Suppressors of the Chloroplast Protein Import Mutant tic40 Reveal a Genetic Link between Protein Import and Thylakoid Biogenesis

Identification of suppressor of tic40 Mutants

To identify mutant plants in which the chloroplast protein import defect displayed by tic40 is suppressed, ∼40,000 tic40-4 seeds were mutagenized using EMS. The M1 plants were grown to maturity to produce the M2 generation of seeds, and the progeny of ∼10,000 M1 plants was screened for putative suppressor of tic40 (stic) mutants. Because young tic40 seedlings display a reduced size and clear pale-green mutant phenotype (Chou et al., 2003; Kovacheva et al., 2005), it was assumed that suppression of tic40 would result in improved growth and a darker green phenotype. Thus, M2 seeds were germinated and grown in vitro for 10 d and then screened visually for seedlings that appeared significantly larger and greener than the tic40-like seedlings. This led to the identification of several putative stic mutants, which were thoroughly characterized genetically to exclude false positives, and further backcrossed to tic40-4 to remove unwanted background mutations.

A subset of seven mutants carrying mutations that were transmitted in a Mendelian fashion were selected for further characterization and for identification of the mutations responsible for the suppression effect. The selected stic mutants suppressed the tic40 mutation to a similar degree, as they had comparable phenotypes (Figures 1A and 1B). Moreover, all were found to be semidominant: The F1 progeny from their backcrosses to tic40 displayed phenotypes that were intermediate between tic40 single mutants and stic tic40 double homozygotes (Supplemental Figure 1), while the F2 progeny from these crosses segregated in a 1:2:1 ratio for tic40-like, intermediate, and full-suppressor-type plants, respectively. Rough mapping of the mutations (see “Molecular Identification of the stic1 and stic2 Loci” section below) revealed that they fall into two allelic groups, identifying two loci, with five mutants belonging to group 1 (called stic1-1 to stic1-5) and two mutants belonging to group 2 (called stic2-1 and stic2-2).

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Figure 1.

Suppression of the tic40 Phenotype by the stic Mutations.

(A) and (B) Suppressor mutants selected for further characterization and mapping. Photographs showing representative 10-d-old, in vitro-grown (A) and 25-d-old, soil-grown (B) M5 generation seedlings for each selected mutant line. The plants were grown side-by-side under identical conditions alongside appropriate controls. I = 1A-1 (stic2-1 tic40-4), II = 1B-1 (stic2-2 tic40-4), III = 2A-3 (stic1-1 tic40-4), IV = 3A-1 (stic1-2 tic40-4), V = 3A-5 (stic1-3 tic40-4), VI = 4B-2 (stic1-4 tic40-4), VII = 4B-4 (stic1-5 tic40-4), VIII = tic40-4, and IX = the wild type.

(C) and (D) Chloroplast ultrastructure in the stic mutants. Transmission electron microscopy analysis of chloroplasts from cotyledons of 10-d-old, in vitro-grown seedlings of the following genotypes: wild type, tic40-4, and representative stic1 tic40 (stic1-1 tic40-4) and stic2 tic40 (stic2-1 tic40-4) double mutants.

(C) Micrographs of typical chloroplasts are shown for each genotype, which were selected on the basis of a thorough analysis of three different plants per genotype. Bars = 2 µm.

(D) To provide a quantitative measure of the extent of thylakoid development in the different genotypes, numbers of interconnecting stromal lamellae associated with each granum were counted. The presented data are from the analysis of a total of 60 grana proportionately representative of three typical chloroplasts from three different plants per genotype. Values are means and error bars correspond to the se of the mean (n = 60).

(E) and (F) Analyses of chloroplast protein import efficiency in the stic mutants. In vitro import of radiolabeled Rubisco small subunit (SSU) precursor protein into chloroplasts isolated from 12- to 14-d-old plants of the following genotypes: wild type, tic40, and representative stic1 tic40 (stic1-1 tic40-4) (E) and stic2 tic40 (stic2-1 tic40-4) (F) double mutants. Top: Phosphor screen images of representative chloroplast protein import time-course experiments. Times shown indicate minutes (min) after the start of each import reaction. Positions of the precursor (p) and imported mature (m) forms of SSU are shown. Bottom: The amount of accumulated mature SSU protein at each time point was determined by quantifying the relevant band intensities in the top image and in two further similar experiments. Values were each expressed as a percentage of that obtained for wild-type chloroplasts at the final time point in each case. Values shown are mean percentages derived from three independent experiments; error bars show se of the mean (n = 3).

The stic Mutations Suppress the Cellular and Organellar Structural Defects and Chloroplast Protein Import Defects of tic40

The tic40 mutant phenotype is characterized by a reduced number of chloroplasts per cell and by larger chloroplasts with a swollen, more spherical morphology as well as an elevated content of plastoglobules relative to wild-type organelles (Chou et al., 2003; Kovacheva et al., 2005). Furthermore, thylakoid ultrastructure in tic40 appears disorganized, with fewer and less stacked thylakoids (Chou et al., 2003; Kovacheva et al., 2005). To assess whether these defects were visibly reduced in the stic mutants, we employed light and transmission electron microscopy. Light micrographs of whole cotyledon cross sections revealed a disorganized architecture with reduced cell density in tic40 (Supplemental Figure 2). Remarkably, as exemplified by the data shown for stic1-1 tic40 and stic2-1 tic40, all of the stic mutants displayed a noticeable improvement in cellular architecture, with increased cell density and a more uniformly extended abaxial epidermal cell layer (Supplemental Figure 2). Transmission electron micrographs showed that the thylakoids are more developed in the stic mutants than in tic40, appearing more densely packed (Figure 1C) and having a significantly greater number of interconnections (Figure 1D). The increased cell density and improved thylakoid organization of the stic mutants, compared with tic40, most likely contribute to the larger size and greener appearance of the corresponding plants. However, the size and shape of the chloroplasts, the increased number of plastoglobules, and the number of chloroplasts per cell was not significantly different in the stic mutants relative to tic40 (Figure 1C; Supplemental Figure 2).

