Ramesh Patel

Functional Specialization amongst the Arabidopsis Toc159 Family of Chloroplast Protein Import Receptors

The initial stages of preprotein import into chloroplasts are mediated by the receptor GTPase Toc159. In Arabidopsis thaliana, Toc159 is encoded by a small gene family: atTOC159, atTOC132, atTOC120, and atTOC90. Phylogenetic analysis suggested that at least two distinct Toc159 subtypes, characterized by atToc159 and atToc132/atToc120, exist in plants. atTOC159 was strongly expressed in young, photosynthetic tissues, whereas atTOC132 and atTOC120 were expressed at a uniformly low level and so were relatively prominent in nonphotosynthetic tissues. Based on the albino phenotype of its knockout mutant, atToc159 was previously proposed to be a receptor with specificity for photosynthetic preproteins. To elucidate the roles of the other isoforms, we characterized Arabidopsis knockout mutants for each one. None of the single mutants had strong visible phenotypes, but toc132 toc120 double homozygotes appeared similar to toc159, indicating redundancy between atToc132 and atToc120. Transgenic complementation studies confirmed this redundancy but revealed little functional overlap between atToc132/atToc120 and atToc159 or atToc90. Unlike toc159, toc132 toc120 caused structural abnormalities in root plastids. Furthermore, when proteomics and transcriptomics were used to compare toc132 with ppi1 (a receptor mutant that is specifically defective in the expression, import, and accumulation of photosynthetic proteins), major differences were observed, suggesting that atToc132 (and atToc120) has specificity for nonphotosynthetic proteins. When both atToc159 and the major isoform of the other subtype, atToc132, were absent, an embryo-lethal phenotype resulted, demonstrating the essential role of Toc159 in the import mechanism.

The Arabidopsis ppi1 Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic Proteins

The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34. Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1. Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1. Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins.

A Molecular-Genetic Study of the Arabidopsis Toc75 Gene Family

Toc75 (translocon at the outer envelope membrane of chloroplasts, 75 kD) is the protein translocation channel at the outer envelope membrane of plastids and was first identified in pea (Pisum sativum) using biochemical approaches. The Arabidopsis (Arabidopsis thaliana) genome contains three Toc75-related sequences, termed atTOC75-I, atTOC75-III, and atTOC75-IV, which we studied using a range of molecular, genetic, and biochemical techniques. Expression of atTOC75-III is strongly regulated and at its highest level in young, rapidly expanding tissues. By contrast, atTOC75-IV is expressed uniformly throughout development and at a much lower level than atTOC75-III. The third sequence, atTOC75-I, is a pseudogene that is not expressed due to a gypsy/Ty3 transposon insertion in exon 1, and numerous nonsense, frame-shift, and splice-junction mutations. The expressed genes, atTOC75-III and atTOC75-IV, both encode integral envelope membrane proteins. Unlike atToc75-III, the smaller atToc75-IV protein is not processed upon targeting to the envelope, and its insertion does not require ATP at high concentrations. The atTOC75-III gene is essential for viability, since homozygous atToc75-III knockout mutants (termed toc75-III) could not be identified, and aborted seeds were observed at a frequency of approximately 25% in the siliques of self-pollinated toc75-III heterozygotes. Homozygous toc75-III embryos were found to abort at the two-cell stage. Homozygous atToc75-IV knockout plants (termed toc75-IV) displayed no obvious visible phenotypes. However, structural abnormalities were observed in the etioplasts of toc75-IV seedlings and atTOC75-IV overexpressing lines, and toc75-IV plants were less efficient at deetiolation than wild type. These results suggest some role for atToc75-IV during growth in the dark.

Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis thaliana

Toc64/OEP64 was identified biochemically in pea as a putative component of the chloroplast protein import apparatus. In Arabidopsis, three paralogous genes (atTOC64-III, atTOC64-V and atTOC64-I) encode Toc64-related proteins, and these have been reported to localize in chloroplasts, mitochondria and the cytosol, respectively. To assess the role of the atToc64-III protein in chloroplast protein import in an in vivo context, we identified and characterized Arabidopsis knockout mutants. The absence of detectable defects in toc64-III single mutants raised the possibility of redundancy, and prompted us to also identify toc64-V and toc64-I mutants, cross them to toc64-III, and generate double- and triple-mutant combinations. The toc64 mutants were analysed carefully with respect to a variety of criteria, including chlorophyll accumulation, photosynthetic performance, organellar ultrastructure and chloroplast protein accumulation. In each case, the mutant plants were indistinguishable from wild type. Furthermore, the efficiency of chloroplast protein import was not affected by the toc64 mutations, even when a putative substrate of the atToc64-III protein (wheatgerm-translated precursor of the 33 kDa subunit of the oxygen-evolving complex, OE33) was examined. Moreover, under various stress conditions (high light, osmotic stress and cold), the toc64 triple-mutant plants were not significantly different from wild type. These results demonstrate that Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis, and draw into question the functional significance of this component.

The Omp85-Related Chloroplast Outer Envelope Protein OEP80 Is Essential for Viability in Arabidopsis

β-Barrel proteins of the Omp85 (Outer membrane protein, 85 kD) superfamily exist in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Prominent Omp85 proteins in bacteria and mitochondria mediate biogenesis of other β-barrel proteins and are indispensable for viability. In Arabidopsis (Arabidopsis thaliana) chloroplasts, there are two distinct types of Omp85-related protein: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD) and OEP80 (Outer Envelope Protein, 80 kD). Toc75 functions as a preprotein translocation channel during chloroplast import, but the role of OEP80 remains elusive. We characterized three T-DNA mutants of the Arabidopsis OEP80 (AtOEP80) gene. Selectable markers associated with the oep80-1 and oep80-2 insertions segregated abnormally, suggesting embryo lethality of the homozygous genotypes. Indeed, no homozygotes were identified among >100 individuals, and heterozygotes of both mutants produced approximately 25% aborted seeds upon self-pollination. Embryo arrest occurred at a relatively late stage (globular embryo proper) as revealed by analysis using Nomarski optics microscopy. This is substantially later than arrest caused by loss of the principal Toc75 isoform, atToc75-III (two-cell stage), suggesting a more specialized role for AtOEP80. Surprisingly, the oep80-3 T-DNA (located in exon 1 between the first and second ATG codons of the open reading frame) did not cause any detectable developmental defects or affect the size of the AtOEP80 protein in chloroplasts. This indicates that the N-terminal region of AtOEP80 is not essential for the targeting, biogenesis, or functionality of the protein, in contrast with atToc75-III, which requires a bipartite targeting sequence.

Molecular and genetic analyses of Tic20 homologues in Arabidopsis thaliana chloroplasts

The Tic20 protein was identified in pea (Pisum sativum) as a component of the chloroplast protein import apparatus. In Arabidopsis, there are four Tic20 homologues, termed atTic20-I, atTic20-IV, atTic20-II and atTic20-V, all with predicted topological similarity to the pea protein (psTic20). Analysis of Tic20 sequences from many species indicated that they are phylogenetically unrelated to mitochondrial Tim17-22-23 proteins, and that they form two evolutionarily conserved subgroups [characterized by psTic20/atTic20-I/IV (Group 1) and atTic20-II/V (Group 2)]. Like psTic20, all four Arabidopsis proteins have a predicted transit peptide consistent with targeting to the inner envelope. Envelope localization of each one was confirmed by analysis of YFP fusions. RT-PCR and microarray data revealed that the four genes are expressed throughout development. To assess the functional significance of the genes, T-DNA mutants were identified. Homozygous tic20-I plants had an albino phenotype that correlated with abnormal chloroplast development and reduced levels of chloroplast proteins. However, knockouts for the other three genes were indistinguishable from the wild type. To test for redundancy, double and triple mutants were studied; apart from those involving tic20-I, none was distinguishable from the wild type. The tic20-I tic20-II and tic20-I tic20-V double mutants were albino, like the corresponding tic20-I parent. 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 complementation analysis. Thus, atTic20-I and atTic20-IV are the major functional Tic20 isoforms in Arabidopsis, with partially overlapping roles. While the Group 2 proteins have been conserved over approximately 1.2u2003billion (1.2u2003×u200310(9) ) years, they are not essential for normal development.

