AtBAG7, an Arabidopsis Bcl-2–associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response

Abstract

The Bcl-2–associated athanogene (BAG) family is an evolutionarily conserved, multifunctional group of cochaperones that perform diverse cellular functions ranging from proliferation to growth arrest and cell death in yeast, in mammals, and, as recently observed, in plants. The Arabidopsis genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. In the present study we show that an Arabidopsis BAG, AtBAG7, is a uniquely localized endoplasmic reticulum (ER) BAG that is necessary for the proper maintenance of the unfolded protein response (UPR). AtBAG7 was shown to interact directly in vivo with the molecular chaperone, AtBiP2, by bimolecular fluorescence complementation assays, and the interaction was confirmed by yeast two-hybrid assay. Treatment with an inducer of UPR, tunicamycin, resulted in accelerated cell death of AtBAG7-null mutants. Furthermore, AtBAG7 knockouts were sensitive to known ER stress stimuli, heat and cold. In these knockouts heat sensitivity was reverted successfully to the wild-type phenotype with the addition of the chemical chaperone, tauroursodexycholic acid (TUDCA). Real-time PCR of ER stress proteins indicated that the expression of the heat-shock protein, AtBiP3, is selectively up-regulated in AtBAG7-null mutants upon heat and cold stress. Our results reveal an unexpected diversity of the plant's BAG gene family and suggest that AtBAG7 is an essential component of the UPR during heat and cold tolerance, thus confirming the cytoprotective role of plant BAGs.

As nonmotile organisms devoid of an adaptive immune system, plants must adapt rapidly to changing environmental conditions. To combat environmental constraints, plants have developed various defense strategies (1). At the organism level, abiotic stresses such as extreme temperature can induce both structural (e.g., increased leaf thickness) and developmental (e.g., fruit ripening) changes. Adverse environmental conditions also induce changes at the cellular level (2). Recent research has established that the regulation of cell death pathways is relevant to both abiotic and biotic stress responses in plants (3–5). Although core regulators of programmed cell death are conserved in mammals (e.g., the Bcl-2 family and caspases), they have not been identified in plants. The failure to identify plant homologs of mammalian cell-death regulators results, in part, from a lack of conservation at the primary sequence level. We have used functional genomic approaches in conditional yeast strains and bioinformatics approaches to address this issue and to identify the Arabidopsis Bcl-2–associated athanogene (BAG) gene family (5, 6).

The BAG proteins are a broadly conserved gene family with homologs spanning wide evolutionary distances including yeast, animals, and plants. The first identified mammalian BAG gene (BAG1) was discovered in a screen of a mouse embryo cDNA library using recombinant human Bcl-2 protein as bait to identify binding partners to Bcl-2 (7). BAG1 enhanced cell survival synergistically with Bcl-2, suggesting involvement in programmed cell death (PCD) pathways. Subsequent studies uncovered the BAG family and more accurately indicated that BAGs function as molecular cochaperones (8). Members of the BAG family are distinguished by the presence of a conserved C-terminal BAG domain (BD) containing ∼110–130 amino acids. The BD is comprised of three α helices of 30–40 amino acids each; the second and third helices mediate direct interaction with the ATPase domain of heat-shock protein 70 (Hsp70)/heat-shock cognate 70 (Hsc70) chaperones (9, 10). Six BAG family members have been identified in humans and were shown to regulate the function of Hsp70/Hsc70 both positively and negatively and to form complexes with a range of transcription factors (11) to modulate numerous physiological processes including apoptosis, tumorigenesis, neuronal differentiation, stress responses, and cell-cycle progression (reviewed in ref. 12).

Far less is known about the function of BAGs in plants. Initial BLAST searches failed to identify BAG homologs in Arabidopsis because of low sequence identities. Therefore more sensitive methods were used, including Hidden Markov Model-based approaches and profile–profile algorithms (13, 14). With these approaches, seven BAG homologs were identified in Arabidopsis, four of which have domain organization similar to animal members (5). The remaining three members contain a predicted calmodulin-binding motif near the BD, a feature unique to plant Bcl-2–associated athanogene (BAG) proteins (5). Like their mammalian counterparts, the plant BAGs are multifunctional and regulate cytoprotective processes from pathogen attack to abiotic stress and development (5). Our initial studies focused on two structural representative groups of the Arabidopsis BAG family, AtBAG4 (structurally similar to mammalian BAGs) and AtBAG6 (unique to plants). Transgenic tobacco overexpressing AtBAG4 provided enhanced tolerance to several abiotic stress stimuli, including cold, salt, and drought (5). Interestingly, AtBAG6 appears to play a role within basal defense pathways (5). These results suggest that the Arabidopsis BAG family may have developed specialized roles for cell regulation in response to various stress stimuli.