To assess if the improvement in chloroplast development seen in the double mutants was linked to recovery of the tic40 protein import defect described previously (Chou et al., 2003; Kovacheva et al., 2005), we performed in vitro protein import assays with chloroplasts isolated from wild-type, tic40, stic1-1 tic40, and stic2-1 tic40 seedlings and radiolabeled Rubisco small subunit (SSU) precursor protein (Figures 1E and 1F). For both stic1-1 tic40 and stic2-1 tic40, the SSU precursor was imported significantly more efficiently than for the tic40 single mutant. The amount of mature protein detected at the final time point in each case was ∼20 to 25% greater in the double mutants than in tic40, confirming that the suppression phenotypes are indeed linked to partial recovery of the tic40 protein import defect.

Molecular Identification of the stic1 and stic2 Loci

To identify the mutations responsible for the suppression effects, the stic mutants were crossed to Arabidopsis plants of the Landsberg erecta ecotype into which the tic40-4 mutation (originally of the Columbia-0 ecotype) had been introgressed. The F2 generations from these crosses were used initially to roughly map the mutations by assessing their linkage to polymorphic markers located on each arm of the five Arabidopsis chromosomes (Konieczny and Ausubel, 1993; Berendzen et al., 2005). This revealed that all five stic1 mutants carry a mutation on chromosome 1, while the two stic2 mutants mapped to chromosome 2 (Supplemental Figure 3). Further fine-mapping and sequencing, using the stic1-4 and stic2-1 alleles only, revealed potential splice-defect mutations in ALB4 (G to A at nucleotide 920 relative to the ATG) for stic1-4 (Figure 2A) and in a gene with unknown function (At2g24020; G to A at nucleotide 670 relative to the ATG) for stic2-1 (Figure 2B). ALB4 is a paralog of the ALB3 thylakoid membrane insertase and has been proposed to cooperate with ALB3 in the insertion, assembly, or stabilization of thylakoid membrane proteins (Benz et al., 2009; Trösch et al., 2015). Both ALB4 and ALB3 are members of the conserved Alb3/Oxa1/YidC family of protein insertases (Luirink et al., 2001; Kuhn et al., 2003; Yi and Dalbey, 2005). STIC2 is a homolog of bacterial ybaB, which encodes a protein implicated in membrane protein biogenesis (Skretas and Georgiou, 2010), as well as in DNA binding (Cooley et al., 2009; Jutras et al., 2012).

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Figure 2.

Molecular and Phenotypic Characterization of the stic Mutants.

(A) and (B) Schematic diagrams of STIC1 (A) and STIC2 (B) indicating the positions of the point mutations identified in the five stic1 mutants (stic1-1 to stic1-5) and the two stic2 mutants (stic2-1 and stic2-2). The T-DNA insertion sites for each stic T-DNA mutant (alb4-1, stic2-3, and stic2-4) as determined in this study are also indicated. Start and stop codons are shown. The gray boxes represent the 5′ and 3′ untranslated regions, whereas the black boxes and interconnecting black lines represent exons and introns, respectively.

(C) and (D) Analyses of STIC protein expression in the stic mutants. Immunoblot analyses of total seedling protein extracts (40 µg per sample) from the different stic1 (C) and stic2 (D) mutants. Extracts from stic1 single mutants and wild-type control seedlings (C), or from stic2 tic40 double mutants and tic40 control seedlings (D), were analyzed. Antisera raised against Tic110, stromal Hsp70 (cpHsp70), ALB4, and STIC2 were used to detect the corresponding proteins, as indicated to the left of each blot.

(E) Visible appearance of the stic mutants. Photographs of representative 28-d-old plants are shown. Double and triple mutant plants in the tic40-4 mutant background are shown next to the tic40-4 single mutant (top), whereas single and double mutant plants in the wild-type TIC40 background are shown next to the wild type (bottom). The plants in each set were grown side-by-side under identical conditions.

(F) Chlorophyll accumulation in the stic mutants. Concentrations of chlorophyll in rosette leaves of the 28-d-old mutant and control plants shown in (E) were quantified using a SPAD-502 meter. The values shown are means derived from measurements of six different plants and indicate nmol total chlorophyll per mg tissue fresh weight. Error bars correspond to se of the mean (n = 6).

