The TOC159 mutant of Arabidopsis thaliana accumulates altered levels of saturated and polyunsaturated fatty acids

Plant Physiology and Biochemistry 87 (2015) 61e72

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Research article

The TOC159 mutant of Arabidopsis thaliana accumulates altered levels of saturated and polyunsaturated fatty acids Meshack Afitlhile*, Morgan Fry, Samantha Workman Department of Biological Sciences, Western Illinois University, Waggoner Hall 311, 1 University Circle, Macomb, IL 61455, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 October 2014 Accepted 24 December 2014 Available online 26 December 2014

We evaluated whether the TOC159 mutant of Arabidopsis called plastid protein import 2-2 (ppi2-2) accumulates normal levels of fatty acids, and transcripts of fatty acid desaturases and galactolipid synthesis enzymes. The ppi2-2 mutant accumulates decreased pigments and total fatty acid content. The MGD1 gene was downregulated and the mutant accumulates decreased levels of monogalactosyldiacylglycerol (MGDG) and 16:3, which suggests that the prokaryotic pathway was impaired in the mutant. The HY5 gene, which encodes long hypocotyl5 transcription factor, was upregulated in the mutant. The DGD1 gene, an HY5 target was marginally increased and the mutant accumulates digalactosyldiacylglycerol at the control level. The mutant had increased expression of 3-ketoacyl-ACP synthase II gene, which encodes a plastid enzyme that elongates 16:0 to 18:0. Interestingly, glycerolipids in the mutant accumulate increased levels of 18:0. A gene that encodes stearoyl-ACP desaturase (SAD) was expressed at the control level and 18:1 was increased, which suggest that SAD may be strongly regulated at the posttranscriptional level. The molar ratio of MGDG to bilayer forming plastid lipids was decreased in the coldacclimated wild type but not in the ppi2-2 mutant. This indicates that the mutant was unresponsive to cold-stress, and is consistent with increased levels of 18:0, and decreased 16:3 and 18:3 in the ppi2-2 mutant. Overall, these data indicate that a defective Toc159 receptor impaired the synthesis of MGDG, and affected desaturation of 16 and 18-carbon fatty acids. We conclude that expression of the MGD1 gene and synthesis of MGDG are tightly linked to plastid biogenesis. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: ppi2-2 mutant MGDG MGD1 16:3 18:0 18:3

1. Introduction Chloroplasts are the site of many biochemical processes including photosynthesis and fatty acid synthesis. The light reactions of photosynthesis take place in the thylakoid membranes. The biogenesis of thylakoid membranes is tightly linked to the development of the chloroplasts from proplastids, and this requires monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), which accounts for 75% of total thylakoid lipids (Kobayashi et al., 2007). During photomorphogenesis, the synthesis of photosystem proteins, lipids and pigments is coupled with their coordinated assembly into functional photosystems (Pogson et al., 1998). Both chlorophylls and carotenoids are required for the assembly and stability of light harvesting complex (LHC) apoproteins (Pogson et al., 1998). Fatty acid synthesis takes place in the stroma yielding palmitate

* Corresponding author. E-mail address: m-afi[email protected] (M. Afitlhile). http://dx.doi.org/10.1016/j.plaphy.2014.12.018 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.

(16:0)-acyl carrier protein (ACP), which is elongated to stearate (18:0)-ACP by 3-ketoacyl-ACP synthase II (KAS II). Therefore, KAS II activity determines the ratio of 16 to 18-carbon fatty acids. The 18:0-ACP is desaturated to oleate (18:1)-ACP by a soluble stearoylACP desaturase (Ohlrogge and Browse, 1995). Thus, 16:0-ACP and 18:1-ACP are the main products of fatty acid synthesis in the chloroplast stroma. In the plastids, acyltransferases transfer 16:0 and 18:1 from ACP to glycerol-3-phosphate, which result in the synthesis of phosphatidylglycerol (PG), MGDG, DGDG, and sulfoquinovosyldiacylglycerol (SQDG) (Ohlrogge and Browse, 1995). The photosynthetic protein-pigment complexes are embedded in the thylakoid membranes, and these membranes also contain MGDG, DGDG, SQDG and PG (Benning, 2008). Therefore, chloroplast lipids are major components of thylakoid membranes and they stabilize €rmann, photosystems and the light-harvesting complex II (Do 2007). The 16:0 and 18:1 are also exported as CoA thioesters from the plastids to the endoplasmic reticulum where these fatty acids are used in the synthesis of extrachloroplast lipids such as

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Abbreviations MGD1 HY5 KAS II ppi2-2 18:0 16:3 18:3

monogalactosyldiacylglycerol synthase 1 long hypocotyl5 3-ketoacyl-ACP synthase II plastid protein import 2-2 stearic acid hexadecatrienoic acid a-linolenic acid

phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) (Ohlrogge and Browse, 1995; Wallis and Browse, 2010). The diacylglycerol (DAG)

that is derived from PC is returned to the plastids, where it is incorporated into MGDG and DGDG by MGD1 and DGD1 synthases, respectively (Benning, 2008). MGDG that is synthesized by the prokaryotic pathway contains hexadecatrienoic acid (16:3) at the sn-2 position. However, MGDG that is synthesized from eukaryotic DAG contains unsaturated 18-carbon fatty acids, and a small amount of 16:0 may be present at the sn-1 position (Wallis and Browse, 2010). The desaturation of 16:0 and 18:1 that are esterified on the glycerol backbone is carried out by membrane-bound fatty acid desaturases (fad). In Arabidopsis, MGDG that is synthesized by the chloroplast pathway is a substrate for FAD5 desaturase (Heilmann et al., 2004), which yields 16:1D7 at the sn-2 position of MGDG. The 16:1D7 is desaturated by FAD6 and FAD7/8 enzymes to yield 16:3D7,10,13. Therefore, the accumulation of 16:3 serves as a relative measure of FAD5 desaturase activity (Heilmann et al., 2004).

