In Vivo Function of Tic22, a Protein Import Component of the Intermembrane Space of Chloroplasts

Molecular Plant  •  Volume 6  •  Number 3  •  Pages 817–829  •  May 2013

Research article

In Vivo Function of Tic22, a Protein Import Component of the Intermembrane Space of Chloroplasts Mareike Rudolfa,2, Anu B. Machettiraa,2, Lucia E. Großa, Katrin L. Weberb, Kathrin Boltec, Tihana Biondaa, Maik S. Sommera, Uwe G. Maierc,d, Andreas P.M. Weberb, Enrico Schleiffa,1 and Joanna Trippa,1 a Department of Biosciences, Molecular Cell Biology of Plants, Center of Membrane Proteomics and Cluster of Excellence Frankfurt, Goethe University, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany b Institute of Plant Biochemistry, Heinrich-Heine-University, Universitätsstr. 1, D-40225 Düsseldorf, Germany c Cell Biology, Philipps University Marburg, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany d LOEWE Center for Synthetic Microbiology (SYNMIKRO), Hans-Meerwein-Str., D-35032 Marburg, Germany

ABSTRACT  Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane (TOC and TIC). It was suggested that both machineries are interconnected by components of the intermembrane space (IMS). That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates. Key words: protein translocation; TOC and TIC; intermembrane space translocon; chloroplast biogenesis; metabolite content.

Introduction Import of preproteins into chloroplasts is facilitated by multi-protein translocon complexes in the outer and inner envelope membrane (TOC/TIC, respectively) and associated proteins in the cytoplasm, the intermembrane space (IMS), and the stroma (Jarvis, 2008; Kessler and Schnell, 2009; Li and Chiu, 2010; Schleiff and Becker, 2011). The individual subunits of those complexes can be classified into (1) receptors for preprotein recognition, (2) membrane-spanning pores for the transfer across the envelope, and (3) scaffolds for the association of soluble factors assisting the targeting and translocation event. Some knowledge has been gained about the associated factors in the cytoplasm and the stroma. It is discussed that chaperones and regulatory proteins such as Hsp70, Hsp90, and 14–3–3 are involved in the delivery of preproteins to the chloroplast surface (May and Soll, 2000; Qbadou et  al., 2006; Fellerer et  al., 2011), where they are recognized by the TOC receptors Toc64 (Qbadou et al., 2006), Toc33, Toc159, and their isoforms Toc34 and Toc90, Toc120, and Toc132, respectively (for detailed information, see Soll and Schleiff, 2004). In the stroma, Hsp70 and Hsp93 are

thought to energize the translocation event (Tsugeki and Nishimura, 1993; Constan et al., 2004; Kovacheva et al., 2007; Shi and Theg, 2010; Su and Li, 2010). Here, Tic40 and Tic110 are discussed as docking sites for their recruitment to the inner envelope (Jackson et al., 1998; Chou et al., 2003; Inaba et al., 2003; Kovacheva et al., 2005; Chou et al., 2006). Contrarily, the transfer of the preproteins from TOC to TIC and therewith their transport across the IMS is by far not understood. The recently supported bimodal energizing of the translocation process and the length requirement of an unfolded polypeptide stretch at the very N-terminus of the preprotein for proper import into chloroplasts suggests the

1 To whom correspondence should be addressed. (E.S.) E-mail schleiff@bio. uni-frankfurt.de. (J.T.) E-mail [email protected]. 2

Both authors contributed equally.

© The Author 2012. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sss114, Advance Access publication 30 November 2012 Received 4 March 2012; accepted 30 September 2012

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  Rudolf et al.  •  Functional Analysis of atTic22-III and atTic22-IV

existence of IMS components that actively facilitate the translocation event (Bionda et  al., 2010; Ruprecht et  al., 2010). Several components have been proposed to be involved in the transfer of preproteins across the IMS, namely imsHsp70, Toc12, Toc64, and Tic22 (Marshall et  al., 1990; Waegemann and Soll, 1991; Schnell et  al., 1994; Kouranov and Schnell, 1997; Kouranov et al., 1998, 1999; Becker et al., 2004; Qbadou et al., 2007). Based on co-immunoprecipitation experiments, it was suggested that these components assemble to a socalled IMS complex (Becker et al., 2004; Qbadou et al., 2007). However, almost all of these factors are currently under debate. First, despite longstanding research, the molecular nature of the putative imsHsp70 is not yet known. Second, contradictory results about the localization of Toc12 exist, as the protein was identified in the outer envelope membrane as well as in the stroma (Becker et al., 2004; Chiu et al., 2010). Third, depletion of Toc64 does not cause a drastic phenotype in vivo, which challenges its actual function in protein import (Rosenbaum-Hofmann and Theg, 2005; Aronsson et al., 2007). Tic22 was originally identified by crosslinking to translocating preproteins (Kouranov and Schnell, 1997; Kouranov et al., 1998). It was found to be attached to both chloroplast membranes in pea (Vojta et al., 2004). The protein appears to function at the same stage of import as Tic20 (Kouranov et  al., 1998), the latter being a pore-forming component of the TIC complex (Chen et al., 2002; Kasmati et al., 2011; KovácsBogdán et al., 2011; Machettira et al., 2011). In Arabidopsis thaliana, two isoforms of Tic22 exist, namely Tic22-IV and Tic22-III (Jackson-Constan and Keegstra, 2001). Co-expression analysis based on global transcriptome data revealed that TIC22-III clusters with the A. thaliana Tic20 isoforms TIC20-I, TIC20-IV, and TOC64-III, while TIC22-IV clusters with the poreforming subunits TOC75-III and TIC110 (Moghadam and Schleiff, 2005). Apart from these few data, our knowledge about Tic22 and especially about the function of the different isoforms in A. thaliana is rather sparse. In here, we demonstrate that plants with a single T-DNA insertion in either of the genes in A.  thaliana show only mild phenotypes, possibly due to a functional redundancy of the two proteins. Analyzing a double mutant, we could observe a more pronounced phenotype, with pale yellow leaves, a reduced amount of thylakoid grana stacks under high-light conditions, and defects in protein import in early developmental stages.