Further sequencing of the other stic1 alleles confirmed the identity of the affected gene, as stic1-1, stic1-3, and stic1-5 all had nonsense mutations in ALB4 (nucleotide substitutions C639T, C930T, and C2102T, respectively, relative to the ATG), while stic1-2 had a missense mutation in ALB4 (G2213A) (Figure 2A). Similarly, the identity of the second gene was confirmed by sequencing of the stic2-2 allele, which revealed another potential splice-defect mutation (G564A) in the At2g24020 gene (Figure 2B). Using RT-PCR, the predicted splicing defects of the stic1-4, stic2-1, and stic2-2 alleles were confirmed, while the nonsense alleles stic1-1, stic1-3, and stic1-5 were shown to have less ALB4 transcript and the missense allele stic1-2 to have more ALB4 transcript relative to the wild type (Supplemental Figure 4). In addition to the obviously aberrant transcripts, all splice-defect mutants produced transcripts of similar size to the relevant wild-type transcript, albeit at reduced levels (Supplemental Figure 4). However, in the stic1-4 mutant, these wild-type-like transcripts correspond to the loss of the first 1, 3, or 12 nucleotides of the fourth exon. Similarly, in the case of stic2-1 and stic2-2, wild-type-like transcripts carried small deletions that led to frame shifts or deletions causing the removal of a few codons and/or missense mutations. For all mutants, although the RT-PCR results suggested that they may still produce some form of the relevant STIC protein, further analysis indicated that, with the exception of stic1-2, this is not the case (see “Analysis of STIC Protein Expression in the stic Mutants” section below).

Confirmation of Suppression by Independent T-DNA Mutants and Complementation

To confirm that STIC1/ALB4 and STIC2 loss of function is responsible for the suppression of tic40 in the stic mutants, we identified T-DNA mutants for each gene. For STIC1/ALB4, we obtained the alb4-1 (SALK_136199) mutant, which was characterized previously by Gerdes et al. (2006) and shown to have a significant knockdown effect on the expression of the ALB4 gene. We confirmed the presence and position of the T-DNA insertion (Figure 2A) as well as the knockdown effect (30.3% ALB4 transcript relative to the wild type in 2-week-old seedlings, se = 7.2%, n = 6). For STIC2, we identified two T-DNA mutants carrying insertions within the open reading frame of the gene: stic2-3 (SALK_001500) and stic2-4 (WiscDsLox 445D01) (Figure 2B). All of these stic1 and stic2 T-DNA mutants were crossed to tic40, and corresponding double homozygous mutants (alb4-1 tic40-4, stic2-3 tic40-4, and stic2-4 tic40-3) were identified. The phenotypes of these T-DNA double mutants were then compared with those of the original stic1 tic40 and stic2 tic40 double mutants and found to be visibly indistinguishable (Supplemental Figures 5A and 5C). This phenotypic similarity was also demonstrated quantitatively by measuring chlorophyll concentrations in the rosette leaves of all genotypes (Supplemental Figures 5B and 5D).

Finally, we confirmed the identity of the STIC1 and STIC2 genes by complementation. The stic1-1 tic40-4 and stic2-2 tic40-4 double mutants were transformed with constructs that drive overexpression of STIC1/ALB4 and STIC2, respectively, each with a C-terminal FLAG tag (Supplemental Figure 6). The resulting transformants were phenotypically similar to tic40 plants (Supplemental Figures 6A and 6C) and were shown to express the respective FLAG-tagged protein (Supplemental Figures 6B and 6D). Thus, we concluded that the phenotypic reversion was caused by the expression of ALB4-FLAG and STIC2-FLAG, respectively.

Analysis of STIC Protein Expression in the stic Mutants

Immunoblot analyses of whole-seedling protein samples from all stic1 mutants showed that none of the stic1 EMS alleles, with the exception of stic1-2, accumulates the full-length STIC1/ALB4 protein (Figure 2C). Because the ALB4 protein expressed in the stic1-2 mutant carries a G397S amino acid substitution due to the stic1-2 missense mutation (Figure 2A), it is evident that this mutation impedes the function of the ALB4 C terminus and that an intact C terminus is important for ALB4 function. As reported previously, the alb4-1 T-DNA mutant expresses the ALB4 protein at a reduced level (Gerdes et al., 2006). It is noteworthy that the antibody used to detect the ALB4 protein was raised against only the 154 most C-terminal amino acids of the protein, so the accumulation of truncated forms of the STIC1/ALB4 protein cannot be ruled out, especially as most of the mutants carry nonsense mutations (Figure 2A).

We also tested the expression of the STIC2 protein by immunoblotting (Figure 2D). Surprisingly, despite the complete lack of intact STIC2 transcript in the stic2-3 and stic2-4 T-DNA mutants (Supplemental Figure 4D), we found that all stic2 alleles accumulate small amounts of a protein that is comparable in size with STIC2 and cross-reacts with the anti-STIC2 antibody raised against the full predicted mature STIC2 protein (amino acids 49 to 182 relative to the start codon) (Figure 2D).

The Arabidopsis genome encodes a close homolog of STIC2, named STIC2-Like (STCL; At4g30620), with which STIC2 shares both structural (similar exon structure; 78% amino acid sequence identity) and functional similarity (Supplemental Figure 7). A phylogenetic analysis of STIC2/STCL-related sequences from different species confirmed that this gene family is ubiquitously distributed in plants and that the two Arabidopsis genes arose from a duplication that occurred in an early ancestor of the Brassicaceae (Supplemental Figure 8 and Supplemental Data Set 1). Given the high level of similarity between the two proteins, the cross-reacting band observed in the stic2 mutants is most likely STCL, not STIC2. Because the intensity of this band in the stic2-1 and stic2-2 mutants is comparable to that observed in the stic2 T-DNA mutants (Figure 2D), we conclude that these point mutants do not accumulate significant amounts of STIC2 protein. This point was further supported by the similar analysis of plants deficient in both STIC2 and STCL (Supplemental Figure 9).

