•AtDjB1 belongs to the J-protein family in Arabidopsis thaliana. Its biological functions in plants are largely unknown.
•In this study, we examined the roles of AtDjB1 in resisting heat and oxidative stresses in A. thaliana using reverse genetic analysis.
•AtDjB1 knockout plants (atj1-1) were more sensitive to heat stress than wildtype plants, and displayed decreased concentrations of ascorbate (ASC), and increased concentrations of hydrogen peroxide (H2O2) and oxidative products after heat shock. Application of H2O2 accelerated cell death and decreased seedling viability in atj1-1. Exogenous ASC conferred much greater thermotolerance in atj1-1 than in wildtype plants, suggesting that a lower concentration of ASC in atj1-1 could be responsible for the increased concentration of H2O2 and decreased thermotolerance. Furthermore, AtDjB1 was found to localize to mitochondria, directly interact with a mitochondrial heat-shock protein 70 (mtHSC70-1), and stimulate ATPase activity of mtHSC70-1. AtDjB1 knockout led to the accumulation of cellular ATP and decreased seedling respiration, indicating that AtDjB1 modulated the ASC concentration probably through affecting the function of mitochondria.
•Taken together, these results suggest that AtDjB1 plays a crucial role in maintaining redox homeostasis, and facilitates thermotolerance by protecting cells against heat-induced oxidative damage.
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J-proteins are defined by the presence of a J-domain of c. 75 conserved amino acid residues. In recent years, a large number of J-proteins have been characterized from a variety of different organisms. DnaJ was originally characterized in Escherichia coli as a 41 kDa HSP (Georgopoulos et al., 1980). Subsequently, members of the J-protein family were found to function as molecular chaperones, alone or in association with HSP70 partners. A characteristic of HSP70 chaperone proteins is a low-level basal ATPase activity (Becker & Craig, 1994). J-proteins can stimulate the ATPase activity of HSP70 many-fold (Mayer & Bukau, 2005). HSP70/J-protein machinery is involved in a variety of essential cellular processes, including protein folding, translocation of polypeptides, degradation of misfolded proteins and the refolding of proteins damaged after exposure of cells to stresses (Walsh et al., 2004; Hennessy et al., 2005; Mayer & Bukau, 2005; Craig et al., 2006).
A total of 120 J-proteins have been identified in the genome of Arabidopsis thaliana (Miernyk, 2001; Rajan & D’Silva, 2009). The very large number of A. thaliana J-proteins with unassigned functions suggests possible roles in plant-specific cellular processes. J-proteins have been reported to localize in different subcellular compartments and are involved in development, signal transduction and resisting environmental stresses in A. thaliana (Sedbrook et al., 1999; Christensen et al., 2002; Guan et al., 2003; Vitha et al., 2003; Suetsugu et al., 2005; Tamura et al., 2007; Glynn et al., 2008; Yamamoto et al., 2008; Kneissl et al., 2009; Yang et al., 2010; Shen et al., 2011). AtDjB1 is a member of the Arabidopsis J-protein family. The open reading frame of AtDjB1 encodes 409 amino acids, and the deduced amino acid sequence of AtDjB1 contains a potential N-terminal mitochondrial-targeting sequence (Kroczynska et al., 1996; Miernyk, 2001). Radioactive recombinant AtDjB1 is imported into intact pea cotyledon mitochondria in vitro, confirming the mitochondrial location of AtDjB1 (Kroczynska et al., 1996). Recombinant AtDjB1 stimulates the ATPase activity of both E. coli DnaK and maize endosperm HSP70 in vitro (Kroczynska et al., 1996). In addition, complementation of an E. coli dnaJ deletion mutant with the AtDjB1 gene rescues the growth ability of E. coli under heat stress conditions (Kroczynska et al., 1996). However, the roles of AtDjB1 in growth and development of plants, and resistance to environmental stresses have not been reported. In this work, we used reverse genetic analysis to define the roles of the AtDjB1 in resisting heat and oxidative stresses in A. thaliana. We found that AtDjB1 facilitates thermotolerance by protecting cells against heat-induced oxidative damage.
Materials and Methods
Plant materials and growth conditions
Seeds of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia-0) were surface-sterilized, plated on Murashige and Skoog (MS) medium containing 1.0 or 3.0% (w/v) sucrose and 0.8% (w/v) agar, and kept at 4°C for 3 d. Plants were grown in a growth chamber under long-day conditions (16 h light : 8 h dark) at 22°C. Two-week-old seedlings were transplanted to soil and cultured under the original growth conditions. Plants were irrigated with 1× Hoagland nutrient solution once a week.
Identification and isolation of AtDjB1 mutants
Seeds of two putative T-DNA insertion mutants for AtDjB1, SALK_049553 (atj1-1) and SALK_065970 (atj1-4), were obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH, USA). Homozygous AtDjB1 mutants were identified by PCR as described by the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu/T-DNA_Genotyping_Procedure.ppt). PCR was with genomic DNA from T3 generation seedlings using AtDjB1-specific primers (atj1-1: LP, 5′-ACGCGGAAGCGAACATACTGA-3′; RP, 5′-TCTGCATGGTGACACTTTGTGG-3′; atj1-4: LP, 5′-ATAGCGCCAGCAGCACCATTA-3′; RP, 5′-TCAGTATGTTCGCTTCCGCGT-3′) and a T-DNA left border primer LBb1. The location of the T-DNA insertion was determined by PCR product sequencing.
