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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Arabidopsis Stromal 70-kD Heat Shock Proteins Are Essential for Plant Development and Important for Thermotolerance of Germinating Seeds^{1,[C],[W],[OA]}

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The 70-kD heat shock proteins (Hsp70s) have been shown to be important for protein folding, protein translocation, and stress responses in almost all organisms and in almost all subcellular compartments. However, the function of plastid stromal Hsp70s in higher plants is still uncertain. Genomic surveys have revealed that there are two putative stromal Hsp70s in Arabidopsis thaliana, denoted cpHsc70-1 (At4g24280) and cpHsc70-2 (At5g49910). In this study, we show that cpHsc70-1 and cpHsc70-2 could indeed be imported into the chloroplast stroma. Their corresponding T-DNA insertion knockout mutants were isolated and designated as cphsc70-1 and cphsc70-2. No visible phenotype was observed in the cphsc70-2 mutant under normal growth conditions. In contrast, cphsc70-1 mutant plants exhibited variegated cotyledons, malformed leaves, growth retardation, and impaired root growth, even though the protein level of cpHsc70-2 was up-regulated in the cphsc70-1 mutant. After heat shock treatment of germinating seeds, root growth from cphsc70-1 seeds was further impaired, indicating that cpHsc70-1 is important for thermotolerance of germinating seeds. No cphsc70-1 cphsc70-2 double mutant could be obtained, suggesting that the cphsc70 double knockout was lethal. Genotype analyses of F₁ seedlings from various crosses indicated that double-knockout mutation was lethal to the female gametes and reduced the transmission efficiency of the male gametes. These results indicate that cpHsc70s are essential for plant development and the two cpHsc70s most likely have redundant but also distinct functions.

In comparison, the function of higher plant plastid Hsp70 is still uncertain. Two plastid Hsp70 proteins, Com70 and IAP70, localized at the outer envelope membrane of pea chloroplasts, have been proposed to be involved in protein import into chloroplasts (Schnell et al., 1994; Kourtz and Ko, 1997). However, their molecular function and identity are still unknown. Nielsen et al. (1997) found that the stromal Hsp70 of pea (Pisum sativum) chloroplasts, S78, could be coimmunoprecipitated with importing precursor proteins, but S78 did not associate with the Toc/Tic (translocon at the outer and inner envelope membranes of chloroplasts) translocon complex that is responsible for protein import into chloroplasts. On the other hand, in vitro binding assays showed physical interactions between S78 and recombinant transit peptides of precursors to the small subunit of Rubisco (prRBCS; Ivey et al., 2000). However, precursors with altered affinities for Hsp70 in their transit peptides are still efficiently imported into chloroplasts (Rial et al., 2003, 2006). Therefore, whether stromal Hsp70s are involved in chloroplast protein import requires further studies. In the green algae Chlamydomonas and Dunaliella, chloroplast stromal Hsp70B has been shown to function in PSII protection and repair during and after photoinhibition (Schroda et al., 1999; Yokthongwattana et al., 2001). Chlamydomonas stromal Hsp70B regulates the assembly state of VIPP1 (vesicle inducing protein in plastid 1), which may be important for thylakoid biogenesis or maintenance (Liu et al., 2005, 2007). It is not known whether higher plant stromal Hsp70 has any similar function.

Genomic surveys revealed that there are 14 genes encoding Hsp70s in Arabidopsis (Sung et al., 2001a). Among them, two genes encode putative plastid stromal Hsp70s, cpHsc70-1 (At4g24280), and cpHsc70-2 (At5g49910). They harbor a predicted chloroplast-targeting transit peptide and a C-terminal motif (PEGDVIDADFTDSK) conserved among plastid Hsp70s (Guy and Li, 1998). Their polypeptide sequences share a 90.7% identity. Although reverse transcription (RT)-PCR analyses indicated that the amplified signal for cpHsc70-1 was very low and the transcripts of cpHsc70-2 was much higher than cpHsc70-1 in all organs (Sung et al., 2001b), according to the public microarray and MPSS databases (Brenner et al., 2000 [MPSS, http://mpss.udel.edu/at/]; Zimmermann et al., 2004 [Genevestigator, http://www.genevestigator.ethz.ch/]; Toufighi et al., 2005 [Botany Array Resource, http://bbc.botany.utoronto.ca/]), both transcripts are quite abundant in almost all tissues, with the level of cpHsc70-1 slightly higher than cpHsc70-2 in most tissues.