Fig. 1. The accumulation of photosynthetic pigments and total protein in the wild type and ppi2-2 mutant of Arabidopsis. Values are means ± SE of 3 or 4 biological replicates. Asterisks above the error bars indicate significant difference (P < 0.05) of each treatment compared to the wild type (Col-0) as determined by Student's t test.

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Fig. 2. Total fatty acids content in the wild type and ppi2-2 mutant of Arabidopsis. Values are means ± SE of 4 or 5 replicates from two independent measurements. Col-0 (CA) and ppi2-2 (CA) are the cold-acclimated wild type and ppi2-2 mutant.

The cytoplasm-synthesized MGD1 synthase, stearoyl-ACP desaturase and FAD5 desaturase have the chloroplast targeting sequences, which indicate their import into plastids might use the general protein import pathway. Chloroplast desaturases that are encoded by FAD6, FAD7 and FAD8 genes lack the chloroplast transit peptides (Falcone et al., 1994; Gibson et al., 1994; Iba et al., 1993); it is not known if these enzymes engage the chloroplast protein import machinery. A recent study has demonstrated that superoxide dismutase, which lacks the classical transit peptide requires an ortholog of atToc120 for its import into the pea chloroplasts (Chang et al., 2014). Plastids evolved from photosynthetic bacteria similar to present day cyanobacteria (Jarvis, 2008; Jarvis and Lopez-Juez, 2013). During this evolution, most of the genes were transferred from the bacterial ancestor to the nuclear genome of the eukaryote host cell. Consequently, plastid biogenesis is dependent on the import of ~3000 nuclear-encoded proteins (Jarvis, 2008; Kessler and Schnell, 2009). Since plastids need a specific set and amount of proteins depending on their functional and metabolic states, plastids provide feedback information to the nucleus so that nuclear gene expression can be adjusted accordingly (Inaba et al., 2011; Jarvis and Lopez-Juez, 2013; Kakizaki et al., 2009). Translocation of the cytoplasm-synthesized preproteins into plastids is mediated by multiprotein complexes in the envelope membranes called translocons at the outer/inner envelope membrane of chloroplasts (TOC/ TIC complexes) (Jarvis, 2008; Kessler and Schnell, 2009). In Arabidopsis, the Toc159 protein family is encoded by four different genes, atTOC159, atTOC132, atTOC120 and atTOC90 (Jarvis, 2008; Kessler and Schnell, 2009). The Toc159 protein family has diverged into atToc159 and atToc132/atToc120, which represent separate receptors that recognize different types of preproteins (Jarvis, 2008; Kessler and Schnell, 2009). In Arabidopsis, atToc159 is highly expressed and abundant in green tissues, while atToc132 is

abundant in the roots (Bauer et al., 2000; Ivanova et al., 2004; Kubis et al., 2004). The atToc159 null mutant called plastid protein import 2-1 or ppi2-1 was isolated and characterized in Arabidopsis thaliana ecotype Wassilewskija (Bauer et al., 2000), while a mutant of A. thaliana ecotype Columbia (ppi2-2) was described a decade later (Kakizaki et al., 2009). Analysis of the ppi2-1 mutant showed that the mutant has undeveloped plastids and lacks organized thylakoid membranes, indicating that the mutation affects chloroplasts biogenesis (Bauer et al., 2000). Both ppi2 mutants have an albino phenotype and are incapable of importing photosynthetic proteins, although the import of housekeeping proteins is not affected (Bauer et al., 2000; Kakizaki et al., 2009). Overall, studies suggest that the atToc159 is a receptor with specificity for photosynthetic proteins, while atToc132/atToc120 is specific for the import of nonphotosynthetic proteins (Bauer et al., 2000; Ivanova et al., 2004; Kakizaki et al., 2009; Kubis et al., 2004; Smith et al., 2004). In our study, we tested whether the ppi2-2 mutant was capable of accumulating normal levels of fatty acids and transcripts of plastidlocalized galactolipid synthesizing enzymes and fatty acid desaturases. Since chloroplasts are the site of fatty acids and glycolipids synthesis, we speculated that the atToc159 receptor, which is highly expressed in green tissues, may play a major role in the import of chloroplast-localized lipid synthesizing enzymes and fatty acid desaturases. 2. Methods 2.1. Plant material and growth conditions All experiments were carried out using the wild type and ppi2 mutant of A. thaliana ecotype Columbia (ppi2-2). In one study, fatty acid composition was measured in the ppi2 mutant of A. thaliana ecotype Wassilewskija (ppi2-1). The ppi2-2 mutant was described

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Fig. 3. Fatty acid compositions in the wild type and ppi2-2 mutant of Arabidopsis. Values are means ± SE of 4 or 5 replicates from two independent measurements. Asterisks above the error bars indicate significant difference (P < 0.05) of each treatment compared to the non-acclimated wild type (Col-0) as determined by Student's t test.

by Kakizaki et al. (2009) and seeds were obtained from Takehito Inaba (University of Miyazaki, Japan). The ppi2-1 mutant was described by Bauer et al. (2000) and seeds were obtained from Matthew Smith (Wilfred Laurier University, Canada). Wild type and mutant seeds were surface sterilized in 95% EtOH, washed in 10% bleach that contained 0.1% Tween-20, and rinsed 5 times in sterile H2O. Surface sterilized seeds were plated on 0.8% agar containing 0.5X Murashige-Skoog (MS) medium and 1% sucrose, and seeds were stratified in the dark at 4
C for 3e5 days. MS plates were moved to a growth chamber set at 22
C with cool fluorescent lighting of 120 mmol m2 s1and 70% relative humidity under 16 h light/8 h dark cycles. For cold acclimation studies, 15day-old mutant and wild type seedlings were transferred to 4
C for 5 d under continuous white light.