RESULTS TIC22-III and TIC22-IV Have a Similar Expression Profile in A. thaliana An initial survey for components of the two translocons in A.  thaliana revealed two genes coding for Tic22 isoforms (Jackson-Constan and Keegstra, 2001). Currently, very little is known about their function. At first, we analyzed the expression profile of the two genes. In many cases, a tissue-specific

expression of import components hints at a functional specialization or at the existence of distinct import complexes as proposed for TOC33/TOC34 or TOC159/TOC132/TOC120/ TOC90 (Gutensohn et  al., 2000; Kubis et  al., 2003, 2004; Moghadam and Schleiff, 2005). qRT–PCR was performed with isoform-specific oligonucleotides on RNA isolated from different developmental stages and tissues of A. thaliana wild-type plants. The expression of both genes was normalized to the expression of UBIQUITIN (Figure 1A). The maximal amount of transcript observed in flowers was 0.10 ± 0.02 for TIC22-III and 0.48 ± 0.04 for TIC22-IV in relation to UBIQUITIN. For better

Figure 1.  Gene Expression and Protein Localization of atTIC22-III and atTIC22-IV. (A) Expression levels were analyzed by quantitative RT–PCR. RNA for preparation of cDNA was isolated from 30 mg of seeds, 25–50 individual plants grown on MS-Medium (day 3, day 12, day 25), or of the respective tissues harvested from a soil-grown plant (roots, rosettes, stem, hypsophyll, siliques, flowers day 48 and day 66). Plants were grown under long-day conditions. PCR reactions were performed in triplicate, whereby cDNA from three independent RNA preparations was used. The data were normalized to the expression of UBIQUITIN, and presented relative to the expression in sample flowers day 48, which was set to 1. (B) Chloroplasts (lane 1) were isolated from the double knockout line tic22-III/tic22-IV complemented with Tic22-III-Strep (panel 1–3) or Tic22IV-Strep (panel 4–6) and incubated with thermolysin (Th, lane 3)  or trypsin (Try, lane 5)  as described in the Methods section. As control, chloroplasts were treated with the buffer for digestion in the absence of the protease (lanes 2, 4). The according fractions were immunodecorated with the indicated antibodies. Please note, all samples of the Tic40 analysis were subjected to the same gel and processed identically.

Rudolf et al.  •  Functional Analysis of atTic22-III and atTic22-IV   

comparison of the profile, values were henceforth normalized to their expression level in flowers. We observed that the expression of both genes increases throughout development (compare day 3, day 8, and day 25), reaching its maximum at day 25 (Figure 1A). The expression in older plants was reduced (day 48, day 66), both in photosynthetic (rosettes) and non-photosynthetic tissues (roots) with the exception of flower tissue (Figure  1A). Thus, both genes are predominantly expressed in younger plants, but with no clear preference for photosynthetic or non-photosynthetic tissues. Moreover, there was no clear difference in the overall expression pattern of both genes as well. The only variation detectable concerns the hypsophyll, where a higher expression in relation to the maximal expression was found for TIC22-IV.

Tic22-III and Tic22-IV Are IMS Components of the Chloroplast The unexpected localization of some of the Tic20 isoforms in A.  thaliana to mitochondria or thylakoids (Machettira et  al., 2011) prompted us to confirm whether both proteins are indeed localized in the chloroplast intermembrane space (IMS). We have complemented the T-DNA insertion line (see below) with constructs expressing Tic22-III and Tic22-IV with a C-terminal Strep-tag. We isolated chloroplasts from these plants and analyzed the localization of the tagged proteins by protease treatment of the organelles with thermolysin and trypsin and subsequent Western blot analysis with Strep-specific antibodies (Figure 1B, lane 1). Under the conditions used, trypsin penetrates the outer membrane, while thermolysin does not (Jackson et al., 1998; Kouranov et al., 1998). The activity of the proteases was controlled by Western blotting using specific antibodies against Toc33 or Tic40. The degradation of the cytosolically exposed GTPase domain of Toc33 clearly confirms the activity of the proteases (Figure 1B, panels 2, 5, lanes 3, 5). In contrast, Tic40, which exposes its functional domain to the stroma (Chou et al., 2003), was largely protected from degradation by both proteases (Figure 1B, panels 3, 6), confirming the integrity of the re-isolated organelles. Both Tic22 proteins were thermolysinresistant (panels  1, 4, lane  3), while they were degraded by trypsin (lane  5). We thus conclude that both Tic22 isoforms reside in the IMS. Tic22 is a protein predicted to possess a canonical N-terminal transit peptide (Kouranov et  al., 1999; Vojta et al., 2007). Consistently, both tagged isoforms migrate at the apparent molecular mass expected for the maturated protein according to transit peptides predicted by ChloroP of 96 and 59 amino acids for Tic22-III and Tic22-IV, respectively (Kouranov et al., 1999; Vojta et al., 2007).

Individual Disruption of TIC22-III or TIC22-IV Genes Does Not Affect Plant Development Two independent GABI T-DNA insertion lines of each gene were analyzed to obtain insights into the specific function of the two isoforms of Tic22 in A.  thaliana (Figure  2A). We obtained lines with an inserted T-DNA in an intronic region