Phenotypic Characterization of the stic1 and stic2 Mutants

Given that the stic1 tic40 and stic2 tic40 mutants are visibly identical and contain similar concentrations of chlorophyll (Figures 2E and 2F), we hypothesized that the STIC proteins act in a common process (e.g., protein targeting to the thylakoids) such that the removal of one or the other protein disrupts this process. To begin to address this possibility, we generated a stic1 stic2 tic40 triple mutant, reasoning that in an alternative scenario where stic1 and stic2 suppress tic40 by disrupting different processes, their suppression effects would likely be additive. The fact that the phenotype and chlorophyll content of the triple mutant were identical to that of each double mutant, as shown in Figures 2E and 2F, supported our hypothesis that STIC1/ALB4 and STIC2 cooperate in a common process.

To determine if the stic1 and stic2 mutations affect chloroplast and plant development in isolation, we outcrossed the mutants to wild-type plants to obtain the single stic1 and stic2 mutants. As shown in Figures 2E and 2F, both single mutants look identical to, and have essentially the same chlorophyll content as, wild-type plants. This suggests that the loss of STIC proteins may be compensated for by redundancy or the operation of parallel pathways or that STIC functions become critical only under certain circumstances. The fact that the stic1-1 mutation was not found to have a visible effect on plant development is in agreement with the findings of Gerdes et al. (2006) and Trösch et al. (2015), who characterized the alb4-1 mutant, but in contrast to the stunted growth phenotype observed by Benz et al. (2009).

We also generated stic1 stic2 double mutant plants by outcrossing the stic1 stic2 tic40 triple mutant to the wild type. Like the stic1 and stic2 single mutants, the double mutant was phenotypically indistinguishable from wild-type plants under normal growth conditions (Figures 2E and 2F). This observation is consistent with the notion that the STIC1/ALB4 and STIC2 proteins act in a common pathway, the disruption of which is potentially compensated for by parallel processes or can be tolerated under normal conditions.

Localization of the STIC1 and STIC2 Proteins

Since the STIC2 protein is predicted to carry a transit peptide, we wished to determine if the protein is indeed targeted to chloroplasts. Thus, we generated a construct for the transient expression in protoplasts of STIC2 fused to a C-terminal YFP tag. A similar construct was made for STIC1/ALB4, although this protein was previously established as a chloroplast protein that associates with the thylakoid membranes (Gerdes et al., 2006). In addition, a free-YFP construct and a previously generated Tic110-YFP fusion construct (Bédard et al., 2007) were used as cytosol and chloroplast envelope markers, respectively. Using confocal microscopy to image transfected protoplasts, these control proteins displayed fluorescence patterns typical of cytosol and envelope localization, respectively (Figure 3A). By contrast, the ALB4-YFP signal closely overlapped with the chlorophyll autofluorescence signal, indicating that the protein likely accumulates in the thylakoids as previously reported (Gerdes et al., 2006). The STIC2-YFP protein also accumulated in the chloroplasts, confirming the TargetP (Emanuelsson et al., 2000) prediction, but the fluorescence signal in this case displayed a different profile, being concentrated in areas of the chloroplasts where the chlorophyll fluorescence signal was least intense, which suggests that the protein accumulates in the stroma (Figure 3A). A similar STCL-YFP fusion displayed the same localization profile (Supplemental Figure 7D).

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Figure 3.

Localization of the STIC1/ALB4 and STIC2 Proteins.

(A) Confocal microscopy analysis. Arabidopsis protoplasts transiently expressing the following proteins were analyzed by confocal microscopy: YFP (Free YFP), Tic110-YFP, STIC1/ALB4-YFP, and STIC2-YFP; all of the YFP fusion proteins carried a C-terminal YFP tag. Representative protoplasts are shown. In each case, YFP fluorescence, chlorophyll autofluorescence, merged YFP and chlorophyll fluorescence, and bright-field images are shown. White arrowheads in the STIC2-YFP micrographs indicate areas of the chloroplasts where the YFP signal is clearly accumulated but a strong chlorophyll signal is lacking. Bars = 12.68 µm.

(B) and (C) Chloroplast subfractionation analysis. Chloroplasts isolated from transgenic seedlings overexpressing C-terminally FLAG-tagged STIC1/ALB4 (B) or STIC2 (C) were separated into stroma (S), envelope (E), and thylakoid (T) enriched fractions prior to immunoblotting (∼15 µg of each sample was analyzed). An anti-FLAG antibody was used to detect the STIC1/ALB4-FLAG and STIC2-FLAG proteins, while antisera raised against Tic110, SSU, LHCP, and ALB3 were used to detect the corresponding reference proteins.

To confirm the localizations of the STIC proteins using an alternative approach, chloroplasts were isolated from seedlings expressing the ALB4-FLAG and STIC2-FLAG fusion proteins described earlier and then subfractionated by sucrose density-gradient centrifugation to obtain stromal, envelope, and thylakoidal fractions. Immunoblotting analysis of the different fractions using antibodies against the FLAG tag and the following control proteins enabled us to assess the localization of the STIC proteins biochemically; the controls were Tic110 (envelope), LHCP (thylakoid), ALB3 (thylakoid), and the small subunit of Rubisco (SSU; stroma). In accordance with the microscopy data, the ALB4-FLAG protein was found exclusively in the thylakoid fraction (Figure 3B), while the STIC2-FLAG protein was detected exclusively in the stromal fraction (Figure 3C). The control proteins largely behaved as expected, although we did detect some Tic110 in the thylakoid fractions, indicating that these were somewhat contaminated with envelopes (Figures 3B and 3C), as is often the case in such subfractionation experiments.

