) and abc1k3 data. Colored squares correspond to the observations (n = 4 for each plant line) and 16

black triangles represent the variables. PC1 and PC2 are first and second principal components, 17

respectively, with their percentage of explained variance. The identified prenylquinones and 18

carotenoids are indicated (confront with Table S1). For abbreviations, see text. 19

20

Figure 4: Comparison of the prenylquinone and carotenoid total leaf content. A. α-tocopherol. B. 21

α-tocopherol quinone. C. γ-tocopherol. D. δ-tocopherol. E. Plastochromanol-8. F. Lutein. Plants grown 22

under normal light conditions (day 0, 150 μE × m-2 × s-1) were exposed to continuous high light (500 23

μE × m-2 × s-1) for 1, 2 and 7 days, respectively. Data are from 4 biological replicates (± SD). 24

25

Figure 5: Western blot analysis on purified chloroplast sub-fractions. Total proteins were extracted 26

from total chloroplast membranes (MEM), plastoglobules (PG) and thylakoids (THY). Proteins were 27

separated by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted using the 28

indicated antibodies raised against: the tocopherol cyclase (VTE1), the two most abundant 29

plastoglobule fibrillins (FBN1a and FBN2, respectively) and the light-harvesting antenna type II 30

chlorophyll a/b-binding protein (LHCb2). The positions of the detected proteins on the membrane are 31

indicated by white triangles. 32



22/23

1

Figure 6: Changes in VTE1 and FBN1a gene expression rate determined by real-time RT-PCR. 2

Total RNA extracted from untreated leaves was used for PCR assays. ACTIN2 expression levels were 3

used for normalization. Significant differences between samples (n=3 for each plant line) were 4

determined by a one-way ANOVA. 5

6

Figure 7: in vitro phosphorylation assays. A. VTE1 phosphorylation by ABC1K3. Recombinant 7

ABC1K3-His6 was synthesized in rabbit reticulocyte lysate (IVT mix) and used for in vitro 8

phosphorylation reactions in the presence or absence of the purified recombinant VTE1-His6. The IVT 9

mix already contains multiple kinases and their phosphorylation targets and can thus serve as negative 10

control. Reactions were supplemented with [γ-33P]ATP and 33P incorporation in the samples was then 11

determined by autoradiography. B. For comparison, the positions of ABC1K3-His6 and VTE1-His6 on 12

the gel were determined by co-migration with the two proteins independently detected by 13

immunoblotting of separate SDS-PAGE gels. C. Phosphor Imager quantification of the relative 14

intensity at the position of the white arrow (corresponding to VTE1-His6). Significant differences 15

between samples (n=4 biological replicates) were determined by a one-way ANOVA. D. To validate 16

the presence of the two proteins in the samples, phosphorylation reactions not supplemented with [γ-17 33P]ATP were analyzed on separate SDS-PAGE gels, transferred on nitrocellulose membranes and 18

stained with Coomassie Blue (top panel). The presence of ABC1K3-His6 and VTE1-His6 was then 19

determined by immunoblotting (lower panels). 20

21

22

Supplemental Data 23

Figure S1: Isolation of ABC1K3 T-DNA insertion mutants. A. Mapping of the T-DNA insertions in 24

the genome of two knock-out lines for ABC1K3. Squares represent exons and lines introns. Light grey 25

regions correspond to the predicted UTRs. B. Genotyping of the mutant line SAIL_918E10. C. 26

Genotyping of the mutant line SALK_128696. Genotyping was performed by PCR analysis using the 27

indicated primers. Reactions were designed in order to detect the ABC1K3 wild-type alleles or the T-28

DNA 5’/3’ flanking sequence. DNA from wild type plants (WT) was used as positive control (C+). 29

Hm. Homozygous. Hz. Heterozygous. Nd. PCR product not detected. D. Determination of ABC1K3 30

gene expression rate by real-time RT-PCR. Total RNA extracted from untreated wild-type and abc1k3 31

(SALK_128696) leaves was used for PCR assays. ACTIN2 expression levels were used for 32



23/23

normalization. Significant differences between samples (n=3 for each plant line) were determined by a 1

one-way ANOVA. 2

3

Figure S2: Comparison between wild-type plants and the two abc1k3 T-DNA insertion mutant 4

alleles SALK_128696 and SAIL_918E10. A. Light dependence of electron transport rate (ETR). B. 5

Non-photochemical quenching (NPQ). Plants were grown under normal light conditions (150 μE × m-2 6