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(lines  –1) and lines with the T-DNA in the first exon of the respective gene (lines –2). For GK387C03 and GK810F06, a single T-DNA insertion was confirmed by restriction of genomic DNA, linker ligation, PCR amplification, and sequencing (Thole et  al., 2009). The two lines were annotated as tic22III-2 and tic22-IV-2, respectively. In contrast, for GK518B03 and GK710E01, we observed an additional T-DNA insertion in the genes AT4G37680 and AT5G46270, respectively (Supplemental Figure  1). Both mutations were heterozygous. We screened for plants with wild-type AT4G37680 and AT5G46270 in the according backgrounds. We annotated the lines with confirmed individual T-DNA insertions in TIC22 genes as tic22-III-1 and tic22-IV-1. The exact genomic loci for the T-DNA insertions were confirmed by genomic PCR for all lines using specific oligonucleotides binding in the gene and the left border (LB) of the T-DNA, respectively (Figure 2B). In each case, the T-DNAs were inserted in a tandem inverted repeat orientation (Table 1). Using gene-specific oligonucleotides, we could further confirm that all lines were homozygous, since we obtained PCR products only when DNA from wild-type was used as a template. Homozygosity was also confirmed by segregation analysis of the insertion while growing the plants on sulfadizin containing MS-media, since the sulfadizin resistance cassette is coded within the T-DNA (Supplemental Table 3). Using RT–PCR, the absence of a functional transcript of the respective genes could be confirmed as well (Figure 2C). Here, we obtained no PCR product when using oligonucleotides for the amplification of the region in which the T-DNA is positioned. However, when oligonucleotides probing for the region up- (III-1 or IV-1) or downstream (III-2 or IV-2) of the T-DNA were used, a transcript was detected. This was not unexpected based on the position of the LB region of the T-DNA. To confirm a reduction at protein level, we performed Western blots with leaf extracts using antibodies raised against recombinant Tic22-IV. The obtained polyclonal antibody is able to detect both isoforms, although at Tic22-III with a considerably lower efficiency than at Tic22-IV (Supplemental Figure 2). This can be explained by the low sequence identity of both isoforms of only 30%. We could demonstrate that Tic22-IV is not detectable in tic22-IV-1 (Figure  2D, lane  4), while a signal is still observed in tic22-IV-2 (lane  5), which might be explained by an increased abundance of Tic22-III in these mutant plants. Mutants with impaired import function usually exhibit a chlorotic phenotype or a reduced plant size and growth rate (e.g. Jarvis et al., 1998). Inspection of the single T-DNA insertion lines did not reveal a visible phenotype (Supplemental Figure 3). However, both genes have a similar expression pattern and both proteins are localized in the IMS (Figure  1). Thus, the absence of a detectable phenotype might be due to a functional redundancy of both proteins. To explore this possibility, we generated a double mutant by crossing the tic22III-1 and tic22-IV-1 plants. These two mutants were chosen because the T-DNA is positioned in the middle of the gene

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Table 1.  Positions of T-DNA Insertions. Mutant –1

Mutant –2

LBleft

LBright

LBleft

LBright

TIC22-III

1729

1761

86

88

TIC22-IV

1507

1541

305

333

The last (LBleft) base in front and the first (LBright) after T-DNA insertion is given counted from the first base of the start codon of the genomic DNA.

and transcripts as well as proteins could not be detected. The resulting double mutant was named tic22-III/IV.

tic22-III/IV Plants Show a Reduced Growth and Photosynthetic Performance under High-Light Conditions The double mutant was viable as judged from the growth of the plants for which the homozygosity of the two mutations was confirmed (Figure  2E and 2F). We did not observe any significant alteration of the expression of TIC or TOC components we probed for, on either the transcript or the protein level (Supplemental Figure 4). We compared the plant growth under normal (140 µmol m–2 s–1), high- (400 µmol m–2 s–1), and low-light conditions (20 µmol m–2 s–1). Visual inspection of the growth behavior revealed no drastic differences between wildtype and mutant plants under normal and low-light conditions (Figure 3A and 3B). Only under high-light conditions, a clear difference between tic22-III/IV and wild-type could be observed. This is in contrast to the typical pale-yellow phenotype of the toc33 mutant plants (plastid protein import impaired 1; ppi1) emerging already under normal and low-light conditions (Figure 3; Kubis et al., 2003). At early stages (represented by day 4), the phenotype of tic22-III/IV is characterized by a retarded growth and pale-yellow leaves comparable to that of ppi1 (Figure 3A). At later stages (day 17), rosettes of tic22-III/ IV plants exhibited a smaller diameter than wild-type due to their delayed development, but with green leaves (Figure 3B). Growth analysis according to Boyes and coworkers (2001) uncovered that the developmental rate of tic22-III/IV plants is not drastically reduced under high and normal light when compared to wild-type. However, the double mutants developed fewer leaves compared to wild-type (Figure 3C). In contrast, ppi1 plants show a more retarded developmental rate under these conditions, while the leaf number is not affected (Figure 3C). Under low-light conditions, the mutants behave comparably to wild-type. Only flowering is slightly affected in tic22 mutants, while it is clearly delayed in ppi1. Having observed a chlorotic phenotype of double mutants, we analyzed its photosynthetic capacity under normal and high light. We analyzed 7-day-old plants, as they are all at the same growth stage under both light conditions (Figure  3C). We did not observe a significant difference of Ф(II) between wild-type and double mutant grown under normal light,

Figure 2.  Genotypic and Expression Analysis of tic22-III and tic22-IV Knockout Lines. (A) Schematic representation of the structure of the TIC22-III and -IV genes and position of T-DNA insertions. Exons are depicted as boxes, introns as lines. Primer binding sites for the PCR in (B) and (C) are indicated. LB, left border of the T-DNA; positions are presented in Table 1. (B) Analysis of T-DNA insertions by PCR. PCR reactions were performed on genomic DNA isolated from wild-type and two independent single knockout lines tic22-III (III-1 and III-2) and -IV (IV-1 and IV-2), respectively. PCR was performed using a primer binding in the left border of the T-DNA insertion (LB) and gene-specific forward (F) or reverse primers (R) in the combinations indicated. (C) RT–PCR analysis of the expression of TIC22-III and -IV in wild-type and T-DNA insertion lines. Actin was used for normalization. Primer binding sites are shown in (A). (D) Western blot with total leaf protein extracts from wild-type and mutant plants. Antibodies used are indicated on the left. An antibody against cytoplasmic Hsp70 was used as a loading control. (E) Analysis of T-DNA insertions by PCR as in (B) using genomic DNA isolated from WT and tic22-III/IV. PCR was performed using a primer binding in the left border of the T-DNA insertion (LB) and gene-specific forward (F) or reverse primers (R) in the combinations indicated. (F) RT–PCR analysis of the expression of TIC22-III and -IV in WT and T-DNA insertion lines as in (C).