2.2. Extraction of plant pigments and total soluble proteins For the extraction of plant pigments, 0.1 g leaf tissues were harvested from the 20-day-old wild type and ppi2-2 seedlings. Leaves were frozen in liquid nitrogen and pulverized using mortar and pestle. Total chlorophylls and carotenoids were extracted in 80% acetone and debri were precipitated by centrifugation at 12,000 rpm for 5 min. Absorbance of the supernatant was measured at 470, 646 and 663 nm using Spectrophotometer (Spectronic 20Dþ, Thermo Scientific), and the concentration of chlorophylls a and b, and total carotenoids were calculated as described by Wellburn (1994). For the extraction of total proteins, 0.1 g leaf tissues were frozen in liquid nitrogen and pulverized. The tissue was extracted in 50 mM K-phosphate buffer, pH 7.5 containing 0.1% Triton X-100, 5 mM EDTA, 2 mM mercaptoethanol and protease inhibitor cocktail that inhibits serine-, cysteine-, aspartic- and metallo-proteases, and

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Fig. 4. The composition of plastid and extraplastid lipids in the wild type and ppi2-2 mutant of Arabidopsis. Values are means ± SE of 3 or 4 replicates from two independent measurements. Asterisks above the error bars indicate significant difference (P < 0.05) of each treatment compared to the non-acclimated wild type (Col-0) as determined by Student's t test.

aminopeptidases (Sigma Chemical Company, St. Louis, MO). A protease inhibitor was added into the extraction buffer just before use. Samples were centrifuged at 12,000 rpm for 15 min and the resulting supernatant was used to estimate the amount of protein as described previously (Bradford, 1976).

2.3. Extraction and separation of lipids Leaf tissues were harvested from the 20-day-old wild type and ppi2-2 seedlings that were grown in MS media at 22
C or coldacclimated. Lipids were extracted without grinding leaf tissues and samples were processed as described previously (Li et al., 2008; Welti et al., 2002). Diheptadecanoyl phosphatidylcholine (PC-17:0, SigmaeAldrich Chemical company) was added as an internal lipid standard. The remaining plant tissues were heated overnight at 105
C and weighed to obtain dry weights. Individual lipids were separated on silica G plates (250 mm in silica thickness, Whatman) as described previously (Wang and Benning, 2011). Plates were dipped in 0.15 M (NH4)2SO4 solution and dried at room temperature in a covered plastic container for at least 2 days. Plates were activated by baking in an oven at 110
C for

2.5 h and cooled at room temperature. Authentic lipids were used as loading standards to identify lipid classes. Phospholipid standards (PG, PE, and PC) were purchased from Avanti Polar Lipids, Inc (Alabaster, AL) and MGDG was purchased from Larodan, Sweden. DGDG was purchased from SigmaeAldrich Chemical Company, St Louis, MO. Loaded plates were developed in a solvent that consist of acetone/toluene/water (91: 30: 7, v/v/v). The developed plate was dried in the fume hood and lipids were visualized by staining briefly with iodine. For total fatty acid composition or individual lipids, fatty acid methyl esters were generated by heating lipid samples at 80
C in 2.5% H2SO4 in methanol. Fatty acid methyl esters in isooctane were analyzed by GC-FID using a fused silica VF-23ms column of 30 m  0.25 mm ID and film thickness of 0.25 microns. The oven temperature program was 80
C (1 min hold), and was increased to 160
C at 16
C/min, then to 220
C at 4
C/min. The carrier gas was hydrogen at 1.6 ml/min. Splitless injection volumes ranged from 0.5 to 1.5 ml at an injection temperature of 240
C.

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Table 1 Fatty acid compositions of plastid and extraplastid lipids in the wild type and ppi2-2 mutant of Arabidopsis. Lipids were extracted from the 20-day-old seedlings that were grown in MS media at 22
C or cold-acclimated at 4
C for 5 days. Values are means ± SE of 3 or 4 replicates from two independent measurements. Fatty acid peak not detected is denoted as n.d. Lipid classes

