A molecular genetic study of Tic20 and Tic22 homologues in Arabidopsis thaliana

In this study Arabidopsis thaliana was used as a plant model to investigate the involvement of Tic20 and Tic22 (translocon at the inner envelope membrane of chloroplasts, 20 kD and 22 kD, respectively) in chloroplast protein import. In Arabidopsis, there are four Tic20 homologues and two Tic22 homologues, all with predicted similarity to the corresponding pea protein (psTic20 or psTic22). Phylogenetic analyses revealed two, evolutionarily conserved sub-types of both Tic20 and Tic22, termed Group 1 and Group 2 in each case. TargetP analysis was used to predict the subcellular localization of all Arabidopsis Tic20 and Tic22 proteins to the chloroplast. Moreover, the TMHMM program was used to identify the transmembrane domains of the atTic20 proteins; all atTic20 homologues have four predicted transmembrane α-helices, like psTic20. To test the TargetP predictions, envelope localization of each protein was tested by transiently expressing YFP fusions in Arabidopsis protoplasts. Furthermore, quantitative RT-PCR (and Genevestigator) revealed that all atTIC20 homologues are expressed throughout development; atTIC20-I expression was highest in photosynthetic tissues, whereas atTIC20-IV expression was strong in nonphotosynthetic tissues and seeds. Quantitative RT-PCR also revealed that atTIC22-IV expression is higher than that of atTIC22-III. To assess functional significance of the six genes in vivo, T-DNA mutants were identified. Homozygous tic20-I-1 and tic20-I-2 plants have an albino phenotype correlated with abnormal chloroplast development. To test for functional redundancy, various tic20 double and triple mutants were studied; apart from those involving tic20-I, these were all phenotypically similar to wild type. In contrast, tic20-I tic20-IV double homozygotes could not be identified, due to gametophytic and embryonic lethality. Redundancy between atTic20-I and atTic20-IV was confirmed by partial complementation of tic20-I by atTIC20-IV overexpression. Additionally, tic22-IV tic22-III double mutants had a pale phenotype in early plant development, indicating redundancy between atTic22-IV and atTic22-III and a role during early chloroplast biogenesis.