In vivo assessment of the significance of phosphorylation of the Arabidopsis chloroplast protein import receptor, atToc33

atToc33 is a transit peptide receptor of the chloroplast outer envelope membrane, and possesses GTPase activity. In vitro, its transit peptide‐ and GTP‐binding properties are abrogated by its phosphorylation at serine 181, which was proposed to represent an important regulatory mechanism. We mutated S181 to alanine (to prevent phosphorylation), and to aspartate and glutamate (to mimic the effects of phosphoserine), and expressed all three proteins in ppi1 (atToc33 knockout) plants using the native promoter. The mutants complemented ppi1 with equal efficiency in respect of all criteria tested, including protein import efficiency and light stress tolerance. The data suggest that atToc33 phosphorylation may not play an important role in vivo.

Nucleotide binding and dimerization at the chloroplast pre-protein import receptor, atToc33, are not essential in vivo but do increase import efficiency

The atToc33 protein is one of several pre-protein import receptors in the outer envelope of Arabidopsis chloroplasts. It is a GTPase with motifs characteristic of such proteins, and its loss in the plastid protein import 1 (ppi1) mutant interferes with the import of photosynthesis-related pre-proteins, causing a chlorotic phenotype in mutant plants. To assess the significance of GTPase cycling by atToc33, we generated several atToc33 point mutants with predicted effects on GTP binding (K49R, S50N and S50N/S51N), GTP hydrolysis (G45R, G45V, Q68A and N101A), both binding and hydrolysis (G45R/K49N/S50R), and dimerization or the functional interaction between dimeric partners (R125A, R130A and R130K). First, a selection of these mutants was assessed in vitro, or in yeast, to confirm that the mutations have the desired effects: in relation to nucleotide binding and dimerization, the mutants behaved as expected. Then, activities of selected mutants were tested in vivo, by assessing for complementation of ppi1 in transgenic plants. Remarkably, all tested mutants mediated high levels of complementation: complemented plants were similar to the wild type in growth rate, chlorophyll accumulation, photosynthetic performance, and chloroplast ultrastructure. Protein import into mutant chloroplasts was also complemented to >50% of the wild-type level. Overall, the data indicate that neither nucleotide binding nor dimerization at atToc33 is essential for chloroplast import (in plants that continue to express the other TOC receptors in native form), although both processes do increase import efficiency. Absence of atToc33 GTPase activity might somehow be compensated for by that of the Toc159 receptors. However, overexpression of atToc33 (or its close relative, atToc34) in Toc159-deficient plants did not mediate complementation, indicating that the receptors do not share functional redundancy in the conventional sense.

In vivo Studies on the Roles of Tic55-Related Proteins in Chloroplast Protein Import in Arabidopsis thaliana

The Tic55 (Translocon at the inner envelope membrane of chloroplasts, 55 kDa) protein was identified in pea as a putative regulator, possibly linking chloroplast protein import to the redox state of the photosynthetic machinery. Two Tic55 homologs have been proposed to exist in Arabidopsis: atTic55-II and AtPTC52 (Protochlorophyllide-dependent Translocon Component, 52 kDa; has also been called atTic55-IV). Our phylogenetic analysis shows that atTic55-II is an ortholog of psTic55 from pea (Pisum sativum), and that AtPTC52 is a more distant homolog of the two. AtPTC52 was included in this study to rule out possible functional links between the proteins in Arabidopsis. No detectable mutant phenotypes were found in two independent T-DNA knockout mutant plant lines for each Arabidopsis protein, when compared with wild-type: visible appearance, chlorophyll content, photosynthetic performance, and chloroplast protein import, for example, were all normal. Both wild-type and tic55-II mutant chloroplasts exhibited deficient protein import when treated with diethylpyrocarbonate, indicating that Tic55 is not the sole target of this reagent in relation to protein import. Furthermore, ptc52 mutant chloroplasts were not defective with respect to pPORA import, which was previously reported to involve PTC52 in barley. Thus, we conclude that atTic55-II and AtPTC52 are not strictly required for functional protein import in Arabidopsis.

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

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.