Genotype

Fatty acids composition (mol%) 16:0

PC 0 5 PE 0 5 PG 0 5 MGDG 0 5 DGDG 0 5 SQDG 0 5

16:1

16:2

16:3

18:0

18:1

18:2

18:3

Col-0 ppi2-2 Col-0 ppi2-2

27.5 29.5 25.0 26.8

± ± ± ±

3.4 1.1 1.4 2.7

2.3 5.6 1.0 3.1

± ± ± ±

1.0 1.6 0.4 1.3

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

4.4 7.4 3.2 10.1

± ± ± ±

0.6 1.0 0.2 5.9

4.0 3.9 5.1 6.6

± ± ± ±

0.5 0.2 0.3 1.2

27.9 21.3 32.4 24.0

± ± ± ±

3.2 1.6 2.1 7.7

33.7 32.0 33.1 29.3

± ± ± ±

1.7 1.0 1.0 3.7

Col-0 ppi2-2 Col-0 ppi2-2

32.3 40.7 27.0 34.6

± ± ± ±

0.6 5.1 0.4 4.6

1.8 7.6 1.3 7.2

± ± ± ±

0.1 2.6 0.2 2.4

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

4.8 10.0 3.2 10.1

± ± ± ±

0.4 3.6 0.3 3.4

2.1 2.6 2.8 4.1

± ± ± ±

0.1 0.7 0.1 1.2

30.8 15.6 35.9 11.4

± ± ± ±

0.8 2.8 2.1 5.6

28.0 23.4 29.8 27.5

± ± ± ±

0.9 1.6 1.4 2.4

Col-0 ppi2-2 Col-0 ppi2-2

29.9 36.4 28.9 34.9

± ± ± ±

1.3 4.4 0.9 2.8

18.3 13.7 13.8 10.6

± ± ± ±

5.4 0.5 1.1 0.3

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

8.0 10.4 19.1 23.5

± ± ± ±

2.7 0.7 0.7 5.5

4.7 3.9 9.5 8.7

± ± ± ±

0.6 0.9 1.0 2.2

14.1 13.5 10.4 7.3

± ± ± ±

2.1 3.1 1.5 0.4

25.0 22.2 18.1 23.8

± ± ± ±

1.5 1.9 1.1 2.0

Col-0 ppi2-2 Col-0 ppi2-2

3.1 9.0 2.7 12.4

± ± ± ±

0.2 1.2 0.2 1.2

1.1 3.4 0.4 3.0

± ± ± ±

0.2 0.4 0.1 0.1

1.9 ± 0.1 2.3 ± 0.3 1.1 ± 0.1 n.d.

25.4 ± 0.4 11.4 ± 2.0 25.5 ± 0.3 7.8 ± 0.1

1.0 5.6 0.8 5.7

± ± ± ±

0.1 1.6 0.1 0.3

1.3 2.4 1.7 3.4

± ± ± ±

0.1 0.7 0.1 0.0

6.0 12.5 6.4 19.7

± ± ± ±

0.4 3.5 0.4 1.8

59.9 53.2 61.4 47.9

± ± ± ±

0.6 2.5 0.6 3.4

Col-0 ppi2-2 Col-0 ppi2-2

19.2 21.8 18.5 25.9

± ± ± ±

0.7 0.8 1.3 2.2

6.5 5.4 4.9 7.5

± ± ± ±

1.6 1.0 0.9 1.2

n.d. n.d. n.d. n.d.

1.5 ± 0.1 1.6 ± 0.4 1.4 ± 0.1 n.d.

3.4 8.1 2.3 11.9

± ± ± ±

0.5 1.6 0.1 3.6

1.8 2.9 2.6 4.3

± ± ± ±

0.3 0.8 0.3 2.1

7.2 10.5 7.1 9.2

± ± ± ±

0.5 1.4 0.4 1.3

60.4 49.8 63.0 40.9

± ± ± ±

0.9 2.8 1.7 6.4

Col-0 ppi2-2 Col-0 ppi2-2

40.0 36.0 37.8 34.2

± ± ± ±

1.0 3.9 1.5 4.7

3.9 10.4 2.4 24.5

± ± ± ±

0.4 3.0 0.1 1.5

n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d.

5.7 15.6 4.8 25.2

± ± ± ±

0.8 5.7 0.2 3.3

2.4 ± 0.3 1.8 ± 0.3 3.0 ± 0.2 n.d.

8.6 9.2 8.7 3.0

± ± ± ±

0.6 1.2 0.6 0.4

39.4 26.8 43.1 13.1

± ± ± ±

0.7 6.0 1.0 0.3

2.4. RNA extraction and cDNA synthesis Total RNA was extracted from the leaves using RNeasy Plant Mini kit (Qiagen, Maryland, USA). Prior to cDNA synthesis, 2 mg of total RNA was treated with DNase I (New England Biolabs, Ipswich, MA) and then re-purified using Qiagen kit. RNA concentration and quality were measured before and after DNase treatment using a NanoDrop 1000 (V 3.8.1, Thermo Scientific). The integrity of the RNA was confirmed on 1.2% formaldehyde agarose gel, and 0.5 mg of DNase-treated RNA was used to synthesize first strand cDNA using SuperScript III reverse transcriptase and oligo(dT) primer according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). A 2 mL aliquot of first-strand cDNA was used as the template to amplify genes of interest.

2.5. Primer design and quantitative real-time PCR The published gene sequences were obtained from the Genbank database and gene specific primers were designed using PrimerBLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primers were designed to amplify close to the 30 end of the transcripts and cover an exoneexon junction if possible. The primer-BLAST tool was used to check for the specificity of each of the primer pair sequence against the Arabidopsis database. The best annealing temperatures of each primer pair was established by gradient PCR, and each primer pair yielded only a single PCR product of the expected size. The amplicon lengths ranged from 70 to 140 bp. The gene-specific primer pairs to cDNA for Actin2 were 50 -AGGTATCGCTGACCGTATGA-30 (forward) and 50 -CAATTTTTACCTGCTGGAATGTGC-30 (reverse), IF2a were 50 CAACCACAGAGGACAAGGATT-30 (forward) and 50 -