Rudolf et al.  •  Functional Analysis of atTic22-III and atTic22-IV   

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analyzing the Ф(II)-dependence on the activation light intensity revealed a behavior of the tic22 double mutant which is comparable to that of ppi1 (Figure  4B). The rapid decay of Ф(II) at increasing light intensities (Figure  4B) is consistent with the observed growth phenotype under high-light conditions (Figure  3). A  similar behavior of Ф(II) is observed in plants lacking the plastidic protease SPPA1 (Wetzel et al., 2009), which indicates a defect in chloroplast protein homeostasis in the tic22 double mutant. Consistently, we inspected the ultrastructure of the chloroplasts of 5-day-old tic22 plants (Figure 4C). In plants grown under normal light conditions, we did not observe drastic differences compared to wild-type. In contrast, ppi1 plants showed a reduced stack formation, as previously shown (Jarvis et al., 1998). Chloroplasts of plants grown under high light showed reduced thylakoids in both the tic22-III/IV and ppi1 mutant when compared to wild-type. In line with the observed phenotype, we detected a reduced chlorophyll content in the mutant (Table 2).

tic22-III/IV Chloroplasts Show a Reduced Import Rate

Figure  3.  Phenotypic Analysis of Knockout Plants under Different Light Conditions. (A) Phenotype of 4-day-old plants grown under long-day conditions with light intensities of 20, 140, and 400 µM m–2 s–1, respectively. (B) Phenotype of plants grown for 17 d under conditions as in (A). The scale bar in (A) and (B) shows 5 mm and all figures of one panel are scaled to the same size. (C) Stages of leaf development and inflorescence emergence under different light conditions (LL, NL, HL). Each stage of leaf development (left panel) indicates the formation of a new leaf pair >1 mm in length. The diagram on the right indicates the period from the inflorescence emergence to the opening of flowers. Five plants of each line were analyzed.

while it is reduced for ppi1 as previously reported (Figure 4A; Oreb et al., 2007). Under high-light conditions, we observed a slight reduction in the maximal Ф(II) (Table 2). In contrast,

Next, we analyzed the import capacity of isolated chloroplasts. We isolated chloroplasts from 14-day-old plants because wild-type and double mutant plants were in the same growth stage at this age (Figure 3). We used the radiolabeled model substrate pSSU (precursor of the small subunit of Rubisco), as Tic22 was initially discovered in a crosslink with this precursor (Kouranov et  al., 1998). The import of pSSU into chloroplasts of wild-type and mutant plants showed typical kinetic behavior (Figure 5A). However, the initial import rate of pSSU into the double mutant plants was threefold reduced when compared to wild-type (Figure 5B) and almost similar to wild-type, when the mutant was complemented with a Strep-tagged version of Tic22-IV (Strep-IV). For control, we analyzed the insertion of the outer membrane proteins Oep24 and Toc34 focusing at the 2-min time point, reflecting the initial rate of import. After incubation with chloroplasts, the insertion efficiency was probed by carbonate extraction (Figure 5C). We observed a slight reduction in the import efficiency of Oep24 and Toc34 as well, but in the error range of the experiment (Figure  5D). Thus, the effect on pSSU is specific to the absence of Tic22 and not a result of reduced chloroplast efficiency per se.

tic22-III/IV Chloroplasts Show Metabolic Alterations Next, we analyzed the steady-state metabolite contents in double mutant plants (Table  3 and Supplemental Table  2). We determined the relative amounts of 45 metabolites in wild-type, tic22-III/IV, and ppi1 plants at the age of 7 days or 7 weeks. We analyzed the changes in metabolite content with respect to wild-type considering changes of ≥1.25-fold and with a p-value ≤ 0.05 as significant (Figure 6A). For 15 metabolites, we did not observe any significant alteration (Table 3), while, for 17 metabolites, a difference was observed in the tic22-III/IV

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double mutant (Figure 6A). For nine of these metabolites, the alteration paralleled the change observed in ppi1 (Figure 6A), although changes in ppi1 were in general more drastic. In the tic22 double mutant, 12 of these metabolites are affected in their abundance in the early growth stage, while six are affected at later growth stages. The inspection of the metabolic pathways indicates that carbon metabolism is affected in young plants (Figure  6B) because metabolites such as mannose, maltose, and xylose are elevated in the mutant. Remarkably, the content of fructose, likely resulting from cleavage of sucrose by cytosolic and vacuolar invertases, is decreased, which also holds true for metabolites of the citrate cycle. In addition, in early developmental stages, the amount of α-hydroxyglutarate is reduced as well. This metabolite is produced as a byproduct of the α-oxidation of fatty acids in peroxisomes or by conversion of lysine after protein degradation in chloroplasts and is subsequently transported to mitochondria (Araújo et al., 2010). In older plants, quinate, which is produced from either shikimic acid or dehydroquinate (see Herrmann, 1995), is significantly reduced in the ppi1 and tic22-II/IV double mutant plants (Table  3 and Figure  6A). Although the amounts of shikimic acid are not significantly altered, this could indicate that the flux towards quinate biosynthesis might be reduced, which could be a consequence of reduced production or withdrawal of precursors from the Calvin–Benson cycle. At the same time, tryptophane is enriched, which is also a product of a shikimate-dependent pathway. In addition, glycolate and serine, both intermediates of the photorespiratory pathway, are enriched in older plants, which also points in the direction of altered carbon assimilation in the mutant lines. Although the metabolic changes observed in both mutant lines do not yield a cohesive picture, the observed changes are consistent with a function of Tic22 in chloroplast biogenesis, given that mostly chloroplast-localized pathways or those fed by plastidial metabolites are affected.

Discussion

Figure  4.  Photosynthetic Performance and Chloroplast Ultrastructure of the Double Mutant. (A, B) The time (A) or light-intensity (B) dependence of the effective PSII quantum yield (Ф(II)) determined for WT, tic22 double knockout, and ppi1. Measurements were made on cotyledons of 7-day-old plants. Values shown are means from eight measurements (±  SD). Lines represent the analysis of the data by equation 1 (A) and 2 (B). Values are given in Table 2. (C) Cotyledons of 5-day-old plants grown on MS medium were analyzed by electron microscopy. Representative images of each line are shown.