Transcript abundances of AtDjB1 in wildtype (WT) and two AtDjB1 mutants were determined by reverse transcription PCR (RT-PCR), using a one-step RT-PCR Kit (TaKaRa, Otsu, Shiga, Japan). Total RNA was isolated from 10-d-old seedlings with TRIzol reagent (Invitrogen). For the AtDjB1 full transcript, the last 456 bp fragment in the AtDjB1 coding region was amplified using a forward primer (FP1) and a reverse primer (RP1). For the AtDjB1 partial transcript, a fragment from 391 to 1058 in the AtDjB1 coding region was amplified using FP2/RP2. Actin was used as a control and amplified using FP3/RP3.
To generate pCAMBIA1300–AtDjB1, the AtDjB1 coding region was PCR-amplified with cDNA from A. thaliana (Col-0) seedlings using FP4/RP4. The PCR product was cloned into the binary vector pCAMBIA1300 digested with XbaI/SacI. To generate PAtDjB1:β-glucuronidase (PAtDjB1:GUS), or PmtHSC70-1:GUS, a 1144 bp (or 1207 bp) DNA fragment upstream of the AtDjB1 (or mtHSC70-1) translational start codon was PCR-amplified with genomic DNA from A. thaliana seedlings using FP5/RP5 for the AtDjB1 promoter and FP6/RP6 for the mtHSC70-1 promoter. The PCR fragment was ligated into pCAMBIA1300–GUS digested with PstI/XbaI (or PstI/BamHI). To generate the eGFP (enhanced green fluorescent protein)–AtDjB1 construct, the AtDjB1 coding region was PCR-amplified from a plasmid containing the AtDjB1 coding region using FP7/RP7. To generate the eGFP–mtHSC70-1 construct, the mtHSC70-1 coding region was PCR-amplified with cDNA from A. thaliana seedlings using FP8/RP8. The PCR fragment was fused to the C termini of eGFP in vector pEGAD digested with SmaI/BamHI. The resulting plasmids were introduced into Agrobacterium tumefaciens (GV3101). Transformation of A. thaliana was by the floral-dipping method (Clough & Bent, 1998). Transgenic lines were selected on MS medium containing 25 μg ml−1 hygromycin. For the eGFP–AtDjB1 (or eGFP–mtHSC70-1) construct, transgenic lines were selected on soil by spraying 30 μg ml−1 of the weedicide Basta. T3 progeny homozygotes containing a single copy insertion were used.
Real-time quantitative RT-PCR
Total RNA was isolated from different tissues or seedlings of A. thaliana using TRIzol® reagent (Invitrogen). Real-time quantitative RT-PCR (q-PCR) was performed as described by Zhang et al. (2009). Primer pairs were designed using Primer Express (Applied Biosystems, Carlsbad, California, USA): AtDjB1-specific, FP9/RP9; mtHSC70-1-specific, FP10/RP10; and Actin-specific, FP11/RP11.
Heat stress treatment and chlorophyll measurement
Seeds of different genotypes were plated on separate regions of the same MS plate, with 30 or 50 seeds per genotype and experiment. For the basal thermotolerance assay, 10-d-old seedlings grown at 22°C were exposed to 44°C for 2 h and recovered for an additional 2–5 d in the original growth conditions before calculating the survival rate. For the acquired thermotolerance assay, 10-d-old seedlings grown at 22°C were acclimated at 37°C for 30 min and returned to the original growth conditions for 2 h, before challenge at 45°C for 125 min. Seedlings recovered for an additional 3 d in original growth conditions before calculating survival rate. Plants that were still green and producing new leaves were scored as survived. Total Chlorophyll (Chl) content was estimated as described by Porra et al. (1989).
Membrane system stability was assessed by electrolyte leakage measurement after HS. Green leaves were cut from 10-d-old seedlings grown at 22°C, washed three times with ion-free water, then incubated at 45°C for 2 h in 5 ml of ion-free water. The conductivity of the incubation medium was recorded with a Leici conductivity meter (DDS-IIA, Shanghai, China).
Assay of root vitality
The A. thaliana root vitality was measured with 2,3,5-triphenyltetrazolium chloride (TTC) according to Gong et al. (1997) with modifications. Ten-day-old seedlings grown at 22°C were exposed to 45°C for 1 h. Heat-shocked seedlings were removed from MS medium and cultured in 0.6% (w/v) TTC solution at 22°C for 20 h. After washing three times, 1.0 cm tips of primary roots (total 30 per sample) were cut off, homogenized in 95% (v/v) ethanol, and centrifuged at 4000 g for 5 min. The absorbance of the supernatant at 530 nm was measured spectrophotometrically.
Determination of hydrogen peroxide amount
Hydrogen peroxide (H2O2) amount was measured with xylenol orange according to Choi et al. (2007) with modifications. Ten-day-old seedlings grown at 22°C were heated at 44°C for 2 h and recovered at 22°C for 30 min. Seedlings were harvested and ground in liquid nitrogen, and 1 ml of phosphate buffer (20 mM K2HPO4, pH 6.5) was added to 0.1 g of ground frozen tissue. After centrifugation at 12 000 g for 10 min, 300 μl of supernatant was immediately added to 3 ml of xylenol orange reagent and incubated for 30 min at room temperature before measuring A560 against a blank of 300 μl of distilled water.