AGTTAGATCAGAAGCAAGCGGA-30 (reverse), pyruvate E1a subunit were 50 -TTGCAGACGCTAGTCCACAG-30 (forward) and 50 CTTGAGCTGTGCCTTCGGTA-30 (reverse), KAS II were 50 CCCTTGCTCACTGTTTTGGC-30 (forward) and 50 -TTGGATGAACCCATCCGGTC-30 (reverse), LHCB1 were 50 -AGCTCAAGAACGGAAGATTGG-30 (forward) and 50 -GCCAAATGGTCAGCAAGGTT-30 (reverse), HY5 were 50 -TCGGAGAAAGTCAAAGGAAGC-30 (forward) and50 -TCTGTTTTCCAACTCGCTCAAG-30 (reverse), stearoyl-ACP desaturase were 50 -ACTTAACCGGGCTTTCAGGT-30 (forward) and 50 -CCTTTCTTGGCTCTTGCTTG-30 (reverse), FAD5 were 5 0 -AATCACCCAAAGGTGTGGAGAGC-30 (forward) and 50 -CCGCTAGAGCCAGCGGATGC-30 (reverse), FAD7 were 50 0 0 TGACGAATCTTGGCATCCTATGT-3 (forward) and 5 -GCATCACGAGAGGCAGTGTA-30 (reverse), and FAD8 were 50 CTGCCTTTTCCAATGCTCGC-30 (forward) and 50 -ATTGCAGTCCAACAGGCAGT-30 (reverse). Quantitative real-time PCR was conducted on a StepOnePlus Real-Time PCR System (Applied Biosystems, CA) using Fast SYBR Green Master mix (Life Technologies, Grand Island, NY) to monitor double-stranded DNA synthesis. Each reaction contained 10 mL of 2X SYBR Green Master mix reagent, 2 mL of first-strand cDNA and 0.5 mL of each gene specific primer in a final volume of 20 mL. The thermal cycling conditions were as described in the manufacturer's protocol at 40 cycles of amplification and the annealing and extension temperature of 60
C for all genes. For LHCB1 and pyruvate dehydrogenase E1a genes, the annealing temperature was 55
C and extension 60
C. Amplicon dissociation curves (melting curves) were recorded after cycle 40 at 95
C for 15 s and 60
C for 1 min, then incrementally until 95
C. The transcript level of each gene was normalized to that of Actin2 or initiation factor 2a (IF2a). There were 4 biological replicates for each treatment. The qPCR

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Fig. 5. The ratio of MGDG to bilayer forming lipids in plastids of the wild type and ppi2-2 mutant of Arabidopsis. Values are means ± SE of 3 or 4 replicates from two independent measurements.

3. Results and discussion

mutant had 6-fold decrease in total protein (Fig. 1B). The highly decreased chlorophyll content in the ppi2-2 mutant is consistent with findings from a previous study (Kakizaki et al., 2009). It is well documented that thylakoid membranes and the associated photosystems are enriched in chlorophylls, carotenoids and proteins €rmann, 2007; Pogson et al., 1998). Therefore, the drastic (Do decrease in level of pigments is consistent with the undeveloped thylakoid membranes in the ppi2-2 mutant. Since the synthesis and accumulation of pigments and antenna proteins are an integral part of chloroplast biogenesis, these data indicate that the atToc159 receptor plays a critical role in plastid biogenesis.

3.1. The composition of pigments and total protein in the wild type and ppi2-2 mutant

3.2. Fatty acid composition in the wild type and ppi2-2 mutant

The ppi2-2 mutant, which is defective in the Toc159 receptor, had 18 and 25-fold decreased levels of carotenoids and chlorophylls compared to the wild type (Fig. 1A). Furthermore, the ppi2-2

Both the non-acclimated and cold-acclimated ppi2-2 mutant accumulated 2-fold decreased total fatty acid content when compared to the non-acclimated wild type (P < 0.05; Fig. 2). These

deltaedelta CT method was used in analysis of gene expression among the treatments as described by Pfaffl (2001). 2.6. Statistical analysis Statistical analysis of the data was calculated using the Student's t-test at p < 0.05, using either two samples assuming equal variances or two samples assuming unequal variances.

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data indicate that in the ppi2-2 mutant the synthesis of lipids was partially impaired. Analysis of the individual fatty acids showed that the non-acclimated ppi2-2 mutant accumulates palmitic acid (16:0) and linoleic acid (18:2) at the control levels, which is the non-acclimated wild type (Fig. 3A, B). The ppi2-2 mutant however, accumulates increased levels of stearic acid (18:0) and oleic acid (18:1), and decreased levels of palmitoleic acid (16:1), hexadecatrienoic acid (16:3) and a-linolenic acid (18:3). Interestingly, in the cold-acclimated ppi2-2 mutant the amounts of 16:0 and 18:2 decreased (P < 0.05), and was not mirrored by increased 16:1, 16:3 or 18:3. Since a drastic decrease in total fatty acid content was measured in the ppi2-2 mutant, these data suggest a decrease in de novo fatty acid synthesis in the ppi2-2 mutant. In our previous study, we reported increased levels of 16:3 in a mutant of A. thaliana ecotype Wassilewskija that is null for TOC159 gene, and the mutant was referred to as ppi2-1 (Afitlhile et al., 2013). In view of the measured decreased levels of 16:3 in the ppi2-2 mutant, we re-measured fatty acid composition in the ppi2-1 mutant. Consistent with our previous findings, the ppi2-1 mutant accumulated high levels of 16:0, 18:0, 18:1, 18:2 and decreased levels of 18:3 (Fig. S1). Contrary to our previous findings, the ppi2-1 mutant accumulates decreased levels of 16:3. Therefore, under the normal growth temperature the ppi2 mutants accumulated high levels of 16:0, increased 18:0 and 18:1, and decreased levels of 16:3 and 18:3. Since 16:3 is synthesized exclusively in the plastid-derived MGDG, decreased levels of 16:3 indicate that in plastids of the ppi2 mutants the prokaryotic pathway was impaired. 3.3. Lipid composition in the wild type and ppi2-2 mutant Since the ppi2 mutants accumulate altered levels of saturated and unsaturated fatty acids, we measured polar lipids to determine whether a defective TOC159 gene affected the synthesis of glycerolipids. Depending on the growth temperature, on mol% basis the ppi2-2 mutant accumulated levels of the extraplastid lipid PC that were similar or higher than in the non-acclimated wild type (Fig. 4A). The ppi2-2 mutant however accumulated increased levels of PE irrespective of the growth temperature, and thus indicate that the ER-localized pathway of lipid synthesis was not affected by a defective atToc159 receptor. Interestingly, in the wild type levels of PC were higher than those of PE, which is consistent with prior findings (Essigman et al., 1998). In the non-acclimated ppi2-2 mutant however, levels of PC were lower than those of PE, which indicates that mutation in the TOC159 gene altered homeostasis of the extraplastid lipids. The ppi2-2 mutant accumulated the plastid lipids PG, DGDG and SQDG at wild type levels (Fig. 4B). Compared to non-acclimated wild type, the ppi2-2 mutant accumulated decreased levels of a major thylakoid lipid, MGDG (P < 0.05). In the cold-acclimated wild type, the decreased levels of MGDG was consistent with MGDG being converted to DGDG, a bilayer forming galactolipid. It is well documented that MGDG and DGDG constitute ~50 and 20% of total lipids in the thylakoid membranes, while PG and SQDG makes the €rmann, 2007). Previous studies have shown that remainder (Do 16:3 and 18:3 accounted for 30 and 57% respectively, of the total fatty acids in MGDG (Hendrickson et al., 2006; Kim et al., 2010). Taken together, these data indicate that in the ppi2-2 mutant, the decreased levels of MGDG accounted for the drastic decrease in levels of 16:3 and contributed significantly to the reduced levels of