× s-1) and leaves (n=5) were analyzed at intervals of 24 h. C. Principal component analysis (PCA) 7

performed on the lipid profiles of plants grown under normal light conditions. Prenylquinones and 8

carotenoids were extracted and analyzed by UHPLC-APCI-QTOFMS. Colored squares correspond to 9

the observations (n = 6 for each plant line). PC1 and PC2 are first and second principal components, 10

respectively, with their percentage of explained variance. 11

12

Figure S3: Predicted ABC1K3 coexpressed gene network. A. Gene network for the two genes 13

ABC1K3 and VTE1 estimated using the online tools provided by the ATTED-II database. Octagons 14

represent plastoglobule proteins. Line thickness corresponds to the average correlation rank between 15

two connected genes. Red circles indicate proteins involved in the biosynthesis of secondary 16

metabolites. B. Stability of the coexpression corresponding to the number of samples which support the 17

focused gene-to-gene correlation. The green circle indicates the position of the focused gene pair 18

ABC1K3-VTE1 in the background distribution calculated for the top 300 highly coexpressed gene pairs 19

in the ATTED-II database. C. Detail of the stability of gene coexpression. When the coexpression was 20

supported by many factors, the PCA correlation will not change against subtractions of a few main 21

factors, which indicates strong gene-to-gene functional relationship. 22

23

Table S1: List of the molecules identified from UHPLC-APCI-QTOFMS data acquired in negative 24

ionization mode. 25

26

Table S2: List of all the molecular markers detected by UHPLC-APCI-QTOFMS lipid profiling and 27

relative abundance in analyzed samples. 28

29



0.0

0.5

1.0

1.5

2.0

2.5

3.0

NP

Q(5

00µ

E·m

-2·s

-1)

HL treatment (days)0 1 2 3 4 5 6 7

HL treatment (days)

Fv/

Fm

0.45

0.50

0.55

0.60

0.70

0.75

0.80

0.85

0.65

0 1 2 3 4 5 6 7

WT abc1K3 vte1A

B

Figure 1: Effect of continuous HL exposure on plant photosynthetic parameters. A. PSII activity(Fv/Fm). B. Non-photochemical quenching (NPQ). Leaves (n=5) from plants exposed to normal (150 μE × m-2 × s-1) or high (500 μE × m-2 × s-1) light intensity were analyzed at intervals of 24 h.

1



Figure 2: Changes in chloroplast ultrastructure observed under HL treatment. Transmission electron micrographs of leaves from A. wild-type, B. abc1k3 and C. vte1 plants exposed to normal light intensity (left panels, 150 μE × m-2 × s-1) or to continuous high light (right panels, 500 μE × m-2 ×s-1) for 4 days. Black bars correspond to a length of 1 μm.

2

Day 0 HL +4 days

A

B

C



-1.00

-0.75

-0.50

-0.25

0.00

0.25

0.50

0.75

1.00

-1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00

PC1 (37%)

PC

2 (

34

%)

C18:0

δ-T

γ-T

DMPBQ

α-T

α-TQ

β-Car

LutPQH2

PQ

PC-8

PQ-OH

PC-OH

UQ

UQH2

K

vte1

WT

abc1k3

-1.00

-0.75

-0.50

-0.25

0.00

0.25

0.50

0.75

1.00

-1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00

PC1 (49%)

PC

2 (

23

%)

C18:0

δ-T

γ-T

α-T

α-TQ

β-Car

Lut

PQH2

PQ

PC-8

PQ-OH

PC-OH

UQ

UQH2

K

WT

abc1k3

A

-1.00

-0.75

-0.50

-0.25

0.00

0.25

0.50

0.75

1.00

-1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00

PC1 (45%)

PC

2 (

22

%)

C18:0δ-T

γ-T

α-T

α-TQ

β-Car

Lut

PQH2

PQ

PC-8

PQ-OH

PC-OH

UQ

UQH2

K

DMPBQ

vte1

WT

abc1k3

-1.00

-0.75

-0.50

-0.25

0.00

0.25

0.50

0.75

1.00

-1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00

PC1 (45%)

PC

2 (

25

%)

C18:0

δ-T

γ-T

α-T

α-TQ

β-Car

Lut

PQH2

PQ

PC-8

PQ-OH

PC-OH

UQ

UQH2

K

WT

abc1k3

B

Figure 3: Bi-plots derived from an untargeted principal component analysis (PCA) showing differences between lipid profiles of different plant lines and after HL treatment. A. Plants grown undernormal light conditions (150 μE × m-2 × s-1). B. Plants exposed to continuous high light (500 μE × m-2 × s-1) for 7 days. Inset on the right: Bi-plots from a PCA performed exclusively on wild-type (WT) and abc1k3 data. Colored squares correspond to the observations (n = 4 for each plant line) and black triangles represent the variables. PC1 and PC2 are first and second principal components, respectively, with their percentage of explained variance. The identified prenylquinones and carotenoids are indicated (confront with Table S1). For abbreviations, see text.