Both Tic22 isoforms analyzed are localized in the IMS and have a comparable expression pattern (Figure  1), although the expression of TIC22-III is about fivefold lower than that of TIC22-IV based on the comparison to UBIQUITIN expression. The two isoforms apparently perform at least partially redundant functions, as a significant phenotype could only be observed for the double mutant (Figure 3). The phenotype is specific for the malfunction of the TIC22 genes, as the double mutant can be complemented by expression of either isoform (Supplemental Figure  5). The redundancy is further supported by in vitro import assays when the complementation with one of the isoforms (TIC22-IV) rescued the reduced import rate shown in the double mutant (Figure 5A). The double mutant is still viable and the phenotype is most pronounced under high light, but almost absent under lowlight conditions (Figures 3 and 4). Double mutant plants

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Table 2.  PAM Parameters and Chlorophyll Content. Unit

WT NL

Ф(II)ta

Ф(II)max

–

t(Ф(II)50%) Ф(II)PAR

PAR(Ф(II)50%)

Chlorophyllb

III/IV HL

NL

ppi1 HL

NL

HL

0.78 ± 0.01

0.86 ± 0.01

0.80 ± 0.02

0.70 ± 0.01

0.56 ± 0.02

0.55 ± 0.02

Sec

131 ± 3

142 ± 4

187 ± 7

115 ± 4

190 ± 20

119 ± 7

µE

540 ± 40

102 ± 4

43 ± 1

19 ± 2

108 ± 3

µg mg–1 FW

1.3 ± 0.1

0.79 ± 0.05

14 ± 1 0.55 ± 0.05

a Values from Figure 4A and 4B. b Determined from 10-day-old plants grown under NL, n > 3, shown is average and standard deviation.

reveal a reduced import rate (Figure  5), a reduced photosynthetic activity, a decreased adaptation capacity, as well as changes in the thylakoid morphology, especially when grown under high-light conditions (Figures 3 and 4, and Table  2). Considering its suggested importance in protein import (Kouranov and Schnell, 1997; Kouranov et  al., 1998; Becker et al., 2004; Kalanon et al., 2009), we can conclude that Tic22 functions especially at times of high import rates, as found during plastid transitions early on in plant development or under high light inducing a higher protein turnover by photo-damaging. The metabolic changes observed in double mutant plants (Figure 6 and Table 3) might be explained as a consequence of the reduced import capacity. The observed phenotype is partially overlapping with those observed in the mutant of TOC33, ppi1 (Figure 4; Kubis et al., 2003). However, the tic22 double mutant is viable, which leads to two alternative interpretations. On the one hand, the Tic22 function could have a catalytic character only by accelerating/ coordinating protein translocation. Therefore, its malfunction might be compensated, such as by a direct interaction of the TOC and TIC complex, as suggested in the past (Akita et al., 1997; Nielsen et al., 1997). On the other hand, a third as-yet unidentified isoform might exist, which is still able to complement for the function of Tic22-III and Tic22-IV. Indeed, in the database ARAMEMNON (Schwacke et  al., 2003), a third putative Tic22 is annotated (At5g62650), which shares 12–13% identity and 20–22% similarity with Tic22-III and Tic22-IV, respectively. In contrast, Tic22-III and Tic22-IV share a 30% identity and a 45% similarity. In addition, the annotated protein encoded by At5g62650 is twice as long as the other two proteins, and the gene is expressed predominantly in seeds and senescent leaves according to the expression data deposited in the Arabidopsis eFP Browser (Winter et al., 2007). Thus, further work will have to decide between the two possible explanations for the viability of the double mutant.

METHODS Plant Growth The T-DNA insertion lines tic22-III-1 (GK518B03), tic22-III-2 (GK387C03), tic22-IV-1 (GK710E01), and tic22-IV-2 (GK810F06) were obtained from GABI-Kat (Rosso et  al., 2003). Crossing

was performed as described in Weigel and Glazebrook (2005). Plants used for Western blot analysis, RT–PCR, qRT–PCR, and protoplast transformation were grown under short-day conditions (8-h day/16-h night). For phenotypic analysis, analysis of the photosynthetic performance, and electron microscopy, plants were grown in climate chambers (CLF Plant Climatics, Emersacker, G) under long-day conditions (14-h day/10-h night). For the analysis of plants under different light conditions, light intensities of 140 µmol m–2 s–1 (standard conditions), 400 µmol m–2 s–1 (high light), and 20 µmol m–2 s–1 (low light) were applied. The phenotypic analysis was performed as described (Boyes et al., 2001).

DNA, RNA, and Protein Isolation and Analysis DNA and RNA from wild-type and mutants were isolated as described (Oreb et  al., 2007). For quantitative RT–PCR, RNA was isolated and the reactions were performed in an Mx3000P QPCR cycler (Stratagene, USA) using the SYBR-Green Jump Start Taq Ready Mix (Sigma Aldrich, Germany) according to the manufacturer’s recommendations. Conventional PCR, RT–PCR, and qRT–PCR were performed as described before (Oreb et al., 2007; Bohnsack et  al., 2008). The oligonucleotides used are listed in Supplemental Table 1. For the analysis of the protein level of import components, cell extracts from leaves of 7-dayold plants were prepared. For protein extraction, leaf tissue was ground in liquid nitrogen and boiled in SDS sample buffer.

Protein Expression and Antibody Generation The cDNA encoding atTic22-IV was cloned into pet21d by conventional methods. For expression of his-tagged Tic22-IV, 500  ml 2YT medium with 50  μg  ml–1 ampicillin were inoculated with 25 ml overnight culture of BL21[DE3] cells harboring the desired construct and incubated at 37°C. At OD600 of 0.6–0.8, expression was induced by addition of isopropylβ-D-thiogalactopyranosid (IPTG) to a final concentration of 0.5  mM. After incubation for a further 3  h at 37°C, the bacteria were harvested by centrifugation (5000  g, 10  min, 4°C). Harvested cells were re-suspended in 100 mM Tris/HCl, pH  8.0, 200  mM NaCl, 10  mM β-mercaptoethanol and lysed by 20 000 psi cell pressure in a French press. The cell suspension was subsequently sonified twice for 30  s on ice. Tic22containing inclusion bodies were separated from the soluble

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  Rudolf et al.  •  Functional Analysis of atTic22-III and atTic22-IV

fraction by centrifugation (25 000 g, 30 min, 4°C) and solubilized in 50 mM NaPi, pH 7.7, 150 mM NaCl, 8 M Urea by incubation for 3 h at 20°C. After centrifugation (25 000 g, 30 min, 4°C), the supernatant was incubated with Ni-NTA resin for 1 h at 20°C. The matrix was washed twice with 20 vol. of 50 mM NaPi, pH 7.7, 150 mM NaCl, 8 M Urea, 0.2% (v/v) Triton X-100 and twice with 10 vol. of 50 mM NaPi, pH 7.7, 1 M NaCl, 8 M Urea, 15  mM imidazol. Bound proteins were eluted with 50 mM NaPi, pH 7.7, 150 mM NaCl, 8 M Urea and 500 mM imidazol. Eluted fractions were concentrated and separated by SDS–PAGE. Tic22 was excised and antibodies were produced by the Pineda antibody service, Berlin.