Measurement of ascorbate and thiobarbituric acid reactive substances
The mtHSC70-1 or mtHSC70-2 coding region was amplified with cDNA from A. thaliana seedlings using FP12/RP12 or FP13/RP13. The HSC70-1 or HSP70 coding region was amplified from a plasmid containing the respective gene using FP14/RP14 or FP15/RP15. The four genes above were ligated to SmaI/BamHI-digested pGBKT7 (Clontech, Mountain View, CA, USA). The AtDjB1 or AtDjA2 coding region was amplified from a plasmid containing the respective gene using FP16/RP16 or FP17/RP17, then ligated to SmaI/BamHI-digested pGADT7 (Clontech). The AtDjA3 coding region was amplified from a plasmid containing AtDjA3 using FP18/RP18, then ligated to EcoRI/BamHI-digested pGADT7 (Clontech). The expression vector pGADT7–AtDjB1 was cotransformed into yeast strain AH109 (Clontech) with pGBKT7–mtHSC70-1/mtHSC70-2/HSC70-1/HSP70; and the expression vector pGBKT7–mtHSC70-1 was cotransformed into yeast strain AH109 (Clontech) with pGADT7–AtDjB1/AtDjA2/AtDjA3, by the lithium acetate method (Clontech Yeast Protocols Handbook). Cells were plated onto selective medium without Leu and Trp (SM-LW), and putative transformants were transferred to selective medium lacking Leu, Trp, His and adenine (SM-LWHA). Interaction between 53 protein and SV40 protein, or interaction between Lam protein and SV40 protein was used as a positive and negative control, respectively. Autoactivation was analyzed by β-galactosidase assay (Breeden & Nasmyth, 1985) and by growth experiments on SM-LWHA when the detected gene was cotransformed with the pGADT7 empty vector.
Expression and purification of recombinant proteins
For preparation of glutathione S-transferase (GST)-fusion proteins, the AtDjB1 or mtHSC70-1 coding region was amplified from a plasmid containing the respective gene using FP19/RP19 or FP20/RP20, then subcloned into pGEX-4T-1 (GE Healthcare, Uppsala, Sweden) digested with BamHI/SmaI. E. coli Gode plus were transformed with both recombinant plasmids, grown at 37°C to A600 = 0.6, and induced with 0.1 mM isopropylthio-β-d-galactopyranoside at 7°C for 20 h.
To purify the GST-fusion proteins, induced cells were harvested, frozen, thawed and lyzed by sonication in phosphate-buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 3 mM EDTA. Clarified lysate was mixed with glutathione agarose (Sigma) equilibrated with PBS containing 150 mM NaCl, incubated at 4°C for 2 h, and washed consecutively with 40 bed volumes of PBS containing 1% (v/v) Triton X-100 at 4°C. GST–AtDjB1 fusion protein was eluted with three bed volumes of elution buffer (10 mM 3-(N-Morpholino) propanesulfonic acid (MOPS)/KOH, pH 7.2, 150 mM KCl, 3 mM MgCl2, 10 mM reduced glutathione). To obtain mtHSC70-1, the GST–mtHSC70-1 fusion protein in column was cleaved in thrombin cleavage buffer (25 mM Tris-HCl, pH 6.9, 40 U ml−1 thrombin, 0.1% (w/v) BSA and 1 mM dithioreitol (DTT)) at 4°C overnight, and the digested product was collected.
Production of AtDjB1- or mtHSC70-1-specific antibody
Purified GST–AtDjB1 or mtHSC70-1 protein was used to produce polyclonal rabbit antiserum. The resulting antisera were then purified through a protein A-agarose column. The purified anti-AtDjB1 or anti-mtHSC70-1 antibody could specifically detect eGFP–AtDjB1 or mtHSC70-1 protein, respectively, in seedlings of transgenic A. thaliana harboring eGFP–AtDjB1.
In vitro binding assay
A binding reaction was performed according to Diefenbach & Kindl (2000) with modifications. Briefly, the reaction mixture (40 μl) containing 10 mM MOPS/KOH, pH 7.2, 150 mM KCl, 3 mM MgCl2, 2 mM ADP (adenosine diphosphate), 10 μg of GST–AtDjB1 and 10 μg of mtHSC70-1 was incubated at 20°C for 20 min followed by HS at 37°C for 10 min. Samples were analyzed by 7.5% native-polyacrylamide gel electrophoresis and Coomassie blue staining.