In this report, we show that the two putative plastid Hsp70s were indeed imported into the chloroplast stroma. T-DNA insertion knockout mutants of the two genes were analyzed. Our data indicate that the stromal Hsp70s are important for plant development under both normal and heat-stress conditions.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Arabidopsis cpHsc70-1 and cpHsc70-2 Could Be Imported into the Stroma of Chloroplasts

To verify the plastid localization of Arabidopsis cpHsc70-1 and cpHsc70-2, [³⁵S]Met-labeled cpHsc70-1 and cpHsc70-2 were synthesized by in vitro translation and incubated with isolated pea chloroplasts. As shown in Figure 1 , the precursor form of both cpHsc70-1 and cpHsc70-2 was about 80 kD. After import, a mature protein of approximately 71 kD was produced, suggesting the cleavage of a transit peptide. The imported mature cpHsc70s were present in the soluble fraction of chloroplasts (lane 4) and were resistant to thermolysin (data not shown) and trypsin digestion (lanes 2 and 3). Trypsin can penetrate the outer but not the inner envelope membrane (Jackson et al., 1998), and resulted in complete digestion of the outer membrane protein Toc75 but not the thylakoid membrane protein chlorophyll a/b-binding protein of PSII (CAB; Fig. 1). These data suggested that the two cpHsc70s were located in the chloroplast stroma. It is less likely that they were located in the thylakoid lumen because their transit peptides do not resemble the typical bipartite luminal-targeting sequence (Gutensohn et al., 2006).

View larger version (51K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Arabidopsis cpHsc70s were imported into the stroma of isolated chloroplasts. [35S]Met-labeled precursors of cpHsc70-1 and cpHsc70-2 were incubated with isolated pea chloroplasts under import conditions. After 30 min, chloroplasts were pelleted and digested with the indicated concentration of trypsin. Intact chloroplasts were recovered through a 40% Percoll cushion after the digestion (lanes 1–3), lysed hypotonically, and separated into soluble (sup) and membrane (ppt) fractions (lanes 4 and 5). An equal amount of chloroplasts was loaded in lanes 1 to 3 and the soluble and membrane proteins from the same amount of chloroplasts were loaded in lanes 4 and 5. Samples were analyzed by SDS-PAGE and fluorography. -Toc75 and -CAB are the same set of samples decorated with antibodies against Toc75 and CAB on immunoblots. M, Molecular mass markers; TR, in vitro-translated cpHsc70 precursor proteins before import.

To study the function of cpHsc70s, we obtained their putative T-DNA insertion mutants, SALK_140810 and SALK_095715, from the SALK T-DNA collection (Alonso et al., 2003). The T-DNA insertion sites were confirmed by genomic PCR and DNA sequencing. As shown in Figure 2A , both SALK_140810 and SALK_095715 have a T-DNA insertion in the intron II of cpHsc70-1 and cpHsc70-2, respectively. RT-PCR analyses indicated that both T-DNA insertion lines were null mutants (Fig. 2B). We designated these two mutants as cphsc70-1 (SALK_140810) and cphsc70-2 (SALK_095715). Genomic DNA analyses further indicated that both cphsc70-1 and cphsc70-2 contained T-DNA insertion in a single locus and the variegated-cotyledon phenotype cphsc70-1 (see below) was linked to the T-DNA insertion (Supplemental Fig. S1; Supplemental Table S1).