To assess the possibility that the localization of STIC1/ALB4 is dynamic, enabling colocalization with Tic40 in the envelope at specific developmental stages, we similarly analyzed chloroplasts isolated from plants of different ages, including some that were of approximately the same age as those used for the microscopy (5 weeks old; Supplemental Figure 10). In each case, we failed to detect any ALB4-FLAG protein in the envelope fraction, indicating that the protein is stably confined to the thylakoid membrane.

The accumulation of STIC2 in the stroma is not surprising because the protein does not possess any hydrophobic regions that might form a transmembrane helix to anchor it in a membrane (Krogh et al., 2001). However, a previously reported chloroplast proteome study suggested that some STIC2 protein is associated with the thylakoids (Peltier et al., 2004), presumably as a peripheral protein via protein-protein interactions. The fact that no STIC2-FLAG protein was detected in the thylakoids in our study suggests that either the chloroplast thylakoid fraction analyzed by Peltier et al. (2004) was slightly contaminated with stromal proteins, or the amount of STIC2-FLAG that associates with the thylakoids is so low as to be beyond the limits of detection in our assay.

Chloroplast Morphology and Ultrastructure of the stic Mutants

Because STIC1/ALB4 and STIC2 are both chloroplast proteins, we sought to gain further insight into their functions by characterizing chloroplast ultrastructure in corresponding single mutant plants, which at a macroscopic level are indistinguishable from the wild type. As shown in Figure 4A (upper micrographs), we observed that the cotyledon chloroplasts of stic1 and stic2 seedlings have an unusual morphology: In both cases, the chloroplasts appeared swollen and less appressed to the cell periphery than wild-type chloroplasts. The shape difference was quantified by measuring the length and width of chloroplast cross sections and comparing the length/width ratio with that of the wild type (Figure 4B). In both mutants, this ratio is roughly three-quarters that of wild-type chloroplasts, confirming that they are more spherical.

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Figure 4.

Morphology and Ultrastructure of stic1 and stic2 Mutant Chloroplasts.

(A) Transmission electron micrographs of chloroplasts in cotyledons of 10-d-old, in vitro-grown seedlings of the following genotypes: wild type and representative single stic mutants, stic1-1 and stic2-1. Whole-organelle (top) and thylakoid close-up (bottom) images are shown for each genotype. Bars = 10 and 1.5 µm, respectively. Typical micrographs are shown, which were selected on the basis of a thorough analysis of three different plants per genotype.

(B) Analysis of chloroplast shape. Using the electron micrographs described in (A), chloroplast length and width measurements for a total of n different organelle cross sections were made (values of n are shown in the figure). Roughly equal numbers of chloroplasts from two different seedlings per genotype were analyzed. The length and width values were used to derive ratios, as an indicator of chloroplast shape, and the average ratio (±se) for each genotype is shown.

(C) Analysis of plastoglobule numbers. Using the electron micrographs described in (A), the number of plastoglobules per chloroplast cross section in a total of n different organelles was counted (values of n are shown in the figure). Roughly equal numbers of chloroplasts from three different seedlings per genotype were analyzed. The average number (±se) for each genotype is shown.

Upon observation at higher magnification, the thylakoid membrane networks in the stic mutants appeared to be as developed and complex as those of wild-type chloroplasts (Figure 4A, lower micrographs): Comparable grana of variable thickness were seen in all genotypes, interconnected by networks of stromal lamella membranes, dispersed throughout each chloroplast cross section. Nonetheless, the lamellae were found to be less parallel and tightly packed (i.e., more loosely distributed) in the mutants, possibly due to the rounder shape of the chloroplasts. Interestingly, a significantly higher number of plastoglobules (lipid bodies) were visible in the mutant chloroplasts, as illustrated in Figure 4C, pointing toward a thylakoid biogenesis defect (Rudella et al., 2006).

In relation to stic1, these observations are consistent with those made by Gerdes et al. (2006) and Trösch et al. (2015) using the alb4-1 T-DNA insertion mutant. The appearance of the stic mutant chloroplasts is reminiscent of the previously described swollen chloroplast phenotype of tic40 plants (Kovacheva et al., 2005; Bédard et al., 2007), although in that case the thylakoid membrane system was much less developed. The chloroplast morphology defect is possibly more similar to that observed in the vipp1 knockdown mutant (Zhang et al., 2012), which is believed to have an inner envelope membrane integrity defect.

Specificity of the Suppression Effects of stic1 and stic2

To determine if the suppression effects of the stic mutations are specific to tic40, or more general, we crossed the single stic1-1 or stic2-1 mutants to several other protein import mutants: ppi1, toc75-III-3, hsp93-V, and tic110. All of these mutants display a degree of chlorosis linked to defective import, with the lesions occurring at either the outer membrane (ppi1 is a null mutant for the Toc33 receptor; toc75-III-3 is a point mutant of the Toc75 channel) or the inner membrane (hsp93-V is a null mutant for the Hsp93/ClpC chaperone; tic110 is a heterozygous mutant for a chaperone recruitment scaffold protein) (Jarvis et al., 1998; Kubis et al., 2003; Kovacheva et al., 2005; Stanga et al., 2009; Huang et al., 2011). Double mutants were generated and their phenotypes were compared with those of the corresponding single import mutants (Supplemental Figure 11). Most of the double mutants analyzed were double homozygotes, but because the tic110 mutation is embryo lethal in the homozygous state and causes chlorosis in the heterozygous state (Inaba et al., 2005; Kovacheva et al., 2005), the corresponding double mutants were heterozygous for tic110.