The co-immunoprecipitation (Co-IP) assays used transgenic seedlings harboring eGFP–AtDjB1, which were harvested and ground in liquid nitrogen, and homogenized in ice-cold extraction buffer (20 mM Tris-HCl, pH 7.5, 2 μg ml−1 aprotinin, 2 μg ml−1 leupeptin, 0.7 μg ml−1 pepstatin, 50 mM disodium beta-glycerol phosphate pentahydrate, 5 mM NaF, 200 μM Na3VO4, 2 mM DTT, 500 μM PMSF and 250 mM sucrose). Total protein extract was obtained by centrifugation at 25 000 g for 30 min, and 300 μl of IP buffer (20 mM Tris-HCl, pH 7.5, 2 μg ml−1 aprotinin, 2 μg ml−1 leupeptin, 0.7 μg ml−1 pepstatin, 50 mM disodium beta-glycerol phosphate pentahydrate, 5 mM NaF, 200 μM Na3VO4, 2 mM DTT, 500 μM PMSF, 0.9% NaCl, 1% Triton X-100 and 4 mM ADP) and 10 μl of rabbit anti-mtHSC70-1 polyclonal antibody (or 6 μl of mouse anti-tubulin monoclonal antibody) were added to 300 μl of total protein extract. After agitation for 30 min at 25°C, 20 μl of a 5 : 1 slurry of H2O–protein G-agarose were added, and the mixture was agitated at 25°C for 2 h followed by HS at 30°C for 30 min. The beads were washed three times with ice-cold washing buffer (10 mM Tris-HCl, pH 7.5 and 5 mM EDTA), then resuspended in 30 μl of sample buffer and boiled for 5 min. Proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride membrane. Immunoblot analysis was performed with rabbit polyclonal anti-mtHSC70-1 or anti-AtDjB1 antibody, or mouse monoclonal anti-tubulin antibody, and immune complexes were visualized with an alkaline phosphatase detection system.
ATPase activity assay
ATPase activity was measured using the malachite green procedure according to Baykov et al. (1988) and Kroczynska et al. (1996) with modifications. Briefly, absorbance of the phosphomolybdate plus malachite green complex was measured at 600 nm using a microplate reader (Bio-Rad, Model 550). Standard curves were constructed using KH2PO4, and samples were deproteinized by boiling at 100°C for 10 min. The ATPase assay mixture contained 100 mM Bis-Tris, pH 6.0, 20 mM KCl, 1 mM MgCl2, 2 mM DTT, 0.1 mM ATP and 2 μg of purified mtHSC70-1 (or AtDjB1) protein in a final volume of 0.1 ml. After 10 min at 40°C, reactions were stopped by boiling at 100°C for 10 min. Reaction mixtures were then incubated on ice for 10 min, and then centrifuged for 3 min at 12 000 g. Samples of 0.08 ml from each supernatant were taken for measurement of A600.
Measurement of ATP level
Seven-day-old seedlings (0.065 g) grown at 22°C were boiled at 100°C for 10 min in 1 ml of ion-free water, and then placed on ice for 10 min. After centrifugation at 10 000 g for 5 min at 4°C, 60 μl of the supernatant were transferred to a microplate well and used for the measurement of luminescence. ATP level was measured using a Cellular ATP Kit HTS (BioThema, Handen, Sweden) in a luminometer (Centro LB 960).
Measurement of O2 uptake
Respiration of seedlings was measured as O2 uptake with a Clark-type O2 electrode (Hansatech Instruments Ltd, Oxygraph, Norfolk, UK) in 1.5 ml of an air-saturated 2 mM Ca(NO3)2 solution at 25°C, according to Kania et al. (2003).
Statistical analyses were carried out with Statistica 6.0. The significance of differences were tested at P < 0.05 using ANOVA with post-hoc least significant difference (LSD) or Student’s t-test, as indicated.
Note: primers used for PCR amplifications can be found in Supporting Information Table S1.
To gain insight into the biological function of AtDjB1 in A. thaliana, we analyzed two putative T-DNA insertion mutants carrying a T-DNA in AtDjB1. To confirm the presence of the T-DNA, we used AtDjB1-specific and T-DNA-specific primers to PCR-screen the T3 progeny, and obtained two homozygous mutant lines, atj1-1 and atj1-4. Sequence analysis of the T-DNA flanking regions revealed that atj1-1 and atj1-4 contained a T-DNA insertion in introns 14 and 17 of AtDjB1, respectively (Fig. 1a). RT-PCR showed no detectable full AtDjB1 transcript in either homozygous mutant line. Partial AtDjB1 transcripts were significantly down-regulated in both mutants, especially in atj1-1 (Fig. 1b).
To assess the effect of AtDjB1 knockout on basal thermotolerance, the survival rate of seedlings after HS was compared between WT and AtDjB1 mutants. The survival rate of atj1-1 and atj1-4 seedlings was not different from the WT under normal growth conditions. However, atj1-1 and atj1-4 seedlings exhibited a hypersensitivity to HS (44°C for 2 h). While 94% of WT seedlings were alive after HS, the survival rate after HS was only 8% for atj1-1 seedlings and 60% for atj1-4 seedlings (Fig. 1c,d). The increased survival rate of atj1-4, compared with atj1-1, could be explained by its higher expression level of the partial AtDjB1 transcript. Since atj1-1 was probably a strong mutant allele, we used it for further analyses.
Expression of AtDjB1 in the atj1-1 mutant rescued the thermotolerance phenotype of WT plants
To test whether the thermosensitive phenotype of atj1-1 could be attributed to the loss of AtDjB1 function, we performed complementation analysis. A binary vector containing the complete coding region of the AtDjB1 gene (pCAMBIA1300–AtDjB1) was transformed into atj1-1. Nine independent T3 homozygous rescued mutant lines (atj1-1/AtDjB1) with a single copy of exogenous AtDjB1 were generated. PCR confirmed that the AtDjB1 gene was introduced into all transgenic lines obtained.