View larger version (42K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Confirmation of the T-DNA insertion lines for cpHsc70s. A, Schematic representation for the genomic fragments of cpHsc70-1 (At4g24280) and cpHsc70-2 (At5g49910). Black boxes and lines represent exons and introns, respectively. ATG represents the translation initiation sites. The locations of the T-DNA insertions in cphsc70-1 and cphsc70-2 mutants are illustrated. Positions and directions of primers used in B are also indicated. B, cphsc70-1 and cphsc70-2 are null mutants. Primer sets for cpHsc70-1 are: 1E2-S + 1E8-AS for wild-type copy of cpHsc70-1 DNA and RT-PCR of RNA, and SALK-Lba1 + 1E8-AS for cphsc70-1 T-DNA insertion. Primer sets for cpHsc70-2 are: 2E1-S + 2E8-AS for wild-type copy of cpHsc70-2 DNA and RT-PCR of RNA, and SALK-Lba1 + 2E8-AS for cphsc70-2 T-DNA insertion.

cphsc70-1 Plants Exhibited Variegated Cotyledons, Malformed Leaves, and Growth Retardation

The cotyledons of cphsc70-1 plants were variegated (Fig. 3A ). Furthermore, in the early vegetative stage, the true leaves of cphsc70-1 seedlings often had irregular leaf margins and small lesions (Fig. 3B). The mutant was also smaller than the wild type. At 15 d after germination, the fresh weight of cphsc70-1 plants was about 50% of wild-type plants (Fig. 3D). We also measured root growth on vertical plates. Root length of cphsc70-1 plants was also about 50% of wild type (Fig. 3, C and E). In contrast, there was no visible phenotype in the cphsc70-2 mutant. These results suggest that cpHsc70-1 is important for both shoot and root growth. Because there is only one mutant allele available for cphsc70-1, we tried to complement cphsc70-1 with a cpHsc70-1 genomic fragment to confirm that the mutant phenotypes were caused by the loss of cpHsc70-1. As shown in Figure 3, cphsc70-1 transformed with a 5-kb cpHsc70-1 genomic fragment (transformants designated as cpHsc70-1g) fully recovered the wild-type phenotype.

View larger version (50K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Phenotypes of the cphsc70 knockout mutants. A, cphsc70-1 mutants exhibited variegated cotyledons, which were complemented by a genomic fragment of cpHsc70-1 (cpHsc70-1g). Plants were grown on MS plates for 7 d. B, cphsc70-1 had a smaller size and malformed leaves (white arrowhead). Plants were grown on soil for 28 d. C, cphsc70-1 had shorter roots. Plants were grown vertically on an MS plate for 7 d. D, Growth curves of mutants and wild type. Fresh weights of plate-grown seedlings were measured. Values are the average ± SD of two independent experiments. For each experiment, three batches of 10 seedlings each were measured. E, Average root length of plants shown in C. Values are the average ± SD of three independent experiments. For each experiment, the root length of more than 10 seedlings was measured. F, cpHsc70 protein levels in the mutants and wild type. Total stromal proteins (40 µg) were analyzed by an IEF gel and immunoblotting decorated with anti-S78 antibody.

cpHsc70s Are Essential for Plant Development

As a first step toward investigating whether cpHsc70-1 and cpHsc70-2 had different or redundant functions, we tried to generate a cphsc70-1 cphsc70-2 double mutant. As shown in Table I , in the F₂ progenies of cphsc70-2 flowers crossed with cphsc70-1 pollen, no double-knockout seedling was identified. In the meantime, reduced seed sets in the siliques of F₁ plants were observed (Fig. 4A ). Siliques of F₁ plants from a cross of the reverse direction had the same reduced seed sets (Fig. 4A). In addition, plants with the genotype +/cphsc70-1 cphsc70-2/cphsc70-2 were extremely small (Fig. 4B). No plants with the genotypes cphsc70-1/cphsc70-1 +/cphsc70-2 were identified (Table I). To confirm the absence of plants with this genotype, we crossed the cphsc70-1 single mutant flowers with pollen from plants with the genotype +/cphsc70-1 cphsc70-2/cphsc70-2 (Table II , third cross). Half of F₁ progenies were expected to have the genotype cphsc70-1/cphsc70-1 +/cphsc70-2. However, after analyzing 104 F₁ plants, all of them had the genotype heterozygous for both cphsc70s. This result indicated that plants with only one copy of cpHsp70-2 are not viable. This may suggest that a threshold level of cpHsc70 is critical for plant development, although the cause of this lethality is currently unknown.