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18:3.

3.4. Fatty acid composition in plastid and extraplastid lipids in the wild type and ppi2-2 mutant In the ppi2-2 mutant, the extraplastid lipid PE and plastid lipids PG, MGDG, and DGDG accumulated increased levels of 16:0 compared to the non-acclimated wild type (Table 1). The extraplastid lipids PC and PE, and plastid lipids including SQDG accumulated increased levels of 18:0. In the ppi2-2 mutant however, only plastid lipids accumulated the decreased levels of 18:3. These data indicate that in the mutant the decrease in total 18:3 was contributed by the plastid lipids PG, MGDG, DGDG and SQDG. Interestingly, an increase in 18:0 in plastid lipids did not result in decreased accumulation of 18:1 and 18:2. This is indicative that plastids in the ppi2-2 mutant had normal activities of the cytoplasm synthesized 3-ketoacyl-ACP synthase II, stearoyl-ACP desaturase and FAD6 desaturase. Although the ppi2-2 mutant accumulated decreased levels of MGDG, on mol% basis MGDG in the mutant accumulated increased levels of 16:0, 16:1, 18:0, 18:1 and 18:2 (Table 1). In the ppi2-2 mutant, MGDG had 2e3-fold decreased levels of 16:3. In the wild type, 16:3 and 18:3 accounted for 85% of the total fatty acids in MGDG. In the ppi2-2 mutant however, 16:3 and 18:3 accounted for 65% of the total fatty acids in MGDG, and the levels decreased to 55% in the cold-acclimated mutant. It has been suggested that high levels of fatty acid unsaturation are critical for membrane function at low temperature (Routaboul et al., 2000). In the ppi2-2 mutant the increased accumulation of 16:0 and 18:0, and decreased levels of 16:3 and 18:3 was indicative that under cold-stress the mutant likely had reduced membrane fluidity and might not have adapted well to low temperature.

3.5. Molar ratio of plastid lipids in the wild type and ppi2-2 mutant MGDG is a non-bilayer forming lipid while PG, DGDG and SQDG are bilayer forming plastid lipids (Hendrickson et al., 2006). Consequently, at low temperature MGDG is converted to DGDG and oligogalactolipids (Moellering et al., 2010). In our study, the ratio of MGDG to PG þ DGDG þ SGDG, or MGDG to DGDG decreased 1.2 and 1.6-fold in the cold-acclimated wild type (Fig. 5A, B). In the ppi2-2 mutant however, the ratio of MGDG to PG þ DGDG þ SGDG, or MGDG to DGDG was similar in the non-acclimated and coldacclimated ppi2-2 mutant. These data indicate that the wild type responded to cold-stress by increasing the synthesis of bilayer forming plastid lipids. By contrast, the ppi2-2 mutant appeared to have lost the ability to undergo lipid changes characteristic of exposure to low temperatures. The lack of response to cold-stress by the ppi2-2 mutant is consistent with the increased levels of saturated fatty acids, which do not increase membrane fluidity. These data indicate that plastid lipid remodeling was compromised in the cold-stressed ppi2-2 mutant, which is consistent with the findings of Nakayama et al. (2007). These authors showed that the cold-induced gene expression and the accumulation of plastid cor15am protein were compromised in the ppi2 mutant.

Fig. 6. The expression profiles of LHCB1, pyruvate E1a subunit, KAS II, MGD1, DGD1, HY5, stearoyl-ACP desaturase (SAD), FAD5, FAD7 and FAD8 genes in the wild type and ppi2-2 mutant of Arabidopsis. The mRNA levels were analyzed by quantitative real-time PCR and normalized to the levels of Actin2 or IF2a. The expression levels in non-acclimated wild type (Col-0) were set to 1, and values are means ± SE of 4e6 replicates from two independent measurements. Asterisks above the error bars indicate significant difference (P < 0.05) of each treatment compared to the non-acclimated wild type as determined by student's t test.