As exemplified for the import mutants in combination with stic1-1 (Supplemental Figure 11A), the double mutants appeared in all cases identical to the corresponding single mutants, regardless of the severity of the chlorotic phenotype displayed. To quantitatively assess these observations, chlorophyll concentrations in each pair of single and double mutants were measured and used to calculate average ratios (Supplemental Figure 11B). In all cases, the ratios obtained were approximately equal to 1, indicating no difference between the double and single mutants. A similar result was obtained for the import mutants in combination with stic2-1 (Supplemental Figure 11C). These results contrast markedly with those obtained for the stic tic40 double mutants, where the equivalent chlorophyll content ratio values obtained were ∼1.8 to 1.9. Thus, the results clearly indicate that the stic mutations are not general suppressors of import deficiency. We conclude that the suppression effects mediated by stic1 and stic2 are quite specific for tic40, implying a close functional connection between the STIC proteins and Tic40.

Genetic Interactions between the stic Mutations and Thylakoid Protein Targeting Mutations

Given the similarity between STIC1/ALB4 and ALB3 and the well-established role of the latter in thylakoid protein biogenesis, and the genetic evidence pointing toward roles for the STIC proteins in a common process (Figures 2E and 2F), we wished to investigate if the STIC proteins act in thylakoid protein transport. To begin to address this possibility, further genetic interaction tests were performed, this time with mutants for components of the different thylakoid targeting pathways; i.e., the cpSec, cpTat, and cpSRP pathways. The mutants used in this analysis were as follows: cpsecA1 (null for the cpSecA1 ATPase), hcf106 (null for the chloroplast TatB homolog), cpsrp54-3, cpsrp43-2, and cpftsY-1 or cpftsY-2 (null for the cpSRP54, cpSRP43, and cpFtsY components, respectively, of the cpSRP pathway; Supplemental Figure 12A). Again, we generated double mutants with stic1 and stic2 and compared them to the corresponding single thylakoid targeting mutants (Figure 5; Supplemental Figures 12B to 12D).

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Figure 5.

Specific Genetic Interactions between stic Mutations and cpSRP Pathway Mutations.

Double mutant plants carrying one or the other stic mutation together with a mutation affecting one of three well-characterized thylakoid protein targeting pathways were generated and then compared phenotypically with single mutant control plants. The thylakoid targeting mutants analyzed were as follows: cpsecA1 ([A] and [B]; affecting the cpSec pathway), hcf106 ([A] and [B]; affecting the cpTat pathway), cpsrp54-3 ([C] and [D]), and cpftsY-1 or cpftsY-2 ([E] and [F], respectively) (all affecting the cpSRP pathway). The stic mutant alleles employed in this analysis were as follows: stic1-1 ([A], [C], and [E]), stic2-1 (B), stic2-3 (D), and stic2-4 (F). All plants were initially grown in vitro under standard conditions before being transplanted after 8 to 12 d growth, either to MS medium supplemented with 3% (w/v) sucrose ([A] and [B]) or soil ([C] to [F]). The plants were analyzed after a total of 3 to 4 weeks of growth.

The cpsecA1 and hcf106 mutants have seedling-lethal, albino phenotypes similar to that of the alb3 mutant of the cpSRP pathway, and so cannot survive when grown on soil but can be maintained in vitro on medium containing sucrose (Liu et al., 2010). It was recently shown that the alb4-1 mutation interacts synergistically with the alb3-1 null mutation, producing seedlings that are even more chlorotic and weak than the single alb3 mutant (Trösch et al., 2015). This was interpreted to indicate that there is a degree of functional redundancy between the two affected proteins and that ALB4 participates in the cpSRP pathway or in a parallel pathway that performs a similar role. In marked contrast, no synergistic effects were observed when stic1 and stic2 were combined with the cpsecA-1 and hcf106 mutations, as the double mutants were phenotypically indistinguishable from the corresponding albino single mutants (Figures 5A and 5B). If we assume that the cpSec and cpTat pathways are not completely blocked in these mutants, respectively, then the failure of the stic mutations to accentuate the secA1 and hcf106 phenotypes suggests that the STIC proteins do not participate in these pathways.

The cpsrp54, cpsrp43, and cpftsY mutants are all chlorotic, but they are viable and phenotypically less severe than the albino mutant alb3: The cpsrp54 and cpsrp43 mutants produce rosette leaves that are initially yellow but then become progressively greener (Figures 5C and 5D; Supplemental Figures 12B to 12D) (Amin et al., 1999; Klimyuk et al., 1999; Yu et al., 2012), whereas cpftsY is more severely chlorotic, remains yellow throughout development, and displays stunted growth (Figures 5E and 5F) (Asakura et al., 2008). As shown in Figures 5C to 5F, the stic cpsrp54 and stic cpftsY double mutants were all significantly smaller and more chlorotic than the corresponding single mutants, indicating that both stic mutations interact synergistically with cpsrp54 and cpftsY. By contrast, similar synergistic interactions were not detected in the stic1 cpsrp43 and stic2 cpsrp43 double mutants (Supplemental Figures 12B to 12D), implying that the STIC proteins do not act in the canonical cpSRP43 pathway for LHCP targeting.

Overall, these genetic data strongly support the notion that the STIC proteins act together in a specialized cpSRP pathway that is distinct from the cpSec and cpTat pathways. This conclusion is consistent with the thylakoid morphological defects (Figure 4A) and the accumulation of plastoglobules (Figure 4C) seen in the stic mutants, and with the genetic evidence suggesting that the two proteins act in a common process (Figures 2E and 2F).