The basal thermotolerance of the atj1-1/AtDjB1 lines was tested and compared to atj1-1. Six of the nine atj1-1/AtDjB1 lines obtained had increased viability compared with atj1-1 after the seedlings were exposed directly to 44°C for 120 min. For example, while only 14% of the atj1-1 seedlings were alive after HS, the survival rate after HS was 82% for atj1-1/AtDjB1 line R6, and 71% for line R1 (Fig. 2a,b). Basal thermotolerance rescue in the atj1-1/AtDjB1 lines was confirmed by the test of total Chl content of the seedlings (Fig. 2c,d). In addition, we found that knockout of the AtDjB1 gene decreased also basal thermotolerance in the older plants, compared with WT (Fig. S1). Further, we examined the effect of the AtDjB1 gene on acquired thermotolerance: the atj1-1 seedlings were more sensitive to heat stress (37°C for 30 min, 22°C for 120 min, then 45°C for 125 min) than WT. Complementation of the atj1-1 with the AtDjB1 gene rescued the thermotolerance phenotype of the WT seedlings (Fig. S2).
To ensure that the phenotypic changes observed were caused by the AtDjB1 gene, q-PCR was used to detect the expression of the AtDjB1 gene in seedlings of four genotypes. The level of AtDjB1 expression was very low in atj1-1. Introduction of the AtDjB1 gene into atj1-1 rescued the expression of the AtDjB1 gene, and AtDjB1 expression in R1 and R6 seedlings reached a similar level to that in WT plants (Fig. 2e). Taken together, these results indicated that complementation of the atj1-1 with the AtDjB1 gene rescued the basal and acquired thermotolerance, suggesting a role for the AtDjB1 gene in improving the thermotolerance of A. thaliana seedlings.
As an additional test of thermosensitivity, heat-induced cell death was assayed by trypan blue staining in WT, atj1-1 and R6. Ten-day-old seedlings grown at 22°C were heated at 44°C for 2 h and recovered at 22°C for 30 h. The atj1-1 exhibited increased cell death after HS compared with WT and R6 (Fig. 3a). For confirmation, the amount of electrolyte leakage was compared between seedlings of different genotypes. Under nonHS conditions, there were no significant differences in ion conductivity among WT, atj1-1, R1 and R6. After HS, ion conductivity in atj1-1 was distinctly higher than in WT. However, ion conductivity in R1 or R6 line was not evidently different from the WT (Fig. 3b). After HS, electrolyte leakage compared with nonheat-treated plants was 229% in WT, 302% in R1 and 264% in R6. By contrast, electrolyte leakage in atj1-1 reached 430% of the nonheat-treated control (Fig. 3c). These results indicated that AtDjB1 is important for the stability of the membrane system during HS.
The reliability of the results obtained by trypan blue staining and the conductivity measurements were supported by cell vitality assays. Under nonHS or HS conditions, TTC reduction activity in atj1-1 was obviously lower than in the WT; however, TTC reduction activity in R1 or R6 line was not markedly different from that in the WT (Fig. 3d). After HS, the ability to reduce TTC compared with nonheat-treated plants was 86.3% in WT, 83.8% in R1 and 97.9% in R6. By contrast, TTC reduction activity in atj1-1 was merely 54.1% of the nonheat-treated control (Fig. 3e). The HS prevented the reduction of TTC much more in atj1-1 than in WT, or R1 and R6. Taken together, our data suggested that AtDjB1 knockout accelerated HS-induced cell death, and further supported the role of AtDjB1 in improving the thermotolerance of plants.
AtDjB1 knockout led to heat-induced H2O2 accumulation
To determine the particular function of AtDjB1 in antioxidative stress during HS, heat-induced changes in the concentration of cellular H2O2 and lipid peroxidation product were investigated. Ten-day-old seedlings grown at 22°C were heated at 44°C for 2 h and recovered at 22°C for 30 min. While the leaves of unheated WT were only slightly colored by DAB (a histochemical reagent for H2O2), the leaves of heated WT, atj1-1 and R6 were stained a vivid reddish brown, with the strongest staining in the atj1-1 mutant (Fig. 4a). We further quantified the H2O2 content in WT, atj1-1, R1 and R6 seedlings using a xylenol orange assay. There were no significant differences in the H2O2 content among WT, atj1-1, R1 and R6 lines under nonHS conditions. The HS induced the production of H2O2 in seedlings of all four genotypes. After HS, there was much greater accumulation of H2O2 in atj1-1 compared with the WT, R1 or R6 (Fig. 4b). The results indicated that AtDjB1 probably played an important role in the regulation of the H2O2 concentration.
Given that AtDjB1 knockout led to the accumulation of H2O2 under HS, we hypothesized that AtDjB1 functioned by protecting against cell oxidation caused by HS. To confirm this, lipid peroxidation levels in WT, atj1-1, R1 and R6 seedlings were determined. Seedlings were heated at 45°C for 60 min and then allowed to recover at 22°C for 2 d. After HS, there were much higher amounts of TBARS in atj1-1 seedlings than in WT, R1 or R6 seedlings (Fig. 4c). These findings suggested that AtDjB1 is critical for protection against excess accumulation of H2O2 and cell oxidative damage during HS.
