Evolutionary, molecular and genetic analyses of Tic22 homologues in Arabidopsis thaliana chloroplasts

Abstract

The Tic22 protein was previously identified in pea as a putative component of the chloroplast protein import apparatus. It is a peripheral protein of the inner envelope membrane, residing in the intermembrane space. In Arabidopsis, there are two Tic22 homologues, termed atTic22-III and atTic22-IV, both of which are predicted to localize in chloroplasts. These two proteins defined clades that are conserved in all land plants, which appear to have evolved at a similar rates since their separation >400 million years ago, suggesting functional conservation. The atTIC22-IV gene was expressed several-fold more highly than atTIC22-III, but the genes exhibited similar expression profiles and were expressed throughout development. Knockout mutants lacking atTic22-IV were visibly normal, whereas those lacking atTic22-III exhibited moderate chlorosis. Double mutants lacking both isoforms were more strongly chlorotic, particularly during early development, but were viable and fertile. Double-mutant chloroplasts were small and under-developed relative to those in wild type, and displayed inefficient import of precursor proteins. The data indicate that the two Tic22 isoforms act redundantly in chloroplast protein import, and that their function is non-essential but nonetheless required for normal chloroplast biogenesis, particularly during early plant development.

Figure 1. Phylogenetic analysis of the Tic22 gene family in plants and cyanobacteria.

Homologous sequences identified in whole genome sequence datasets from plants and cyanobacteria were analysed using the program MrBayes. The analysis showed a strong support for an origin of Tic22 in cyanobacteria, and revealed that all investigated plant and algal species include at least one member of the gene family. A gene duplication occurred at least 416–449 million years ago (indicated by a star), and resulted in two paralogous gene copies that have been conserved in all investigated angiosperms. Branch lengths in the two clades formed by the gene duplication are very similar (mean branch length and the 95% Highest Posterior Density [HPD] credibility interval are shown for a selection of branches), which indicates that the evolutionary rates of the two paralogous copies have been similar over an extensive period of time. A partial gene duplication (indicated with a red dot) is inferred to have happened after the split between red algae and other archaeplastida. The partially-duplicated gene of unknown function has been conserved in all investigated land plant species, but has not been found in any algal species. All Arabidopsis sequences are shown in red text. Posterior probability values appear above the branches, and the expected number of changes per site along the branches is indicated by the scale bar.

Figure 2. Expression of the Tic22 genes in different tissues and at different developmental stages.

A. Quantitative RT-PCR analysis of total-RNA from whole seedlings grown for five days in the dark (5dD), or five and 14 days in the light (5dL and 14dL, respectively), as well as from three different tissues of mature plants (rosette leaves, siliques, and roots). RNA samples were representative of ∼10–30 seedlings (5dD, 5dL and 14dL), or 5–25 mature plants (rosettes, siliques and roots). Tic22 data were normalized relative to the control gene, ACTIN2 (At3g18780), and then expressed relative to the atTIC22-IV 5dL value. Data shown are means (± SE) derived from three biological replicates. B. Affymetrix GeneChip data were analysed and retrieved using the Genevestigator V3 analysis tool (https://www.genevestigator.com) , . Presented data were prepared using the Development representation in scatter-plot format. Data from all high-quality ATH1 (22 k) arrays were analysed; this amounted to a total of 7392 samples. Values shown are means. The total number of samples used to derive each data point shown is indicated. Typical ranges of low, medium, and high expression for the array type are shown; medium is defined as the interquartile range (IQR). Stages of development are defined as follows, from left to right: germinating seed, seedling, young rosette, developed rosette, bolting, young flower, developed flower, flowers and siliques, mature siliques, and senescence. Data representations were exported from Genevestigator in portable document format, and then annotated using appropriate graphics software. The genes analyzed were as follows: atTIC22-IV (At4g33350; red); atTIC22-III (At3g23710; blue); atTIC110 (At1g06950; green); atTOC33 (At1g02280; orange); atTOC34 (At5g05000; purple).

Figure 3. Molecular characterization of the Tic22 T-DNA insertion lines.