Chloroplast Isolation from Arabidopsis thaliana and Protease Treatment Isolation of intact chloroplast was carried out as previously described (Aronsson and Jarvis, 2002). For proteolysis, chloroplasts were incubated in 50 mM HEPES/KOH, pH 7.6, 330 mM sorbitol, 3  mM MgCl2, and 0.5  mM CaCl2 supplemented with thermolysin or trypsin (25  μg/50  μg of chlorophyll; final concentration, 80 ng μl–1) for 20 min at 4°C. The protease was subsequently inhibited by the addition of 5  mM EDTA (final) or trypsin inhibitor (6  µg/50  µg chlorophyll; final concentration, 20  ng  µl–1). Intact chloroplasts were recovered by centrifugation through a Percoll cushion (40% Percoll in 50 mM HEPES/ KOH, pH  7.6, 330  mM sorbitol, 3  mM MgCl2). Subsequently, chloroplasts were mixed with SDS sample buffer and subjected to SDS–PAGE. Proteins were visualized by Western blot analysis.

Chlorophyll Fluorescence Measurement by Pulse Amplitude Modulation (PAM) Figure 5.  Isolated Chloroplasts of tic22-III/IV Double-Knockout Plants Are Impaired in Protein Import Compared to Wild-Type. (A) The 35S radiolabeled precursor of the small RubisCO subunit (pSSU) was incubated with isolated chloroplasts of Arabidopsis thaliana wildtype, tic22-III/IV double-knockout (III/IV), and Tic22-IV complementation (III/IV / IV-Strep) plants for the duration of 2, 5, and 10 min (wild-type: lanes 1–3; III/IV: lanes 4–6; III/IV/IV-Strep: lanes 7–9). After chloroplast re-isolation, the samples were subjected to SDS–PAGE and the radioactivity was visualized by autoradiography. For import quantifications, 1% of the translation product was analyzed (TP). Bands corresponding to the different processing states of the small subunit of rubisco (SSU) are marked by arrowheads (white: precursor; black: mature protein). (B) Quantification of at least four independent experiments from (A). Error bars show the standard deviation (wild-type: black circles; tic22III/IV double-knockout: white rectangles; and Tic22-IV complemented double-knockout: gray diamonds (III/IV)). (C) The 35S-radiolabeled outer membrane proteins Oep24 (left) or Toc34 (right) were incubated for 2 min with isolated Arabidopsis thaliana wild-type (lanes 1, 3) and tic22-III/IV double-knockout (III/IV) chloroplasts (lanes 2, 4). 10% of the translation product is shown as input (TP). After carbonate extraction, samples were subjected to SDS–PAGE and radioactivity was visualized by autoradiography. (D) Imported Oep24 and Toc34 was quantified as described for (B) and the import after 2 min was presented normalized to wild-type. Values for pSSU are taken from (B) and shown for comparison.

The effective PSII quantum yield (Ф(II)) was measured by PAM as previously described (Oreb et  al., 2007) using the MaxiImaging-PAM Chlorophyll-Fluorometer (Heinz Walz GmbH, Effeltrich, G). The time or light intensity dependence of Ф(II) Φ(II )max xt was analyzed by Φ(II)t = [E1] (t (Φ(II )50%max) + t )   Φ (II )max xPAR or Φ (II)PAR = Y0 − [E2] (PAR (Φ (II ) 50%max) + PAR) At least five independent plants for each genotype were analyzed and average values and standard deviation are shown.

Chlorophyll Measurement Total leaf extract (50 mg) was homogenized in liquid nitrogen and chlorophyll was extracted by adding 1 ml of 80% acetone with 2.5 mM sodium-phosphate-buffer. The supernatant was diluted 1:10 and the chlorophyll absorption was measured at an excitation (E) of 645  nm, 663  nm, and 750  nm using the spectrophotometer Jasco V630 (Jasco, Gross-Umstadt, G). The chlorophyll concentration was calculated according to Arnon (1949) using equation E3: C (µg/µl) = (E663 – E750) × 8.2 + (E645 – E750) × 20.2.

[E3]