View this table: [in this window] [in a new window]   Table I. Progeny genotypes of the cross cphsc70-2 x cphsc70-1

View larger version (41K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. The two cpHsc70s together are essential for plant development. A, Siliques produced by plants of the genotype +/cphsc70-1 +/cphsc70-2 showed reduced seed sets compared to the wild type. Silique I is from an F1 plant produced by crossing cphsc70-1 flowers with cphsc70-2 pollen. Silique II is from an F1 plant of a cross of the reverse direction. Siliques were bleached overnight before photographs were taken. B, A plant with the genotype +/cphsc70-1 cphsc70-2/cphsc70-2 exhibited severely stunted growth in comparison to cphsc70-1 and cphsc70-2 single mutants and wild type. Plants are 28-d-old plate-grown seedlings. [See online article for color version of this figure.]

Effect of Individual cphsc70 Mutations on Chloroplast Protein Import

We next tested whether the cphsc70 knockout mutants had reduced protein import efficiency. As shown in Figure 5, A and B , chloroplasts isolated from adult mutant plants did not show impaired prRBCS import compared to the wild type. Because cphsc70-1 had the most apparent phenotype in cotyledons, we further compared protein import efficiency of chloroplasts isolated from 7-d-old seedlings, in which cotyledons represent the majority of green tissue. As shown in Figure 5C, cphsc70-1 chloroplasts had a greatly reduced amount of imported mature RBCS. However, noticeably the amount of envelope-associated prRBCS was also greatly reduced. We, therefore, further analyzed association of prRBCS with the chloroplast surface when no or little ATP was present in the import system. As shown in Figure 5D, fewer prRBCS associated with the cphsc70-1 mutant chloroplast surface in the absence of ATP. This result suggested that the cphsc70-1 mutation might have some secondary effects that caused damage to the chloroplast surface when isolated, which resulted in less efficient precursor association with the chloroplast. However, the amount of major translocon components like Toc159, Toc75, and Tic110 was not reduced in cphsc70-1 compared to the wild type (Supplemental Fig. S2). Therefore, the reason for the reduced precursor association of cphsc70-1 chloroplasts is not clear.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. In vitro import assays using chloroplasts isolated from mutants and wild type. A, [35S]Met-labeled prRBCS was imported into chloroplasts isolated from 30-d-old plate-grown plants. At the time point indicated, aliquots of the import reaction were removed and centrifuged through a 40% Percoll cushion. An equal amount of proteins from each sample was analyzed by SDS-PAGE and fluorography. B, Quantification of the amount of imported mature RBCS from the experiment shown in A. C, Import of [35S]Met-labeled prRBCS into chloroplast isolated from 7-d-old seedlings. Import was performed for 15 min in the light with 3 mM ATP. The amount of CAB, as determined by silver staining, was used as a loading control. D, Association of prRBCS with the chloroplast surface in the absence of ATP. Chloroplasts were isolated from 7-d-old seedlings. ATP-depleted precursors and chloroplasts were incubated in the dark for 15 min. The amount of the large subunit of Rubisco (RBCL) was used as a loading control.