To evaluate the contribution of H2O2 in the thermosensitivity of atj1-1 plants, we examined the effects of exogenous H2O2 on cell death and viability of seedlings. A DNA fragmentation experiment indicated that a high concentration of H2O2 promoted cell death (Fig. S3, Methods S1). Further experiments showed that application of 10 mM H2O2, instead of HS treatment, accelerated cell death in atj1-1 seedlings, compared with the WT, R1 or R6 (Fig. 5a). In addition, treatment with exogenous H2O2 decreased the survival rate and total Chl content of seedlings, instead of HS treatment. The decrease in atj1-1 was much greater than the decrease for WT, R1 or R6 (Fig. 5b–d). These results suggested that under HS, the elevated H2O2 concentrations in atj1-1 may have accelerated cell death and decreased thermotolerance.
Accumulation of H2O2 and decreased thermotolerance under HS in atj1-1 were caused by a lower rate of ROS detoxification
To determine whether accumulation of H2O2 under HS in atj1-1 was caused by a higher rate of H2O2 production or a lower rate of H2O2 detoxification, we examined the concentration of endogenous ASC, a H2O2 scavenger. Ten-day-old seedlings grown at 22°C were heated at 37°C for 120 min and allowed to recover at 22°C for 30 min. The concentration of ASC in atj1-1 before and after HS was obviously lower than that in WT (Fig. 6a). It is well known that ASC can reduce H2O2 into H2O (Mittler et al., 2004). Taken together, these data indicated that accumulation of H2O2 under HS in atj1-1 was caused at least in part by a lower concentration of ASC.
Next, the effects of exogenous ASC on thermotolerance were tested in WT, atj1-1, R1 or R6 lines. Under nonHS conditions, application of 0.25 μM ASC did not affect the viability and total Chl content for seedlings of all four genotypes. The HS (44°C for 120 min) significantly decreased survival rate and total Chl content for seedlings of all four genotypes. The decrease in atj1-1 was much greater than the decrease for WT, R1 or R6. Under HS conditions, application of 0.25 μM ASC completely or partly rescued the survival rate and total Chl content in seedlings of all four genotypes (Fig. 6b,c,e). After HS the survival rate for seedlings preincubated with ASC compared with nonincubated seedlings was c. 113% in WT, 161% in atj1-1, 126% in R1 and 111% in R6 (Fig. 6d). After HS, the total Chl content for seedlings preincubated with ASC compared with nonincubated seedlings was 142% in the WT, 360% in atj1-1, 196% in R1 and 121% in R6 (Fig. 6f). The rescue in atj1-1 was much greater than that for WT, R1 or R6. These results suggested that a lower concentration of ASC in atj1-1 was likely to be responsible for decreased thermotolerance. This conclusion is supported by the evidence that two oxidative stress mutants (vtc1 and vtc2), which are ASC-deficient, exhibited decreased thermotolerance and heat-induced oxidative damage (Larkindale et al., 2005).
AtDjB1 interacted with mtHSC70-1
Exactly how AtDjB1 regulates the ASC concentration is not known. To date, the only demonstrated ‘activity’ for any J-domain protein in any system is as a chaperone or a chaperone cohort with HSP70. Recombinant AtDjB1 stimulates the ATPase activity of both E. coli DnaK and maize endosperm HSP70 in vitro (Kroczynska et al., 1996). However, the HSP70 partner of AtDjB1 in A. thaliana remains unclear. More than 120 J-proteins and at least 17 HSP70 proteins have been identified in the A. thaliana genome (Sung et al., 2001; Rajan & D’Silva, 2009). Different J-proteins bind to distinct HSP70s in specific chaperone pairs (Diefenbach & Kindl, 2000; Hennessy et al., 2005). The mechanisms for cellular regulation of specific interactions could occur at the level of colocalization of binding partners to specific organelles or tissues in eukaryotes, or at the level of coexpression under certain special conditions. According to the result of mitochondrial localization of AtDjB1 protein (Kroczynska et al., 1996), we anticipated that the partner of the AtDjB1 would be the A. thaliana mitochondrial-located isoform of HSP70. To address this possibility, we evaluated the ability of AtDjB1 to interact with two putative A. thaliana mitochondrial HSP70s (mtHSC70-1 and mtHSC70-2) (Sung et al., 2001) by a yeast two-hybrid assay. In addition, the ability of the AtDjB1 to interact with two A. thaliana cytosolic isoforms of HSP70 (HSC70-1 and HSP70), and the ability of mtHSC70-1 to interact with two A. thaliana cytosolic J-proteins (AtDjA2 and AtDjA3) were also analyzed. All yeast (AH109) transformants grew normally on SM-LW. The capacity of the yeast to grow on SM-LWHA, and β-galactosidase activity were used as interaction reporters. We found that His auxotrophy was restored on SM-LWHA when AtDjB1 was cotransformed with mtHSC70-1 (Fig. 7a,b). However, the cells did not grow on SM-LWHA when AtDjB1 was cotransformed with mtHSC70-2, HSC70-1 or HSP70 (Fig. 7a), or when mtHSC70-1 was cotransformed with AtDjA2 or AtDjA3 (Fig. 7b). The results indicated that AtDjB1 interacted specifically with mtHSC70-1. The yeast showed no positive reaction by X-Gal filter assay and growth experiment on SM-LWHA when AtDjB1, AtDjA2, AtDjA3, mtHSC70-1, mtHSC70-2, HSC70-1 or HSP70 was cotransformed with pGADT7 empty vector (Fig. S4), indicating that none of the detected genes autoactivated.