Rudolf et al.  •  Functional Analysis of atTic22-III and atTic22-IV   

825

Table 3.  Metabolites Analyzed in this Study. Unit*

WT

III/IV

ppi1

7 days

7 weeks

7 days

7 weeks

7 days

7 weeks 26.0 ± 3.0

Asparagine

ng

24.0 ± 3.0

30.0 ± 5.0

44.0 ± 6.0

35.0 ± 4.0

45.0 ± 5.0

Aspartate

µg

2.7 ± 0.5

1.3 ± 0.3

2.6 ± 0.8

1.6 ± 0.5

1.5 ± 0.1

1.5 ± 0.2

Cysteine

ng

5.2 ± 0.8

8.5 ± 1

6.0 ± 3.0

11.0 ± 3.0

17.0 ± 4.0

19.0 ± 1.0

Fructose

µg

1.12 ± 0.04

5.0 ± 1.0

0.79 ± 0.02

3.0 ± 0.5

0.37 ± 0.03

2.6 ± 0.6

Fumarate

µg

15.5 ± 0.8

25.7 ± 0.9

12.2 ± 0.6

34.0 ± 2.0

7.2 ± 0.2

30.0 ± 2.0 14.0 ± 1.0

GABA

ng

12.4 ± 0.2

14.0 ± 2.0

12.0 ± 2.0

7.0 ± 2.0

36 ± 1.0

Gluconate

ng

3.0 ± 0.3

2.6 ± 0.6

5.2 ± 0.6

4.0 ± 1.0

6.7 ± 0.3

2.6 ± 0.8

Glucose

µg

3.1 ± 0.4

7.0 ± 1.0

3.8 ± 0.4

4.3 ± 0.7

3.1 ± 0.1

4.4 ± 0.8

Glutamate

µg

4.1 ± 0.4

4.0 ± 0.3

3.8 ± 0.7

3.8 ± 0.7

4.0 ± 0.6

3.7 ± 0.5

Glycerate

ng

120 ± 10

100 ± 40

78.0 ± 8.0

100 ± 20

76.0 ± 5.0

100 ± 30 0.62 ± 0.02

Glycerol

µg

0.51 ± 0.06

0.53 ± 0.03

0.36 ± 0.08

1.3 ± 0.3

0.44 ± 0.04

Glycine

µg

6.7 ± 0.4

0.2 ± 0.1

6.0 ± 2.0

0.07 ± 0.03

1.63 ± 0.03

0.2 ± 0.2

Glycolate

µg

0.19 ± 0.02

0.25 ± 0.02

0.18 ± 0.01

0.6 ± 0.1

0.22 ± 0.02

0.20 ± 0.02

Isocitrate

µg

1.7 ± 0.1

1.85 ± 0.01

1.1 ± 0.2

1.8 ± 0.3

0.31 ± 0.03

2.3 ± 0.3

Isoleucine

ng

34.0 ± 4.0

100 ± 30

38.0 ± 4.0

80.0 ± 30

35.0 ± 5.0

100 ± 30

Lactate

µg

0.9 ± 0.1

1.2 ± 0.1

0.56 ± 0.08

2.2 ± 0.3

1.74 ± 0.05

0.82 ± 0.07

Leucine

ng

20.0 ± 4.0

100 ± 50

29.0 ± 1.0

80.0 ± 5.0

30.0 ± 7.0

80.0 ± 30.0

Lysine

ng

15.0 ± 6.0

17.0 ± 6.0

13.0 ± 4.0

12.0 ± 7.0

10.0 ± 1.0

23.0 ± 1.0

Malate

µg

3.5 ± 0.2

6.0 ± 1.0

3.5 ± 0.7

6.3 ± 0.5

2.4 ± 0.1

7.5 ± 0.8

Maleate

µg

0.20 ± 0.03

0.34 ± 0.03

0.28 ± 0.01

0.24 ± 0.03

0.18 ± 0.03

0.25 ± 0.02

Malonate

ng

0.4 ± 0.2

9.1 ± 0.9

1.2 ± 0.3

11.0 ± 2.0

0.4 ± 0.1

8.3 ± 0.6

Maltose

µg

0.09 ± 0.02

7.0 ± 3.0

0.16 ± 0.01

7.0 ± 3.0

0.20 ± 0.02

7.0 ± 2.0

Mannitol

µg

0.14 ± 0.02

2.7 ± 0.7

0.22 ± 0.01

1.8 ± 0.6

0.17 ± 0.03

1.6 ± 0.5

Mannose

ng

3.7 ± 0.6

35 ± 6.0

7.3 ± 0.6

25.0 ± 4.0

8.0 ± 1.0

34.0 ± 6.0 25.0 ± 5.0

Methionine

ng

26.0 ± 4.0

18.0 ± 4.0

26.0 ± 4.0

20.0 ± 10.0

28.0 ± 4.0

Myo-inositol

µg

0.83 ± 0.02

4.4 ± 0.4

0.84 ± 0.07

3.5 ± 0.2

1.0 ± 0.1

3.3 ± 0.4

Ornithine

µg

2.8 ± 1.0

3.0 ± 0.8

1.6 ± 1.0

1.2 ± 0.3

8.0 ± 1.0

3.2 ± 0.5

Oxalate

µg

2.3 ± 0.2

7.3 ± 0.9

1.3 ± 0.3

8.0 ± 1.0

1.1 ± 0.1

8.5 ± 0.9

Phenylalanine

ng

57.0 ± 5.0

82.0 ± 9.0

110 ± 10

80.0 ± 20.0

55.0 ± 3.0

84.0 ± 4.0

Proline

ng

0.6 ± 0.3

1.3 ± 0.4

1.0 ± 0.6

4.0 ± 1.0

0.9 ± 0.3

1.7 ± 0.3

Quinate

ng

1.1 ± 0.1

1.1 ± 0.2

0.6 ± 0.1

0.45 ± 0.04

0.27 ± 0.06

0.50 ± 0.02

Raffinose

ng

0.14 ± 0.09

500 ± 400

0.09 ± 0.05

200 ± 100

8.0 ± 1.0

400 ± 200

Serine

µg

1.1 ± 0.1

1.1 ± 0.1

1.7 ± 0.1

1.6 ± 0.1

1.6 ± 0.1

2.0 ± 0.1

Shikimate

µg

0.37 ± 0.02

0.17 ± 0.03

0.38 ± 0.03

0.20 ± 0.07

0.27 ± 0.04

0.14 ± 0.03

Sorbitol

µg

0.23 ± 0.06

0.07 ± 0.02

0.25 ± 0.06

0.26 ± 0.07

0.3 ± 0.1

0.07 ± 0.03

Succinate

µg

0.19 ± 0.01

0.30 ± 0.07

0.11 ± 0.01

0.50 ± 0.03

0.10 ± 0.01

0.36 ± 0.03

Sucrose

µg

9.6 ± 0.9

23.0 ± 2.0

9.1 ± 0.6

22.0 ± 2.0

10.7 ± 0.6

22.0 ± 4.0

Tryptophane

µg

0.03 ± 0.01

0.07 ± 0.02

0.04 ± 0.01

0.3 ± 0.1

0.03 ± 0.01

0.08 ± 0.01

Tyrosine

ng

22.0 ± 4.0

230 ± 70

22.0 ± 2.0

200 ± 100

24.0 ± 2.0

300 ± 200

Valine

µg

0.09 ± 0.02

0.23 ± 0.05

0.14 ± 0.01

0.21 ± 0.08

0.11 ± 0.02

0.21 ± 0.05

Xylose

ng

27.0 ± 2.0

180 ± 20

37.0 ± 1.0

130 ± 10

17.0 ± 2.0

110 ± 10

α-alanine

µg

1.43 ± 0.07

1.1 ± 0.1

2.3 ± 0.2

1.2 ± 0.1

1.4 ± 0. 2

1.00 ± 0.06 280 ± 60

α-hydroxyglutarate

ng

23.0 ± 3.0

320 ± 40

14.0 ± 1.0

270 ± 30

7.0 ± 1.0

α-ketoglutarate

ng

42.0 ± 4.0

32.0 ± 6.0

47.0 ± 3.0

22.0 ± 8.0

24.0 ± 4.0

34 ± 2.0

β-alanine

ng

9.0 ± 1.0

3.5 ± 2.0

7.0 ± 3.0

1.9 ± 0.9

19 ± 2.0

2.9 ± 0.6

* Normalized to mg fresh weight; the p-values for the change are listed in Supplemental Table 2.

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  Rudolf et al.  •  Functional Analysis of atTic22-III and atTic22-IV