3



00 1 2 7

HL treatment (days)

μg

/gF

W40

30

20

10

0.00 1 2 7

HL treatment (days)

μg

/gF

W

1.2

0.8

1.0

0.6

0.4

0.2

0.00 1 2 7

HL treatment (days)

μg

/gF

W

5.0

3.0

4.0

2.0

1.0

0.00 1 2 7

HL treatment (days)

μg

/gF

W

1.5

1.0

0.5

0.00 1 2 7

HL treatment (days)

μg

/gF

W

2.5

2.0

1.5

1.0

0.5

00 1 2 7

HL treatment (days)

μg

/gF

W

80

60

40

20

A

DC

FE

B

WT abc1K3 vte1

Figure 4: Comparison of the prenylquinone and carotenoid total leaf content. A. α-tocopherol. B. α-tocopherol quinone. C. γ-tocopherol. D. δ-tocopherol. E. Plastochromanol-8. F. Lutein. Plants grown undernormal light conditions (day 0, 150 μE × m-2 × s-1) were exposed to continuous high light (500 μE × m-2 × s-1) for 1, 2 and 7 days, respectively. Data are from 4 biological replicates (± SD).

4



WT

MEM PG THY

abc1k3

MEM PG THY

vte1

MEM PG THY

VTE1

FBN2

FBN1a

LHCb2

200

11697

66

45

29

20

KDa

LHCb2

FBN1a

FBN2VTE1

Figure 5: Western blot analysis on purified chloroplast sub-fractions. Total proteins were extracted fromtotal chloroplast membranes (MEM), plastoglobules (PG) and thylakoids (THY). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted using the indicated antibodies raisedagainst: the tocopherol cyclase (VTE1), the two most abundant plastoglobule fibrillins (FBN1a and FBN2, respectively) and the light-harvesting antenna type II chlorophyll a/b-binding protein (LHCb2). The positions of the detected proteins on the membrane are indicated by white triangles.

5



Figure 6: Changes in VTE1 and FBN1a geneexpression rate determined by real-time RT-PCR. Total RNA extracted from untreated leaveswas used for PCR assays. ACTIN2 expression levels were used for normalization. Significantdifferences between samples (n=3 for each plant line) were determined by a one-way ANOVA.

6

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

VTE1 FBN1a

Exp

ress

ion

rate

a

c

d

b

a

ad

VTE1 FBN1a

WT

abc1k3

vte1

Exp

ress

ion

leve

l(R

elat

ive)



7

Figure 7: in vitro phosphorylation assays.A. VTE1 phosphorylation by ABC1K3. Recombinant ABC1K3-His6 was synthesized in rabbit reticulocyte lysate (IVT mix) and used for in vitro phosphorylation reactions in the presence or absence of the purified recombinant VTE1-His6. The IVT mix already contains multiple kinases and their phosphorylation targets and can thus serve as negative control. Reactions were supplemented with [γ-33P]ATP and 33P incorporation in the samples was then determined by autoradiography. B. For comparison, the positions of ABC1K3-His6and VTE1-His6 on the gel were determined by co-migration with the two proteins independently detected by immunoblotting of separate SDS-PAGE gels. C. Phosphor Imager quantification of the relative intensity at the position of the white arrow (corresponding to VTE1-His6). Significant differences between samples (n=4 biological replicates) were determined by a one-way ANOVA. D. To validate the presence of the two proteins in the samples, phosphorylation reactions not supplemented with [γ-33P]ATP were analyzed on separate SDS-PAGE gels, transferred on nitrocellulose membranes and stained with Comassie Blue (top panel). The presence of ABC1K3-His6 and VTE1-His6 was then determined by immunoblotting (lower panels).

ABC1K3 VTE1

Western Blot

0.00

Rel

ativ

e In

tens

ity

0.25

0.50

0.75

1.00

IVT mix ABC1K3-His6 ABC1K3-His6+ VTE1-His6

VTE1-His6

a a a

b

A

C

B

DIVT mix ABC1K3 VTE1 ABC1K3 + VTE1

ABC1K3

VTE1

97

66

KDa

45

45

66

ABC1K3

VTE1

Phosphorylation assay

IVT mix ABC1K3 VTE1 +

33P incorporation

66

KDa

45

29



2 abc1k3 regulates vitamin e metabolism 3 corresponding ... · 1/23 1 running title 2 abc1k3...

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