Previously, it was shown that mammalian BAGs localize either to the nucleus or to cytoplasm. In plants BAG localization is more diverse (nucleus, vacuole, mitochondria, cytoplasm), and in this study we show that the Arabidopsis BAG member AtBAG7 resides in the endoplasmic reticulum (ER). The ER is essential for the maturation of proteins, performing processes such as correct folding, disulfide bond formation, and protein assembly. The synthesis of steroids, lipids, and choles-terol also are ER-specific processes. Several physiological conditions including nutrient deprivation, alterations in redox balance, and changes in calcium homeostasis can perturb these activities, causing an abundance of unfolded proteins, ER stress, and eventually cell death (15). To alleviate the detrimental effects of an accumulation of misfolded protein, eukaryotes have developed an ER–nucleus signaling pathway termed the “unfolded protein response” (UPR) (15, 16). The UPR serves to restore normal function of the cell by halting protein translation and activates signaling pathways that lead to increased production of molecular chaperones involved in protein folding. In contrast to yeast and mammals, the underlying mechanisms of the UPR in plants are not well documented. Recent data, however, suggest that the UPR is important in plants, particularly in response to both abiotic and biotic stimuli where it triggers a variety of processes, including attenuation of protein translation, activation of the ER-associated degradation pathway, or the induction of ER chaperones (17–23).

Here we show that AtBAG7 localizes to the ER and is involved in the maintenance of the UPR. Consistent with the premise that BAG proteins and ER chaperones are involved in stress adaptation and PCD pathways in plants, AtBAG7 knockouts were hypersensitive to known ER stress inducers such as heat, cold, and tunicamycin (Tm), a chemical inducer of ER stress. Furthermore, with bimolecular fluorescence complementation (BiFC), AtBAG7 was shown to interact in vivo with immunoglobulin-binding protein (BiP), an Hsp70 paralog and known component of the UPR. Real-time analysis of a range of ER stress proteins demonstrated selective up-regulation of the Hsp AtBiP3 in AtBAG7-null mutants subjected to heat and cold stress. These findings indicate that AtBAG7 is a functional homolog of the BAG family with a unique ER location and is an important cytoprotective cochaperone for ER stress responses as part of the UPR.

Results

A Member of the Arabidopsis BAG Family Is Localized to the ER.

Although there has been extensive research on BAG genes in mammals, far less is known about plant BAG function. In a previous study we used structural criteria rather than primary sequence to search the Arabidopsis genome for BAG-like proteins. Seven genes were identified, two of which, AtBAG4 and AtBAG6, have demonstrated involvement in plant PCD regulatory pathways and thus have functional similarity to the mammalian BAG genes (5, 24). In this study we focused on AtBAG7 (AT5G62390). The coding region of atbag7 was fused to the C terminus of Enhanced-YFP in the 35S promoter-driven expression cassette, pEarleygate104 (25). Microprojectile bombardment into onion epidermal cells was performed, and YFP-fusion expression was visualized by confocal microscopy. Upon visualization, YFP-AtBAG7 colocalized with the ER-specific dye, ER-Tracker (Invitrogen), and with Arabidopsis wall-associated kinase 2 red fluorescent protein (AtWAK2-RFP), a known ER marker (Fig. 1) (26). This localization was confirmed in green tissue using recombinant Agrobacterium harboring pBAG704. Three days after infiltration, the leaves were stained with ER-Tracker, and the YFP-AtBAG7 fusion was visualized by confocal microscopy. Interestingly, the localization of AtBAG7 to the ER was not predicted by the computer-based localization programs pSORT and signalP, both of which suggested a nuclear localization. However, we cannot exclude the potential translocation of AtBAG7 to other areas of the cell, including the nucleus, given the appropriate signal.

Fig. 1.

The N-terminal YFP-AtBAG7 fusion localizes to the ER. Onion epidermal cells were microprojectile bombarded with YFP-AtBAG7 (A–C) or cobombarded with YFP-AtBAG7 + AtWAK2-RFP (D–F). Two days after bombardment, cells were stained with ER Tracker (A–C) or visualized directly (D–F) by confocal microscopy. (A) YFP-AtBAG7 overlay. (B) ER-Tracker overlay. (C) YFP/ER-Tracker overlay. (D) YFP-AtBAG7 overlay. (E) RFP-AtWAK2 overlay. (F) YFP/RFP overlay.

AtBAG7 Binds the Hsp70 Paralog, BiP.