Figure 6.  Affected Metabolites in the tic22 Double-Knockout Lines. (A) Metabolites, for which an alteration of abundance was observed are shown and the change for the two genotypes with respect to the wildtype from 7-day or 7-week-old plants is indicated. (B) The metabolic relation of the different metabolites identified to be altered in the tic22 double mutant is shown. The reactions in chloroplasts (green), peroxisomes (blue), or mitochondria (yellow) are framed. The observed change in the double mutant is shown as in (A) under each metabolite. For clarity, not all reaction intermediates are shown. Ala, alanine; E4P, erythrose-4P; F6P, fructose-6P; G6P, glucose-6P; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; R5P, ribulose-5P; Val, valine; X5P, xylulose-5P.

Electron Microscopy For the analysis of the chloroplast ultrastructure, 5-day-old seedlings grown under long-day conditions (14-h day/10-h night) with light intensities of 140 or 400 µmol m–2 s–1 were harvested and fixed in 4% (v/v) glutaraldehyde, 50 mM cacodylate, pH 7.4. After fixation in 1% (w/v) OsO4, seedlings were dehydrated in a graded ethanol series (30–100% (v/v)), infiltrated gradually in Epon, polymerized at 60°C. Ultrathin cell sections were stained with 2% (w/v) uranyl acetate and 0.5% (w/v) lead citrate. Transmission electron micrographs were either taken on a JEOL 2100 TEM operated at 80 kV equipped with a fast-scan 2K × 2K CCD camera F214 (TVIPS, Gauting, Germany) or on a Zeiss CEM 902 operated at 80 kV in combination with a wideangle Dual Speed 2K CCD camera (TRS, Moorenweis, Germany).

Chloroplast Isolation and In Vitro Import Arabidopsis thaliana wild-type and Tic22-III/IV doubleknockout plants for in vitro import time-course experiments were grown for 14 d on MS-Gelrite plates (2% (w/v) sucrose; 1  Murashige-Skoog salts incl. vitamins; 0.3% (w/v) Gelrite)

under long-day conditions (14-h day/10-h night) with standard light intensities of 140  µmol  m–2  s–1). The constructs of pSSU, Oep24 were described before (Bionda et  al., 2010; Ulrich et al., 2012). psToc34FL was amplified from cDNA and cloned into pSP65. Isolation of chloroplasts was performed according to Aronsson and Jarvis (2011). For each import reaction with a final volume of 200 µl, 107 chloroplasts were used. At the given time points, 50 µl of the sample was overlayed onto a cushion of 500 µl 35% (v/v) Percoll in MES-MS buffer (50 mM MES/KOH, pH 6, 300 mM sorbitol, 3 mM MgSO4) for re-purification of intact organelles. After 4 min of centrifugation at 4200 g, the pellets containing the intact chloroplasts were washed with MES-MS buffer, pH  6, and re-suspended in SDS-Urea containing buffer. To discriminate between transient association and membrane insertion of Oep24 and Toc34, the re-purified chloroplasts were treated with 100 µl carbonate buffer (0.1  M Na2CO3, 1  mM EDTA, pH  11.4) for 30 min on ice and subsequently centrifuged at 100 000 g for 30  min. The resulting pellet was re-suspended in SDS-Urea buffer. Half of each sample was loaded onto an SDS-gel for

Rudolf et al.  •  Functional Analysis of atTic22-III and atTic22-IV   

analysis. Radiolabeled bands were visualized by phosphorimaging via Typhoon 9400 and quantified using the software ImageQuant 5.2 (both from GE Healthcare).

Analysis of the Metabolic Content Preparation of samples for metabolic analysis was performed as described in Fiehn (2007). In brief, leaves of 7-day-old or 7-week-old plants grown under long-day conditions were ground in liquid nitrogen. 50 mg of leaf powder was used for extraction with 1.5 ml of a pre-chilled (–20°C) mixture of H2O/ methanol/chloroform (1:2.5:1). 50 µM ribitol was added as an internal standard. Following incubation under gentle agitation for 6 min at 4°C on a rotating device, samples where centrifuged for 2 min at 20 000 g at RT. 25–50 µl of the supernatant were dried and used for further analysis by gas chromatography/electron-impact time-of-flight mass spectrometry, as previously described (Lee and Fiehn, 2008). Metabolite content is expressed relative to the internal standard ribitol. All samples were analyzed with three independent biological replicates.

SUPPLEMENTARY DATA Supplementary data are available at Molecular Plant Online.

FUNDING This work was supported by grants from Deutsche Forschungsgemeinschaft (DFG, SFB807-P17 to E.S., TR985/1–1 to J.T., SFB593-TPB9 to U.G.M.), the Center of Membrane Proteomics Frankfurt (CMP), the Cluster of Excellence ‘Macromolecular Complexes’ and Volkswagenstiftung to E.S.

Acknowledgments We thank Ken Keegstra and John Froehlich for providing antibodies against Tic40 and Hsp93. We thank Klaus Dieter Scharf, Oliver Mirus, and Sandra Mißbach for helpful suggestions for the experimental design and manuscript preparation. No conflict of interest declared.

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