Many Hsp70s have been shown to be important for protection of organisms against heat stress. Expression of Arabidopsis cpHsc70-1 and pea stromal Hsp70 S78 were 5 and 9 times higher, respectively, after heat shock (Marshall and Keegstra, 1992; Busch et al., 2005). We, therefore, investigated if cpHsc70s were involved in plant thermotolerance. In acquired thermotolerance tests (Fig. 6A ), in which plants were acclimated at 37.5°C before they underwent heat shock at 44.5°C, the survival rate of the two cphsc70 mutants was similar to the wild type. In comparison, the hot1 mutant (SALK_099583c), which has a T-DNA insertion in the gene-encoding cytosolic ClpB (At1g74310), was totally killed. Interestingly, both cphsc70-1 and cphsc70-2 exhibited pale-green leaves after recovery. When the chlorophyll content was determined, both mutants had a reduced amount of total chlorophylls compared to the wild-type and cpHsc70-1g plants (Fig. 6B). For basal thermotolerance tests, seedlings were directly heated at 44.5°C for 30 or 40 min, and then allowed to recover for 7 d. Almost all seedlings, mutants and wild type alike, were killed by the 40-min heat shock, but all survived the 30-min heat shock (Fig. 6C). Again the cphsc70 mutants had a reduced chlorophyll content after the recovery (Fig. 6D).

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Acquired and basal thermotolerance analyses. A, Seedlings were heated to 37.5°C for 1 h, returned to 22°C for 2 d, heated to 44.5°C for 1 h, and then allowed to recover for 7 d. B, Quantification of seedling chlorophyll contents after the 7-d recovery as shown in A. Group I did not undergo any heat treatment. Group II was only acclimated at 37.5°C for 1 h and did not undergo the 44.5°C 1-h heat shock. C, Seedlings 7-d-old were directly heated at 44.5°C for the amount of time indicated. After the heat shock treatments, seedlings were allowed to recover for 7 d. D, Quantification of chlorophyll contents of seedlings that went through the 30-min heat shock treatment in C. [See online article for color version of this figure.]

View larger version (49K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. cphsc70-1 had reduced seed thermotolerance. A, Seeds were sown on MS plates. After a 3-d cold stratification, plates were heated at 44.5°C for 150 min. Plates were then placed vertically and seeds were allowed to germinate for 7 d. Unheated controls were the same as those shown in Figure 3C. B, Root length of plants from heated seeds as shown in A, and unheated control seeds as shown in Figure 3C, was measured. The ratio of heated to unheated plants of the same genotype was plotted. The average of two independent experiments ± SD is shown. [See online article for color version of this figure.]

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

cphsc70-1

However, in the cphsc70-1 mutant, cpHsp70-2 was up-regulated to a level higher than the two cpHsc70s combined in the wild type (Fig. 3F; Supplemental Fig. S2) and cphsc70-1 still showed clear growth defects. This result suggests that cpHsc70-1 may have some specific functions that cannot be substituted by cpHsc70-2. Therefore it is most likely that the two cpHsc70s have overlapping but distinct functions. The two cpHsc70s share more than a 95% identity in both the ATPase domain and the substrate-binding domain, but only a 63% identity in the C-terminal 5-kD subdomain. The C terminus of Hsc70s has been shown to be important for interacting with cochaperones like Hop (Hsp70/Hsp90 organizing protein). It is possible that the two cpHsc70s may interact with different cochaperones, which lead them to different substrates.