Distinguishing features of mammalian BAG proteins include the presence of a conserved BD and the ability to bind Hsp70 (7, 8, 10). Previously we showed that a member of the Arabidopsis BAG family, AtBAG4, is able to bind an Arabidopsis Hsp70 (5). Therefore, we considered whether AtBAG7 also could interact with Hsp70. Because our initial localization studies indicated that AtBAG7 resides in the ER, we concentrated on the ER-localized Hsp70 paralog, Arabidopsis luminal binding protein BiP2 (AT5G42020). To determine whether AtBAG7 and AtBiP2 interact, we performed an in vivo BiFC assay. The coding region of GFP was split (amino acids 1–174 and 175-end) and cloned independently as N-terminal fusions to AtBiP2 and AtBAG7 to generate pBIP2080 and pBAG7096, respectively. Once generated, these constructs were cobombarded into onion epidermal cells and analyzed for interaction (GFP fluorescence) by confocal microscopy. As shown in Fig. 2, strong GFP expression was observed in samples comprising pBiP2080 and pBAG7096. However, cotransformation of pBAG7096 and an empty vector control did not result in a fluorescent signal. These results demonstrate that AtBAG7 interacts with AtBiP2 in vivo. This interaction was confirmed further by yeast two-hybrid analysis (Fig. S1).

Fig. 2.

AtBAG7 binds to the Hsp70 paralog, BiP. Onion epidermal cells were microprojectile bombarded with AtBAG7-N(GFP), AtBIP2-C(GFP), and RFP-AtWAK2. Two days after bombardment, cells were visualized by confocal microscopy. (A) AtBAG7-N(GFP) + AtBIP2-C(GFP) overlay. (B) RFP-AtWAK2 overlay. (C) A and B overlay.

AtBAG7 Is Involved in Maintenance of the Unfolded Protein Response.

Recently a role for the ER as a cellular sentinel to detect and alleviate stress as part of plant adaptation pathways has been recognized (27). During ER stress, BiP and other Ca²⁺-dependent folding proteins, including calreticulin (CRT) and calnexin (CNX), are expressed as part of the UPR to create an optimal environment for proper protein folding (17, 19, 21, 27–31). Because AtBAG7 interacts with a major mediator of ER stress, AtBiP, we considered whether it plays a role within the UPR. We induced ER stress and a UPR in AtBAG7 knockouts (Salk_065883 and Salk_058247) using Tm, an inhibitor of glycosylation (18, 20). Five-day-old wild-type and AtBAG7-knockout seedlings grown on solid Murashige and Skoog (MS) medium were transferred to liquid MS with or without 0.5 μg/mL Tm, were incubated for 6 hours, and were llowed to recover for 5 days in the absence of Tm. In comparison with wild-type plantlets and untreated controls, treated AtBAG7 knockouts exhibited pronounced growth retardation and chlorosis after 5 days (Fig. 3). These results are consistent with a role for AtBAG7 within the UPR.

Fig. 3.

Tunicamycin accelerates cell death in AtBAG7 knockouts. Seven-day-old wild-type and AtBAG7-mutant plantlets were submerged in 1/2 MS liquid containing, 0.5 μg mL⁻¹ Tm for 6 h, washed twice in 1/2 MS containing no antibiotic, and allowed to recover on 1/2 MS agar. (A) Wild-type MS-treated plantlets. (B) Wild-type Tm-treated plantlets. (C) AtBAG7Δ MS-treated plantlets. (D) AtBAG7Δ Tm-treated plantlets. Plants were analyzed for phenotypic differences 5 days posttreatment.

AtBAG7 Plays an Integral Role in Abiotic Stress Tolerance.

Previously we demonstrated that members of the Arabidopsis BAG family play a role in the regulation of PCD pathways induced by abiotic stress, including cold and salt, as well as in pathways induced by biotic challenge (5). Several biotic and abiotic stresses, including excessive heat and cold, are known to induce ER stress (19, 21, 22, 32). Because AtBAG7 could play a role within ER stress pathways, including the UPR, we investigated whether AtBAG7 impacts plant tolerance to abiotic stimuli that induce ER stress. Five-day-old Arabidopsis BAG7-knockout mutants were subjected to heat or cold stress, and phenotypes were assessed following a recovery period of 4 days. As a positive control for heat sensitivity, AtBiP2-knockout plants were analyzed also, because AtBiP2 is a known component of heat-shock responses (32). As shown in Fig. 4, both AtBAG7 and AtBiP2 mutants were more sensitive than wild-type controls to heat treatment(50 °C for 30 min). In addition to heat stress, AtBAG7 knockouts also were more sensitive than wild-type plants to severe cold stress (−20 °C for 90 min) (Fig. 4). These results correlate with previous reports that Arabidopsis BAGs function in cytoprotection. Therefore, AtBAG7 is localized within the ER and is involved in plant PCD pathways, particularly in response to abiotic stress (5, 33).

Fig. 4.