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3.6. Expression of nuclear genes that encode plastid proteins in Arabidopsis The majority of plastid proteins are nuclear-encoded and posttranslationally imported into plastids. However, defective plastids transmit repressive signals to the nucleus, which result in the downregulation of photosynthesis-related genes (Inaba et al., 2011; Jarvis and Lopez-Juez, 2013; Kobayashi et al., 2014). Studies suggest that nuclear-localized photosynthesis genes are repressed because protein import into defective plastids is impaired (Bauer et al., 2000; Kakizaki et al., 2009; Smith et al., 2004). In fact Kakizaki et al. (2009) demonstrated that the accumulation of the cytoplasm-synthesized plastid proteins was proportional to their transcript levels. These authors proposed that nuclear gene expression could be used as a relative measure of the efficiency of protein import into plastids. 3.6.1. Expression of genes that encode photosynthesis and nonphotosynthesis proteins in the wild type and ppi2-2 mutant Transcription of a nuclear gene that encodes chlorophyll a/b light-harvesting antenna protein (LHCB1) was repressed in the ppi2-2 mutant and upregulated in the cold-acclimated wild type relative to the non-acclimated wild type (Fig. 6A). In the ppi2-2 mutant, a gene that encodes pyruvate dehydrogenase E1a subunit was expressed at wild type levels, regardless of the growth temperature. These gene expression data are consistent with the previous findings in the ppi2 mutants, which led to the hypothesis that the atToc159 receptor was required for the import of photosynthesis-related proteins (Bauer et al., 2000; Kakizaki et al., 2009; Smith et al., 2004). 3.6.2. Expression of galactolipid synthesis genes in the wild type and ppi2-2 mutant The expression of both MGD1 and DGD1 genes are induced by light and the lipids MGDG and DGDG are required in chloroplast biogenesis (Kobayashi et al., 2014). The cytoplasm-synthesized MGD1 synthase has a chloroplast transit peptide, which suggests that the import of MGD1 synthase into plastids could be mediated by the TOC complex. The MGD1 gene was downregulated in the ppi2-2 mutant relative to non-acclimated wild type (Fig. 6B) and levels of MGDG were decreased in the mutant (Fig. 4B). These data are consistent with our previous findings in the ppi2-1 mutant (Afitlhile et al., 2013). Studies suggest that the activity of MGD1 synthase was regulated by thioredoxin (Shimojima et al., 2013; Yamaryo et al., 2006), which is a light-dependent system that operates in the chloroplast and activates many enzymes including those in the Calvin cycle. Since the ppi2 mutants have undeveloped plastids and are impaired in photosynthesis (Bauer et al., 2000; Kakizaki et al., 2009), it seems reasonable to assume that in the ppi2 plastids the thioredoxin system might not be fully functional. Therefore, the decreased accumulation of MGDG in the ppi2 plastids might be related to oxidative-inactivation of MGD1 synthase due to the loss of photosynthetic activities. A recent study showed that both MGD1 and DGD1 genes were downregulated in mutants impaired in chloroplast biogenesis, and like ppi2, these mutants do not accumulate chlorophylls (Kobayashi et al., 2014). In our study, although MGD1 gene was downregulated in the ppi2-2 mutant, the DGD1 gene was expressed at the control level, which was the nonacclimated wild type (P > 0.05; Fig. 6B). The expression of DGD1 gene was mirrored by levels of DGDG that were at the control levels (Fig. 4B). It has been suggested that the DGD1 gene was a direct target of long hypocotyl5 (HY5) protein, and thus the DGD1 gene was positively regulated by HY5 transcription factor (Kobayashi et al., 2014). In our study, HY5 gene was upregulated in the ppi22 mutant and this was mirrored by a statistically insignificant