STIC2 Interacts with the C Termini of ALB4 and ALB3 in Vitro

We wished to investigate whether the STIC proteins can interact physically with each other. To address this possibility, we employed in vitro pull-down experiments using recombinant proteins expressed in bacteria (Figures 6A to 6C). For this work, we used an N-terminally tagged form of the STIC1/ALB4 C terminus (His-ALB4C; residues 345 to 498 of the preprotein), representing most of the stromal C-terminal domain (Falk et al., 2010), and an N-terminally tagged form of the mature STIC2 protein (GST-STIC2; residues 49 to 182 of the preprotein) (Figure 6A, left side). Equal amounts of soluble lysate from bacteria expressing each protein were mixed together, and then the His-ALB4C protein was affinity purified. The GST-STIC2 protein copurified efficiently with His-ALB4C in the absence of significant amounts of any other protein, providing strong evidence for a direct interaction between STIC2 and the C terminus of STIC1/ALB4 (Figure 6A, left side). By contrast, when His-ALB4C was replaced in the assay by the His-tagged LTD stromal targeting factor (Ouyang et al., 2011), significant GST-STIC2 copurification was not detected (Figure 6B). This demonstrated that GST-STIC2 does not associate nonspecifically with the affinity resin used to purify the His-tagged proteins, confirming that the detected copurification (Figure 6A) reflects a genuine protein-protein interaction.

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Figure 6.

Physical Interactions between STIC2 and the STIC1/ALB4 and ALB3 Proteins.

(A) to (C) In vitro pull-down analysis. Experiments were performed using lysates of E. coli cells expressing recombinant GST-STIC2 ([A] to [C]), His-ALB4C (A), His-ALB4C-G397S (A), His-LTD (B), and/or His-ALB3C (C). In each experiment, equal amounts of lysate containing GST-STIC2 and one of the His-tagged proteins were incubated together, and then the His-tagged protein was purified using Ni-NTA resin prior to analysis by SDS-PAGE. In each gel, individually purified protein samples from the relevant lysates were included as reference controls (left side, with angled labels); the asterisks indicate a truncated form of the GST-STIC2 protein that appeared in the lysates to a variable degree. The flow-through (FT) samples contained proteins not bound by the Ni-NTA resin, whereas the imidazole elution fractions (E1–E4) contained proteins that were bound by the resin. All of the gels were stained with Coomassie blue. For all samples, 10 μL was loaded per lane.

(D) Co-IP analysis. Experiments were performed with chloroplasts isolated from 12- to 14-d-old, in vitro-grown wild-type plants overexpressing either STIC1/ALB4-FLAG (left side) or STIC2-FLAG (right side). Chloroplasts were solubilized with DDM directly after isolation (left side in each case) or after treatment with 0.25 to 0.50 mM of the membrane-permeable cross-linker DSP (right side in each case) and subjected to anti-FLAG immunoprecipitation. All samples were analyzed by immunoblotting. Amounts equivalent to ∼25 µg of protein for solubilized chloroplast lysate (SL) and for the co-IP flow-through (FT) were loaded, whereas one-third or one-sixth of elution (E) samples was loaded for the STIC1/ALB4-FLAG and STIC2-FLAG samples, respectively. Antisera raised against STIC1/ALB4, STIC2, ALB3, Tic40, and LHCP or FNR were used to detect the corresponding proteins.

The stic1-2 mutant carries a glycine-to-serine missense mutation (G397S) that affects the stromal domain of STIC1/ALB4 employed in the in vitro pull-down experiments. In view of the evidence indicating that STIC1/ALB4 and STIC2 act in a common pathway (Figure 2), we reasoned that a consequence of this mutation might be to perturb the interaction between the STIC proteins. To address this possibility, we introduced the G397S mutation into the pull-down construct (His-ALB4C-G397S) and used it in further pull-down studies with the GST-STIC2 protein (Figure 6A, right side). Although identical amounts of His-ALB4C-G397S and His-ALB4C were employed, in simultaneous experiments conducted in parallel, substantially reduced binding of GST-STIC2 was observed with His-ALB4C-G397S relative to wild-type His-ALB4C (on average, 17% over the four elutions, se = 4%). These data support the notion that STIC2 interacts specifically with the C terminus of STIC1/ALB4 and indicate that the Gly-397 residue is important for this interaction.

The functional differences between the ALB3 and ALB4 proteins are believed to be mainly attributable to their different stromal C termini. While ALB3 contains two conserved motifs (Motifs II and IV) important for association with the LHCP targeting factor cpSRP43, ALB4 lacks these domains (Falk et al., 2010). Gly-397 of ALB4 has been proposed to be part of Motif III that is present in both ALB4 (residues 397 to 414) and ALB3 (residues 386 to 403) (Falk et al., 2010). Thus, it is possible that this motif is also important for STIC2 interaction with ALB3. Therefore, to determine if the C terminus of ALB3 can also directly interact with GST-STIC2, we conducted further pull-down studies using an N-terminally tagged form of the ALB3 C terminus (His-ALB3C; residues 361 to 462 of the preprotein). As shown in Figure 6C, His-ALB3C also efficiently pulled down the GST-STIC2 protein, indicating that STIC2 interacts not only with STIC1/ALB4 but also with ALB3, further supporting the notion that STIC2 plays a role in protein targeting to the thylakoids.