The yeast two-hybrid assay results were supported by an in vitro binding experiment. GST–AtDjB1 fusion protein was incubated with the mtHSC70-1 protein at 20°C for 20 min followed by HS at 37°C for 10 min. Association of GST–AtDjB1 with mtHSC70-1 resulted in a change in electrophoretic mobility. Compared with GST–AtDjB1 or mtHSC70-1 alone, binding was indicated by an upward bandshift (Fig. 7c). This further confirmed the interaction of AtDjB1 with mtHSC70-1.
To determine if this interaction existed in vivo, transgenic plants harboring eGFP–AtDjB1 were used to Co-IP assay. The mtHSC70-1 protein was immunoprecipitated using rabbit polyclonal anti-mtHSC70-1 antibody and protein G-agarose. As a control, the tubulin protein was immunoprecipitated using mouse monoclonal anti-tubulin antibody and protein G-agarose. After washing, immunoblots were probed with rabbit polyclonal anti-AtDjB1 antibody. The eGFP–AtDjB1 fusion protein was pulled down by mtHSC70-1 (Fig. 7d), but not by tubulin protein (Fig. 7e), suggesting that AtDjB1 and mtHSC70-1 can function in the same complex. Together with the yeast two-hybrid and in vitro binding results, our data indicate that AtDjB1 and mtHSC70-1 interact in vivo.
AtDjB1 and mtHSC70-1 had overlapping tissue-specific expression and subcellular localization
To determine if AtDjB1 and mtHSC70-1 colocalize in A. thaliana, we investigated tissue-specific expression of AtDjB1 and mtHSC70-1 using two approaches. First, q-PCR was used to examine the expression of AtDjB1 and mtHSC70-1 in different tissues. Both AtDjB1 and mtHSC70-1 were constitutively expressed in all tissues examined with highest expression in reproductive (AtDjB1) (Fig. 8a) or root tissues (mtHSC70-1) (Fig. 8b). Then the GUS reporter assay was used to monitor the detailed tissue-specific expression pattern of two genes. GUS expression driven by the AtDjB1 or mtHSC70-1 promoter is shown in Fig. 8(c,d). Both proteins showed a similar expression pattern: PAtDjB1:GUS and PmtHSC70-1:GUS were ubiquitously expressed in A. thaliana.
To further investigate the interaction between AtDjB1 and mtHSC70-1, we determined the subcellular localization of the two proteins using the GFP reporter method. eGFP–AtDjB1 was detected at the mitochondria of root cells of the transgenic plants (Fig. 8e–h). eGFP–mtHSC70-1 was also found to localize to the mitochondria of protoplasts of the transgenic plants (Fig. 8i–l). Taken together, our data indicate that AtDjB1 and mtHSC70-1 had overlapping tissue-specific expression and subcellular localization.
AtDjB1 knockout led to the accumulation of cellular ATP and decreased seedling respiration
To explain the mechanism by which the mtHSC70-1/AtDjB1 chaperone-pair functions, both the basal ATPase activity of recombinant mtHSC70-1 chaperone protein and the extent of stimulation by the recombinant AtDjB1 co-chaperone protein were determined. The mtHSC70-1 or AtDjB1 alone had a low-level activity of ATPase, and the association of AtDjB1 with mtHSC70-1 stimulated the hydrolysis of ATP by 3.2-fold (to AtDjB1) or 5.5-fold (to mtHSC70-1). As a control, BSA protein had virtually zero basal ATPase activity, and there was no detectable stimulation to mtHSC70-1 or AtDjB1 (Fig. 9a). Further, we found that the ATP content was significantly increased in atj1-1 compared with WT and R6 (Fig. 9b). These results suggest that the decreased ATPase activity in atj1-1 was probably responsible for the accumulation of ATP. In relation to mitochondrial function, the effect of AtDjB1 knockout on respiration was measured as O2 uptake. Respiration of seedlings was greatly reduced in atj1-1 compared with WT and R6 (Fig. 9c). We suggest that the accumulation of ATP (as a product of respiration) caused by knockout of AtDjB1 feedback inhibited the mitochondrial electron transport chain (ETC), leading to decreased respiration.
J-proteins act as molecular chaperones, and are involved in many cellular processes, including development, signal transduction and resistance to environmental stresses. Emerging evidence indicates that J-proteins are also involved in thermotolerance. Li et al. (2007) reported that AtDjA2 and AtDjA3 function in improving thermotolerance of A. thaliana. Yang et al. (2009) demonstrated that TMS1, a thermosensitive male sterile J-protein, plays an important role in the thermotolerance of pollen tubes. The data of the present study reveal the role of AtDjB1 in resisting heat stress (Figs 1, 2, S1, S2). A BLAST search showed that homologs of AtDjB1 also exist in some crop plants, such as castor bean, tomato and grape. Thus the present study will aid in the understanding of thermotolerance in crop plants and in ecosystems subject to high and fluctuating temperatures. Although the fact that overexpression of AtDjB1 did not confer added thermotolerance in comparison with WT (Fig. S5) suggests that a genetic engineering approach using this gene would not be an effective method for increasing thermotolerance of crops, it is possible that allelic variation exists for the homologous gene in other plants and may in some cases account for variations in observed thermotolerance.
