A stromal Hsp100 protein is required for normal chloroplast development and function in Arabidopsis

Abstract

Molecular chaperones are required for the translocation of many proteins across organellar membranes, presumably by providing energy in the form of ATP hydrolysis for protein movement. In the chloroplast protein import system, a heat shock protein 100 (Hsp100), known as Hsp93, is hypothesized to be the chaperone providing energy for precursor translocation, although there is little direct evidence for this hypothesis. To learn more about the possible function of Hsp93 during protein import into chloroplasts, we isolated knockout mutant lines that contain T-DNA disruptions in either atHSP93-V or atHSP93-III, which encode the two Arabidopsis (Arabidopsis thaliana) homologs of Hsp93. atHsp93-V mutant plants are much smaller and paler than wild-type plants. In addition, mutant chloroplasts contain less thylakoid membrane when compared to the wild type. Plastid protein composition, however, seems to be largely unaffected in atHsp93-V knockout plants. Chloroplasts isolated from the atHsp93-V knockout mutant line are still able to import a variety of precursor proteins, but the rate of import of some of these precursors is significantly reduced. These results indicate that atHsp93-V has an important, but not essential, role in the biogenesis of Arabidopsis chloroplasts. In contrast, knockout mutant plants for atHsp93-III, the second Arabidopsis Hsp93 homolog, had a visible phenotype identical to the wild type, suggesting that atHsp93-III may not play as important a role as atHsp93-V in chloroplast development and/or function.

Figure 1.

Characterization of the insertional mutations in atHsp93-V and atHsp93-III knockout lines. A, Schematic depicting the structure, from the start codon to the stop codon, of the atHSP93-V gene. Exons are represented by gray boxes; introns are symbolized by thin lines. The approximate locations of the T-DNA insertions in the two knockout mutant lines described in this report are indicated. The second T-DNA insertion shown (in the last exon) is found in the line used for all subsequent analyses. B, RT-PCR analysis for atHSP93-V. Primers specific to either the 5′ end of the atHSP93-V gene, upstream of the T-DNA insertion, or the 3′ end of the gene, downstream of the T-DNA insertion, were used on mRNA isolated from wild-type (WT) and atHsp93-V knockout (KO) mutant plants. C, Immunoblot analysis for Hsp93 proteins. Total leaf protein from 4-week-old soil-grown plants (wild-type [lane 1] and atHsp93-V mutant [lane 2] lines) was extracted by boiling tissue samples in SDS and β-mercaptoethanol. Protein extract equivalent to equal amounts of starting fresh weight was separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Hsp93 proteins. Intact chloroplasts were isolated from 4-week-old wild-type (lane 3) and atHsp93-V mutant (lane 4) plants that had been grown on plates. Total chloroplasts equivalent to 10 μg of chlorophyll were separated by electrophoresis and immunoblotted with antiserum against Hsp93 proteins. A possible truncated protein produced by the atHSP93-V gene in mutant plants is indicated (*). D, Schematic for the atHSP93-III gene (from the start codon to the stop codon), indicating the location of a T-DNA insertion in the knockout mutant line. E, RT-PCR analysis for the atHSP93-III gene. Primers specific to the region of the gene upstream of the T-DNA insert (5′ primers) or to the region of atHSP93-III downstream of the insert (3′ primers) were used to analyze mRNA isolated from either wild-type (WT) or atHsp93-III knockout (KO) plants.

Figure 2.

atHsp93-V knockout mutant plants are much smaller and paler than wild-type plants, but atHsp93-III mutant plants appear similar to the wild type. Wild-type (A and B), atHsp93-V knockout mutant (C and D), and atHsp93-III mutant (E and F) plants were grown on soil in 12-h days (12 h light:12 h dark; approximately 70–80 μmol m⁻² s⁻¹). Individual plants were photographed at 2 weeks (E), 3 weeks (A and C), and 4 weeks (B, D, and F) after germination. The atHsp93-III mutant individuals (E and F) were grown at a different time than the other plants shown in this figure.

Figure 3.

The leaves of atHsp93-V mutant plants contain reduced levels of chlorophyll. Chlorophyll was extracted in 80% acetone from wild-type (WT; square) and atHsp93-V mutant (KO; triangle) plants and quantified as described in “Materials and Methods.” Values shown are the average of four measurements.

Figure 4.

Chloroplasts isolated from atHsp93-V mutant plants are slightly smaller and contain less thylakoid membrane in comparison to wild-type chloroplasts. Leaf tissue was isolated from soil-grown wild-type (A and C) and atHsp93-V mutant (B and D) plants at 6 d (A and B) and at 4 weeks (C and D) after germination and prepared for transmission electron microscopy as described in “Materials and Methods.” Scale bar = 1 μm.

Figure 5.

Endogenous levels of various chloroplastic proteins are unaffected in the atHsp93-V knockout line. Chloroplasts were isolated from 4-week-old plate-grown wild-type (WT) and atHsp93-V knockout (KO) mutant seedlings. Total chloroplast protein equivalent to 10 μg chlorophyll was separated by SDS-PAGE and analyzed by immunoblotting, using antibodies against the proteins listed. Toc, Translocon at the outer envelope membrane of chloroplasts; Tic, translocon at the inner envelope membrane of chloroplasts. The number following the Toc or Tic designation refers to the molecular mass of the specified component. S78, A stromal Hsp70 molecular chaperone; AOS, allene oxide synthase, an enzyme in the jasmonic acid biosynthetic pathway; FtsZ1, a plastid division protein; IEP35, an integral protein of the chloroplast inner envelope membrane; BCCP, a protein involved in lipid biosynthesis; PC, a thylakoid lumen protein.

Figure 6.

Chloroplasts isolated from atHsp93-V mutant plants are able to import a variety of precursor proteins, although not as efficiently as do wild-type chloroplasts. ³⁵S-labeled prSS (a stromal protein), ³⁵S-labeled prLHCP (a thylakoid membrane protein), ³⁵S-labeled prPC (a thylakoid lumen-localized protein), and ³⁵S-labeled tp110-110N (an inner membrane-localized import component) were imported into chloroplasts (25 μg chlorophyll) isolated from wild-type or atHsp93-V mutant plants. After 10 or 20 min at room temperature in the light, import was stopped, and intact chloroplasts were recovered, by centrifuging the reactions through a 40% Percoll cushion. Equivalent amounts of chlorophyll from each sample were analyzed by SDS-PAGE and fluorography. The amount of radiolabeled precursor proteins imported into the chloroplasts was quantified using a phosphoimager. Values presented depict the amount of precursor imported into atHsp93-V mutant chloroplasts as a percentage of the amount imported into wild-type chloroplasts and are the average of at least two independent experiments.

Figure 7.

The rate of import of prSS into atHsp93-V mutant chloroplasts is decreased in comparison to import into chloroplasts isolated from wild-type plants. A, ³⁵S-labeled prSS was imported into chloroplasts isolated from wild-type (WT) or atHsp93-V knockout (KO) mutant plants. At the times indicated, aliquots equivalent to 25 μg of chlorophyll were removed from the import reactions. Import was stopped, and intact chloroplasts were recovered, by centrifuging the aliquots through a 40% Percoll cushion. Equivalent amounts of chlorophyll from each sample were analyzed by SDS-PAGE and fluorography. TP, 1/10 volume of radioactive translation product added to each sample. SS, Mature form of prSS. B, Results from A (wild type [WT] = square; atHsp93-V knockout [KO] = triangle) were quantified using a phosphoimager. Values depicted are the average of three independent import reactions.