BiFC Analysis of the Interaction between ALB3, ALB4, and STIC2

To provide in vivo corroboration of the interactions observed in vitro (Figures 6A to 6C), we employed bimolecular fluorescence complementation (BiFC) in transfected Arabidopsis protoplasts (Lee et al., 2008). Thus, the STIC coding sequences were inserted into vectors that generate fusions to the N-terminal or C-terminal parts of yellow fluorescent protein (nYFP and cYFP, respectively). Various nYFP and cYFP fusion vector pairs were then cotransfected and imaged by confocal microscopy. In this system, YFP fluorescence is only observed when the nYFP and cYFP parts are brought into close proximity by the interaction of the fused proteins (in this case, the STIC1/ALB4, STIC2, and ALB3 proteins), enabling reconstitution of a complete, functional YFP protein (Lee et al., 2008). As a positive control for transfection, the ALB4-YFP plasmid was used on its own (Figure 7); this control produces a stronger signal than BiFC because it does not depend on the interaction of two proteins.

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Figure 7.

In Vivo Analysis of Interactions between STIC2 and the STIC1/ALB4 and ALB3 Proteins.

BiFC analysis of protein-protein interactions was performed by studying Arabidopsis protoplasts coexpressing proteins fused to complementary N-terminal (nYFP) and C-terminal (cYFP) fragments of the YFP protein. The images shown are representative confocal micrographs of protoplasts expressing STIC1/ALB4 with a complete YFP tag (ALB4-YFP; this served as a positive control) or the following fusion protein pairs: STIC2-nYFP with ALB4-cYFP, ALB4-G397S-cYFP, ALB3-cYFP, or PSI-D-cYFP (the latter served as a negative control); STIC2-cYFP with ALB3-nYFP; and PSI-D-nYFP with PSI-D-cYFP (which demonstrated that the negative control protein PSI-D-cYFP can be expressed and is competent to reconstitute YFP when associated with an appropriate interaction partner). For each protoplast, YFP fluorescence, chlorophyll autofluorescence, merged YFP and chlorophyll fluorescence, and bright-field images are shown. Bars representing 5 or 10 μm are shown for each set of images.

Protoplasts that were transfected with both STIC2-nYFP and ALB4-cYFP constructs produced a clear fluorescence signal that overlapped with the chlorophyll signal, as observed in the positive control (Figure 7). Neighboring untransfected protoplasts or protoplasts transfected with only one of the plasmids did not produce a fluorescence signal, although chlorophyll fluorescence was observed (Supplemental Figure 13). In the reciprocal experiment, protoplasts doubly transfected with STIC2-cYFP and ALB4-nYFP produced an identical signal. When ALB4 was replaced with the mutated form (G397S), fluorescence signals were also observed (Figure 7); detection of this interaction may reflect the ability of mutant ALB4 to residually bind STIC2 (Figure 6B) and be aided by the overexpression of both interaction partners. Alternatively, if ALB4-G397S-cYFP homooligomerizes with endogenous ALB4 or heterooligomerizes with ALB3, interaction of STIC2-nYFP with the endogenous proteins may be sufficient to result in fluorescence due to close proximity of the YFP moieties. Clear fluorescence signals were also observed for both STIC2-ALB3 combinations (STIC2-nYFP with ALB3-cYFP; STIC2-cYFP with ALB3-nYFP; Figure 7), while no signals were observed in the negative control, STIC2-nYFP with PSI-D-cYFP (Figure 7). The PSI-D-cYFP fusion is a valid negative control here, as it was shown to be expressed and able to interact with PSI-D-nYFP in chloroplasts (Figure 7; Supplemental Figure 13) (Xia et al., 1998; Kudla and Bock, 2016).

Overall, our BiFC data confirm the data obtained from the in vitro pull-down assays and provide clear in vivo support for the interaction of STIC2 with both ALB4 and ALB3.

STIC2 Can Be Cross-Linked to Both ALB4 and ALB3 in Coimmunoprecipitation Experiments

To further corroborate the positive interaction results from the in vitro pull-down and in vivo BiFC studies, a complementary approach was used: Anti-FLAG coimmunoprecipitation (co-IP) from chloroplasts isolated from wild-type plants that overexpress ALB4-FLAG or STIC2-FLAG proteins (Figure 6D). Using freshly isolated chloroplasts solubilized with the nonionic detergent dodecyl maltoside (Figure 6D, left side of each panel), the ALB4-FLAG and STIC2-FLAG proteins were efficiently immunoprecipitated, eluted from the anti-FLAG resin with SDS buffer, and detected with ALB4 and STIC2 antisera, respectively. However, no significant interaction between the STIC proteins was detectable, nor was there any significant interaction between the STIC proteins and ALB3 or Tic40. The photosynthetic proteins LHCP and ferredoxin-NADP reductase (FNR) used as controls in these assays were also absent from the eluates.

Our failure to detect interactions in our initial studies prompted us to repeat the experiments following treatment with the membrane-permeable chemical cross-linker dithiobis succinimidyl propionate (DSP), which can facilitate the detection of transient or weak interactions that might otherwise be lost during the purification procedures (Figure 6D, right side of each panel). In these experiments, the control proteins LHCP and FNR were again absent from the eluted fractions, providing a clear indication of specificity. No interactions between the STIC proteins and Tic40 were detected, but on this occasion, the ALB3 protein was coimmunoprecipitated with STIC2-FLAG and with ALB4-FLAG in accordance with the results of Trösch et al. (2015) (Figure 6D). Moreover, the STIC2 protein copurified with STIC1-FLAG, and the STIC1/ALB4 protein copurified with STIC2-FLAG, in the reciprocal experiments, confirming that the two STIC proteins can indeed interact physically with each other.