It is interesting that there are also two putative stromal Hsp70s in the fully sequenced genomes of other land plants, such as moss (Physcomitrella spp.), poplar (Populus spp.), rice (Oryza sativa), and sorghum (Sorghum bicolor; Supplemental Table S2), but green algae only harbor a single cpHsp70. A second copy of cpHsc70 may be beneficial to cope with the more stressful and variable environment on land. However, phylogenetic analyses (Fig. 8 ) suggested that land-plant cpHsc70s were duplicated recently during evolution because the two copies of cpHsc70s could not be grouped into two protein subfamilies among different plant families. In most cases, the two copies from the same family are more similar to each other than to cpHsc70s from other families, although the two monocot cpHsc70s have a more divergent relationship. Experiments are required to first define the elements for specific expression of the two genes and then to perform promoter swap experiments to determine the functional specificity/redundancy of the two proteins.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Phylogenetic analysis of plastid Hsp70s. The amino acid sequences of plastid Hsp70s were aligned (Supplemental Fig. S3) and used to construct a gene tree by the Neighbor-Joining method with PHYLIP program version 3.67 (Felsenstein, 1989). The Dayhoff-PAM model was employed to compute the distance matrix, and Synechocystis DnaK2 (SynDnaK2) was set as an out-group. The branch length is proportional to the difference between protein sequences at the beginning and end of the branch. The bootstrap values out of 1,000 trees were given beside the branches. Syn, Synechocystis sp.; At, Arabidopsis; Br, Brassica rapa; Cm, Cyanidioschyzon merolae; Cr, Chlamydomonas reinhardtii; Ds, Dunaliella salina; Mt, Medicago truncatula; Os, Oryza sativa; Pg, Pennisetum glaucum; Pp, Physcomitrella patens; Ps, Pisum sativum; Pt, Populus trichocarpa; Sb, Sorghum bicolor; Vc, Volvox carteri. More information on accession numbers and sources of sequences is given in Supplemental Table S2.

Heat shock proteins have been shown to play important roles in helping cells to cope with environmental stress. The cytosolic Hsp100/ClpB plays major roles in acquired thermotolerance (Hong and Vierling, 2000, 2001; Nieto-Sotelo et al., 2002; Hong et al., 2003; Katiyar-Agarwal et al., 2003). Cytosolic Hsp70 and plastid ClpB and small heat shock proteins have also been implicated in heat sensitivity of plants (Lee and Schöffl, 1996; Sung and Guy, 2003; Wang and Luthe, 2003; Miroshnichenko et al., 2005; Myouga et al., 2006; Yang et al., 2006; Lee et al., 2007). However, no such role has been proposed for plastid Hsp70s. Here we have provided genetic evidence to show that Hsp70s in the plastid stroma are important for thermotolerance of germinating seeds, indicating that plastid physiology is important for seeds to endure heat stress. Plastids are involved in the assimilation of nitrogen and in the syntheses of lipids and many hormones, which are important for seed germination and root growth. It is therefore reasonable to have chaperones protecting the biosynthesis enzymes during heat stress. Accumulation of toxic intermediates due to heat inactivation of the enzymes may also have contributed to the inhibition of root growth observed. For acquired and basal thermotolerance in seedlings, we could not detect a difference in the survival rate between the mutants and the wild type. However, both cphsc70 mutants had reduced chlorophyll contents during heat shock recovery (Fig. 6, B and D). These results suggest that cpHsc70s may play a role in protection of the photosystems during heat shock and/or recovery. In green algae, the chloroplast Hsp70B has been shown to participate in the protection and repair of the PSII during and after photoinhibition (Schroda et al., 1999; Yokthongwattana et al., 2001). Therefore the involvement of stromal Hsp70s in the maintenance of chloroplast photosystems during stress conditions may be a general mechanism in plants. The mild phenotypes in seedling thermotolerance of the Arabidopsis cphsc70 mutants could be due to the presence of two homologous genes. It would be interesting to reduce the total level of cpHsc70s by antisense or RNAi techniques, coupled with inducible promoters to circumvent the essential nature of the proteins to further analyze the function of stromal Hsp70s in stress protection, chloroplast protein import, thylakoid formation, or even other novel functions.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Growth Conditions