Under optimal growth conditions, cellular ROS, such as singlet oxygen, superoxide anion, hydroxyl radical and H2O2, are mainly produced at low amounts in organelles such as cytoplasm membrane, chloroplast, mitochondria and peroxisome. They can act as signaling molecules that regulate plant growth, development, stress adaptation and programmed cell death (Apel & Hirt, 2004; Mittler et al., 2004; Ślesak et al., 2007). However, during stresses, their rate of production is dramatically elevated (Neill et al., 2002; Kacperska, 2004; Miller & Mittler, 2006). Elevated concentrations of ROS have detrimental impacts on cellular structures and macromolecules, such as lipids, proteins and DNA, resulting in oxidative damage at the cellular level (Suzuki & Mittler, 2006; Zhang et al., 2008). There is increasing evidence for a key role of ROS as a trigger of cell death (Desikan et al., 1998; Houot et al., 2001; Yao et al., 2001; Pinto et al., 2002). Transgenic plants with perturbed amounts of cellular antioxidant enzymes demonstrated the important role of H2O2 in plant cell death events (Gechev et al., 2004; Murgia et al., 2004; Vandenabeele et al., 2004). The data of the present study showed that knockout of AtDjB1 led to greater accumulation of H2O2 and oxidative product (Fig. 4) after HS, and that atj1-1 plants were more sensitive to oxidation stress than WT, R1 and R6 plants (Fig. 5), indicating that AtDjB1 could play an important role in maintaining H2O2 homeostasis, and that elevated concentrations of H2O2 under HS in atj1-1 could be responsible for accelerated cell death and decreased thermotolerance. This conclusion was supported by the mitochondrial localization of AtDjB1 (Fig. 8). Mitochondria are the sites of high-rate energy metabolism, and link redox metabolism to ATP synthesis through the ETC, which are the sites of continuous generation of ROS. ROS are released when electron carriers become over-reduced (Møller, 2001). Therefore, the primary line of defense against oxidative stress in mitochondria involves mechanisms to keep ETC oxidized, that is, by balancing substrate oxidation with ATP requirement (Finkemeier et al., 2005).
Does AtDjB1 play a role in the regulation of H2O2 concentration during HS by blocking H2O2 excess production or by increasing H2O2 detoxification? It is well known that ASC plays a key role in the ROS-scavenging mechanism. ASC can reduce H2O2 to H2O (Mittler et al., 2004) or react directly with O2− (Bartoli et al., 2000). Thus, we determined the content of endogenous ASC and the effect of exogenous ASC on plant thermotolerance: endogenous ASC content was lower in atj1-1 plants than in WT, and exogenous ASC conferred much greater thermotolerance in the atj1-1 than in WT (Fig. 6), indicating that AtDjB1 acted by modulating ASC concentrations. However, exogenous ASC did not completely protect the atj1-1 mutant from the effects of the heat treatment (Fig. 6c,e), suggesting that the AtDjB1 protein may have one or more functions besides regulating ASC concentrations. Taken together, our data suggest that AtDjB1 maintained H2O2 homeostasis, at least in part, by modulating ASC concentrations, and possibly other antioxidants; and that AtDjB1 facilitated thermotolerance, at least in part, by protecting cells against heat-induced oxidative damage, and possibly preventing heat-induced protein aggregation. AtDjB1 is a homolog of Mdj1p, a yeast mitochondrial-targeted J-protein. Mdj1p is involved in protein folding, and cooperates with yeast mtHSP70 to play an important role in the prevention of heat-induced protein aggregation (Westermann et al., 1996). The data of the present study showed that AtDjB1 was targeted to mitochondria (Fig. 8), associated with mitochondrial HSC70-1 (Fig. 7) and stimulated its ATPase activity (Fig. 9a). As expected, knockout of AtDjB1 resulted in the accumulation of cellular ATP (Fig. 9b) and inhibition of respiration (Fig. 9c). It is known that the last step of ASC synthesis is linked to the ETC (Bartoli et al., 2000). Thus we suggest that a lower rate of respiration in atj1-1 is likely to lead to decreased concentration of ASC. Based on our data and previous knowledge, we propose a model for explaining how AtDjB1 functions in heat acclimation (Fig. 10). AtDjB1, in association with mtHSC70-1, functions as an ATPase. Knockout of AtDjB1 leads to the accumulation of cellular ATP, which feedback inhibits ETC. The decreased ETC rate results in the decrease of ASC concentration and the accumulation of H2O2. A higher concentration of H2O2 in atj1-1 is likely to be responsible for decreased thermotolerance. Thus AtDjB1 plays a crucial role in thermotolerance of plants by protecting cells against oxidative damage caused by HS. Our finding provides new insights into the network of genes managing ROS concentrations, and provides evidence for the involvement of mitochondria in tolerance of plants to oxidative stress and heat stress. It should be mentioned that under nonstressed conditions, knockout of AtDjB1 led to delayed development in all growth stages (data not shown), indicating that AtDjB1 was also involved in the regulation of plant growth and development.
We thank Dr Jan A. Miernyk (Plant Genetics Research Unit, United States Department of Agriculture, Agricultural Research Service, Columbia, MO, USA) for HSC70-1 and HSP70 genes, and ABRC for distributing the AtDjB1 insertion mutant seeds. This work was supported by Key Research Special Funds of the Ministry of Agriculture, China (grant 2009ZX08009-017B) and the Natural Science Foundation of China (grant 31070257, 30470161 and 30870201) and the Natural Science Foundation of Hebei Province, China (grant C2009000280).