A. Diagrams showing the structure of each gene and the locations of T-DNA insertions. Protein-coding exons are represented by black boxes, untranslated regions by white boxes, and introns by thin lines between the boxes. Locations of RT-PCR primers are shown by arrows beneath each gene model. T-DNA insertion sites are indicated precisely, but insertion sizes are not to scale. ATG, translation initiation codon; Stop, translation termination codon; p(A), polyadenylation site; LB, T-DNA left border. B. Genomic DNA samples extracted from wild type and putative homozygous mutants were analysed by PCR. Appropriate T-DNA- and atTIC22-gene-specific primers were employed. Two different primer combinations were used in each case: the first (“T”) comprised one T-DNA border primer and one gene-specific primer (LB+reverse: tic22-1V-1, tic22-IV-2, tic22-III-2; LB+forward, tic22-III-1); the second (“G”) comprised two gene-specific primers flanking the T-DNA insertion site. The PCR products were resolved by agarose gel electrophoresis, and visualized by staining with SYBR Safe. Amplification using “T” indicated the presence of the mutant allele, whereas amplification using “G” indicated the presence of the wild-type allele; amplification with the former but not the latter demonstrated that the plant was homozygous mutant. The genotype names are shortened as follows: “22-IV-1″ indicates tic22-IV-1; “22-IV-2″ indicates tic22-IV-2; and so on. Sizes of the amplicons are indicated at right (in kb). C. Expression of Tic22 genes in wild-type and mutant plants was analysed by RT-PCR. Locations of the amplification primers are shown in A. Similar analysis of atTOC33 and of the translation initiation factor gene eIF4E1 (At4g18040) was used to normalize loading. Amplicon sizes are indicated at right (in kb). RNA samples were from whole, 10-day-old homozygous plants grown in vitro, and were representative of ∼20–30 seedlings. PCR amplifications were performed over 25 cycles.

Figure 4. Phenotypic analysis of various tic22 single- and double-mutant plants.

A, B. Homozygous plants of the indicated genotypes were grown side-by-side, in vitro, for five days (A), or for 14 days prior to transferral to soil and growth for an additional 10 days (B). Representative plants are shown. C. Chlorophyll concentrations in 5, 7, 10 and 24-day-old plants of the indicated genotypes are shown. The plants were grown in vitro or on soil as described in A, under identical conditions. Values shown are means (±SE) derived from measurements of 12–24 different samples in each case. Units are nmol chlorophyll a+b per mg fresh weight, but the data have been normalized to the wild-type value at each developmental time-point.

Figure 5. Analysis of plastid ultrastructure in the tic22 single and double mutants.

A. Cotyledons of 5-day-old plants were analysed by transmission electron microscopy. On average, ∼20 whole-chloroplast micrographs from each of three (two in the case of tic22-IV-2 tic22-III-1) independent plants per genotype (a minimum of 50 chloroplasts per genotype) were analysed for each genotype, and used to select the representative images shown. Size bar = 1.0 µm. B. Cross sectional area (µm²) of chloroplasts from the cotyledons of wild type and the tic22 mutants was determined by the analysis of the micrographs described in A. At least 50 chloroplasts were measured for each genotype.

Figure 6. Chloroplast protein levels in the tic22 double mutants.

A. Equal protein samples (20 µg) from 5-day-old homozygous, MS-grown plants of the indicated genotypes were analysed by immunoblotting using the antibodies shown. LHCP, light-harvesting chlorophyll-binding protein; OE33, oxygen-evolving complex 33 kD subunit; FNR, ferredoxin-NADP reductase; CPO, coproporphyrinogen oxidase; H3, histone H3. B,C. Chloroplast protein bands in A (as well as those from other, similar experiments) were quantified using Aida software (Raytest), and then the data were normalized using equivalent H3 data. Values shown are means (±SE) derived from 3–8 independent measurements.

Figure 7. Analysis of chloroplast protein import efficiency in the tic22 double mutants.

A. Chloroplasts were isolated from 14-day-old, in vitro-grown plants of the indicated genotypes, and used in protein import assays with [³⁵S]-methionine-labelled Rubisco SSU preprotein. Import was allowed to proceed for 3, 6 and 10 minutes, as indicated, and then samples were analysed by SDS-PAGE and fluorography. TM indicates an aliquot of the SSU translation mixture equivalent to 10% of the amount added to each assay; p and m indicate the precursor and mature forms of SSU, respectively. B. Mature protein bands observed in A were quantified using ImageQuant software, and then the data were expressed as percentages of the value for the final, wild-type time-point. These data, together with those from two additional, similar experiments, were used to calculate the mean (±SD) values shown (n = 3).