All Arabidopsis (Arabidopsis thaliana) plants used in this study were of the ecotype Columbia. Sterilized Arabidopsis seeds were plated on 0.3% Gelrite-solidified 1x Murashige and Skoog (MS) medium containing Gamborg's B5 vitamin and 0.5% Suc. After a 3-d cold stratification, seeds were grown in growth chambers under a 16-h photoperiod with a light intensity approximately 80 µmol m^–2 s^–1 at 22°C. Soil-grown Arabidopsis plants were grown on a 9:1:1 mixture of peat, vermiculite, and perlite at 22°C under a constant light with an intensity approximately 120 µmol m^–2 s^–1, except all plants used for cross-experiments were grown on soil to maturity under a 16-h photoperiod with a light intensity approximately 120 µmol m^–2 s^–1 at 22°C. For growing pea seedlings (Pisum sativum ‘Little Marvel’), imbibed seeds were grown on vermiculite for 9 to 12 d under a 12-h photoperiod at 20°C with a light intensity of approximately 150 µmol m^–2 s^–1.

Identification of cphsc70-1 and cphsc70-2 T-DNA Insertion Mutant Lines

Mutant lines SALK_140810 (cphsc70-1), SALK_095715 (cphsc70-2), and SALK_099583c (hot1) were obtained from the SALK T-DNA collections (Alonso et al., 2003) via the Arabidopsis Biological Resource Center; they were screened by PCR amplification of genomic DNA because cphsc70-1 had lost kanamycin resistance and cphsc70-2 showed heterogeneous growth on kanamycin plates. Primers used for amplifying the wild-type copy of cpHsc70-1 were 1E2-S (5'-gattcacagaggacagctac-3') and 1E8-AS (5'-tgaatctcctgatgaagcac-3'). For amplifying the wild-type copy of cpHsc70-2, the primers used were 2E1-S (5'-gtcctttcactactccaacc-3') and 2E8-AS (5'-ctgaggatgatgtgtcagac-3'). To identify the T-DNAs inserted in cpHsc70-1 and cpHsc70-2, the SALK Lba1 primer (5'-tggttcacgtagtgggccatcg-3') was combined with 1E8-AS and 2E8-AS, respectively. The T-DNA insertion sites were verified by sequencing the PCR products. Primer pairs used for checking cpHsc70-1 and cpHsc70-2 transcripts by RT-PCR were 1E2-S + 1E8-AS and 2E1-S + 2E8-AS, respectively. To test the expression of the cpHsc70 protein in each mutant, total stromal proteins were analyzed in IEF gels (pI 3–7) and immunoblots decorated with the anti-S78 antibody.

Vector Construction and Plant Transformation

Using genomic DNA extracted from wild-type Arabidopsis seedlings as the template, a 5-kb genomic fragment containing the promoter and coding regions of cpHsc70-1 was amplified by PCR with primers H70A-PS (5'-aaaagctttcgtaaaggcttgtaagc-3') and H70At-AS (5'-gaaggtagatggtcaccggtgaagcgag-3'). The PCR product was first cloned into the pGEM-T vector (Promega), and then subcloned into the binary vector pCambia1390. The resulting plasmid was named pCambia1390/cpHsc70-1g and was transformed into Agrobacterium GV3101. The cphsc70-1 mutant plants were infected by the floral spray method (Chung et al., 2000). Transgenic plants harboring the introduced cpHsc70-1 genomic fragments were screened on MS media containing 50 mg L^–1 hygromycin.