increase in the expression of the DGD1 gene (Fig. 6B). Therefore, these gene expression data are consistent with the proposed role of HY5 in transcriptional activation of the DGD1 gene. 3.6.3. Expression of genes that encode fatty acid desaturases in the wild type and ppi2-2 mutant Consistent with the published literature, the content of 18:0 was low in wild type Arabidopsis. The ppi2-2 mutant however, accumulated increased amounts of 18:0 and 18:1 (Fig. 3), and the increase in levels of these fatty acids was reflected in plastid and extraplastid lipids (Table 1). In plastids, the amounts of 18:0 and 18:1 are determined by the activities of 3-ketoacyl-ACP synthase II (KAS II) and stearoyl-ACP desaturase, respectively. The KAS II gene encodes a plastid enzyme that elongates 16:0 to 18:0, thus KAS II activity determines the ratio of 16 to 18-carbon fatty acids (Ohlrogge and Browse, 1995). In our study, the KAS II gene was upregulated in the ppi2-2 mutant relative to the wild type (Fig. 6A), and levels of 18:0 were increased in the mutant (Fig. 3B). The desaturation of 18:0 to 18:1 is carried out by a stromal enzyme, stearoyl ACP desaturase. In the non-acclimated ppi2-2 mutant, there was a statistically insignificant increase (P > 0.05) in the expression of stearoyl-ACP desaturase gene relative to the nonacclimated wild type (Fig. 6C), and this correlated with increased levels of 18:1 in the non-acclimated mutant (Fig. 3B). These data indicate that plastids in the ppi2-2 mutant had enzyme activities that were required in the synthesis of 18:0 and 18:1. However, the accumulation of 18:0 in the ppi2-2 mutant suggests a possible decrease in the activity of stearoyl-ACP desaturase. In our previous study, we reported that in the non-acclimated ppi2-1 mutant, stearoyl-ACP desaturase gene was downregulated and levels of 18:1 were decreased (Afitlhile et al., 2013). The reason for these differences is unclear, although it may reflect the different genetic background of the ppi2 mutants. Whereas the ppi2-2 mutant is of the Columbia (Col-0) background, the ppi2-1 mutant is of the Wassilewskija (Ws) ecotype. A prior study showed that Col-0 was genetically divergent from other ecotypes, including Ws (Barth et al., 2002). It has also been reported that the toc132 knockout mutants in Col-0 and Ws give different phenotypes (Ivanova et al., 2004; Kubis et al., 2004). Nevertheless, it is well documented that in the chloroplast, activity of stearoyl-ACP desaturase is dependent on the enzyme being reduced by electrons from photosystem I (Shanklin and Cahoon, 1998). Since the ppi2 mutants lack organized thylakoid membranes and are impaired in photosynthesis, it is likely that PSI and PSII may not be fully functional in the ppi2 mutants. This would result in highly decreased electron flow through the photosystems, which could potentially impair the reduction of stearoyl-ACP desaturase. The decreased activity of stearoyl-ACP desaturase would account for the increased accumulation of 18:0 in the ppi2 mutants. In Arabidopsis, MGDG that is synthesized by the prokaryotic pathway contains 16:0, which is a substrate of the nuclear-encoded FAD5 desaturase. In non-acclimated ppi2-2 mutant the FAD5 gene was upregulated (Fig. 6C) and there was a 3-fold increase in levels of 16:1 in MGDG (Table 1). In the ppi2-2 mutant however, the levels of 16:3 in MGDG decreased 2e3-fold relative to the non-acclimated wild type. The synthesis of 16:3 requires three activities, which are FAD5, FAD6 and FAD7 or FAD8 desaturases. Therefore, if one or more of these activities was limiting, the levels of 16:3 would be decreased. In the ppi2-2 mutant, the FAD7 and FAD8 genes were expressed at the control level, which is the non-acclimated wild type (P > 0.05; Fig. 6C). The FAD7 or FAD8 desaturase also use lipid substrates in the chloroplasts to desaturate linoleic acid (18:2) into linolenic acid (18:3). The downregulation of FAD7 gene in the coldacclimated wild type and ppi2-2 mutant was consistent with the function of FAD7 desaturase at the normal growth temperature. In

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the ppi2-2 mutant, the cold-inducible FAD8 gene was expressed at the control levels. However, FAD8 gene was upregulated in the cold-acclimated wild type and there was a small increase in levels of 18:3 in the plastid lipids MGDG, DGDG and SQDG (Table 1). Overall, in the ppi2-2 mutant the expressions of FAD5, FAD7 and FAD8 genes were not correlated with levels of 16:3 or 18:3. It is likely that fatty acid desaturases may be tightly regulated at the posttranscriptional level.

4. Conclusions A defective TOC159 gene resulted in drastic decrease in the accumulation of pigments, proteins and total fatty acid content. Glycerolipids in the TOC159 mutant accumulated increased levels of 16:0 and 18:0, and decreased 18:3. In the ppi2-2 mutant, levels of MGDG, a major plastid lipid were highly reduced, which resulted in decreased levels of 16:3. In the mutant, levels of PC were lower than those of PE, which suggest altered homeostasis in extraplastid lipids. Since the prokaryotic pathway was impaired in the ppi2-2 mutant, it is likely that PC was being converted into diacylglycerol, which was then exported from the ER to plastids to be used in the synthesis of plastid lipids. Interestingly, both LHCB1 and MGD1 genes were downregulated in the ppi2-2 mutant, which suggest a possible co-regulation of photosynthesis and galactolipid synthesis genes. By contrast, DGD1 and FAD genes were not downregulated in the ppi2-2 mutant. A plausible explanation was that DGD1 and FAD genes were not regulated by the same retrograde plastid signals that repressed LHCB1 and MGD1 genes. The expressions of DGD1 and KAS II genes were positively correlated with the accumulation of DGDG and 18:0, respectively. These data suggest that DGD1 and KAS II may be strongly regulated at the transcriptional level. Since MGDG is a major plastid lipid and levels of MGDG were highly decreased in the ppi2-2 mutant, our data suggest that the synthesis of MGDG was tightly linked to plastid biogenesis. Overall, these data indicate that a defective atToc159 receptor did not result in the downregulation of genes that encode DGD1 synthase, KAS II enzyme, and fatty acid desaturases. However, the ppi2-2 mutant accumulated the increased levels of 18:0 and decreased 16:3 and 18:3.

Contributions MA conceived and designed the study, directed and carried out experiments, analyzed and interpreted the data and wrote the manuscript. MF and SW carried out the lipid and gene expression studies, respectively, under the direction of MA. The authors read and approve the final manuscript.

Acknowledgments This project was supported by a Western Illinois University College of Arts and Sciences Undergraduate research grant (MF), Graduate professional development grant (SW) and a University Research Council grant (MA). We are grateful to Professor David Hildebrand (University of Kentucky) for the measurement of fatty acids, and Dr. Winthrop Phippen (WIU) for allowing us to use his growth chamber. We thank Jason Tuter (former graduate student in Biology) and Dr. Sue Hum-Musser for helping with the analysis of qPCR data. We thank Drs. Christopher Jacques and Sue HumMusser in the Biological Sciences for proofreading the manuscript. The authors wish to thank Haley Patterson, Gina Meier and Tracy Gongora for their contribution to this project.

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