In Vitro Translation and Protein Import Assays

T3 promoter fused cpHsc70-1 and cpHsc70-2 linear DNAs were amplified by PCR with pfu DNA polymerase (Stratagene) using plasmids containing full-length cDNA as templates and primers containing the T3 promoter sequence. Primers used for amplifying T3:cpHsc70-1 were 5'-ccaattaaccctcactaaagggagccaccatggcatcttcagccgcccaa-3' and 5'-gtctctattggctgtctgtgaagtcag-3'. For amplifying T3:cpHsc70-2, the primers used were 5'-ccaattaaccctcactaaagggattctcatttccaaccatgg-3' and 5'-ttctctaattgctgtctgtgaagtcag-3'. [³⁵S]Met-labeled precursors of cpHsc70-1 and cpHsc70-2 were in vitro translated in a TNT reticulocyte lysate system (Promega), with the addition of PCR-generated T3:cpHsc70-1 and T3:cpHsc70-2 DNA templates. [³⁵S]Met-prRBCS synthesis, pea and Arabidopsis chloroplast isolation, and import assays were conducted as described (Perry et al., 1991), except the grinding buffer for Arabidopsis chloroplast isolation was modified to 50 mM HEPES-KOH (pH 8.0), 330 mM sorbitol, 2 mM EDTA, and 0.5% bovine serum albumin. Trypsin digestion of chloroplasts and separation of chloroplasts after digestion into soluble and membrane fractions were performed as described (Jackson et al., 1998; Hung et al., 2004). For import in the absence of ATP (Fig. 5D), ATP was removed from the in vitro-translated precursors by gel filtration (Perry et al., 1991) and chloroplasts were depleted of internal ATP by incubation in the dark at 4°C for 1 h. Import was performed under a green-safe light at room temperature for 15 min. Quantification of gel bands was performed using the Fuji FLA5000 phosphoimager (Fujifilm). Antibodies against Tic and Toc proteins were prepared as described (Chou et al., 2003; Tu et al., 2004).

Heat-Stress Treatments and Measurement of Chlorophylls

Thermotolerance assays of seeds and seedlings on plates were performed according to Charng et al. (2006) with minor modifications. To make the heat treatment more uniform, heat shock was conducted by heating tape-sealed agar plate in water bath in the dark. Plates were then cooled down to room temperature by floating on tap water, and kept in the dark at room temperature for 2 h to avoid possible additional effects of light stress. Seedlings were allowed to recover for an additional 7 d in the original growth conditions prior to calculating the survival rate. Plants that were still green and producing new leaves were scored as survived. For the acquired thermotolerance test, 5- to 7-d-old seedlings were initially acclimated at 37.5°C for 1 h and returned to the original growth condition for 2 d, then challenged at the lethal temperature 44.5°C for 1 h. Total chlorophyll was determined by the method of Lichtenthaler (1987). Root length was measured using pictures of seedlings and the software ImageJ (National Institutes of Health image).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AL161561 (cpHsc70-1) and AB024032 (cpHsc70-2).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Genomic Southern analyses of the T-DNA in cphsc70 mutants.

Supplemental Figure S2. cphsc70-1 had a higher amount of cpHsc70-2 but a normal amount of chloroplast translocon proteins.

Supplemental Figure S3. Alignment of the amino acid sequences of plastid Hsp70s used for constructing the phylogenetic tree.

Supplemental Table S1. Progeny segregation of the heterozygous cphsc70-1 mutant.

Supplemental Table S2. Plastid Hsp70s used for the phylogenetic analysis.

	ACKNOWLEDGMENTS

 
We thank Dr. Chung-Yen Lin for advice on phylogenetic analyses, Dr. Ken Keegstra for the antibody against S78, Dr. Hwa Dai for the monoclonal antibody against maize mitochondrial porin, and Dr. Neil Hoffman for the antibody against CAB. We thank the Arabidopsis Biological Resource Center and the Salk Institute for providing the cphsc70-1, cphsc70-2, and hot1 mutants. We also thank Dr. Harry Wilson for English editing, Dr. Yi-Fang Tsay for helpful discussions, and Drs. Shih-Long Tu, Yee-yung Charng, and Tien-Shin Yu for critical reading of the manuscript.

Received December 4, 2007; accepted January 6, 2008; published January 11, 2008.

	FOOTNOTES

 
¹ This work was supported by the National Science Council (grant no. NSC–95–2321–B–001–004 to H.M.L.) and the Academia Sinica of Taiwan.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hsou-min Li (mbhmli{at}gate.sinica.edu.tw).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

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www.plantphysiol.org/cgi/doi/10.1104/pp.107.114496

^* Corresponding author; e-mail mbhmli{at}gate.sinica.edu.tw.

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