AtBAG7 knockouts are susceptible to heat and cold stress. Five-day-old wild-type, AtBiP2 –mutant (Salk_073202), and AtBAG7-mutant (Salk_058247) Arabidopsis plants were grown on 1/2 MS, 2% sucrose and subjected to heat treatment at 50 °C for 30 min or cold treatment at −20 °C for 90 min. For complementation of the heat-sensitive phenotype TUDCA was added 1 day before heat treatment, 4-day-old wild-type and AtBiP2- and AtBAG7-knockout (Salk_073202 and _058247, respectively) plantlets grown on 1/2 MS media were transferred to 1/2 MS media containing 0.5 mM TUDCA. (A) Wild-type untreated plantlets. (B) AtBiP2Δ untreated plantlets. (C) AtBAG7Δ untreated plantlets. (D) Wild-type heat-treated plantlets. (E) AtBiP2Δ heat-treated plantlets. (F) AtBAG7Δ heat-treated plantlets. (G) Wild-type cold-treated plantlets. (H) AtBiP2Δ plantlets pretreated with TUDCA, no heat stress. [To serve as a comparative control, wild-type plantlets were used in each of the plates above (Top Row).] (I) AtBAG7Δ plantlets pretreated with TUDCA, no heat stress*. (J) AtBAG7Δ cold-treated plantlets. (K) AtBiP2Δ plantlets pretreated with TUDCA, heat stressed*. (L) AtBAG7Δ plantlets pretreated with TUDCA, heat stressed*. Photographs were taken after a 5-day recovery period.

Chemical Chaperones Revert the AtBAG7-Knockout Heat-Sensitive Phenotype During Heat Stress.

During heat stress, unfolded proteins accumulate within the ER, resulting in ER stress and eventual cell death (34). To overcome the abundance of unfolded proteins and to alleviate ER stress, cells increase expression of folding proteins and chaperones including BiP as part of the UPR. Tauroursodexycholic acid (TUDCA), a chemical chaperone that stabilizes protein conformation and promotes proper folding, alleviates ER stress in plants and mammals (22, 35). In light of our BiFC and abiotic stress results indicating a role for AtBAG7 within the UPR, we attempted to complement the heat-sensitive AtBAG7 knockouts with the addition of TUDCA. One day before heat treatment, 4-day-old wild-type, AtBiP2, and AtBAG7 plantlets grown on MS medium were transferred to MS medium containing 0.5 mM TUDCA. As shown in Fig. 4 and consistent with previous studies performed on AtBiP2 mutants, the survival rate of ER-stressed (i.e., heat-stressed) AtBAG7 knockout plants was increased significantly by pretreatment with TUDCA (Fig. 4). The alleviation of the AtBAG7 sensitive phenotype by the chemical chaperone TUDCA is consistent with AtBAG7 chaperone activity during abiotic-induced ER stress.

During Abiotic Stress BiP3 Transcript Levels Are Increased in the AtBAG7-Null Mutant.

The previous results demonstrate a chaperoning activity for AtBAG7 during stress and maintenance of the UPR. Numerous studies have shown increased levels of ER chaperones, including BiP and Ca²⁺-dependent ER folding proteins, during ER stress and the UPR, (22, 30, 31, 36). Therefore, using real-time PCR, we considered whether AtBAG7 expression also is induced upon ER stress. In addition to AtBAG7, we analyzed the expression of several ER stress markers in the AtBAG7-mutant genotype. ER stress was induced in wild-type and AtBAG7-knockout mutants by heat treatment (50 °C for 30 min) or cold treatment (−20 °C for 90 min), and the expression patterns of AtBAG7 and the ER stress markers BiP1/BiP2, BiP3, CNX, CRT, protein disulfide isomerase, and Bax inhibitor (Bi-1) were quantified by real-time PCR (Table S1) . Consistent with previous studies, the real-time data demonstrated increases in chaperone expression for both heat and cold treatment in wild-type plants (22, 30, 31, 36). The real-time analysis failed to demonstrate an increase in AtBAG7 transcript for either heat or cold treatment. This finding suggests that AtBAG7 activity is regulated posttranscriptionally. Interestingly, expression of BiP1/BiP2, CRT, protein disulfide isomerase, and BI-1 was not affected in the AtBAG7-null mutants (Fig. S2), BiP3 expression, however, was induced earlier and to higher levels following both heat and cold stress (Fig. S3). These data provide further evidence of a role for AtBAG7 within heat and cold stress pathways.

Discussion

The BAG family is a multifunctional group of cytoprotective proteins that are distinguished by a common region, the BD, which mediates interaction with Hsp70 proteins (7, 10–12). BAG genes are broadly conserved, are found in yeasts, plants, and animals, and function as adapter proteins/cochaperones, forming complexes with signaling molecules (e.g., Siah) and molecular chaperones (e.g., Hsc70-interacting protein, Hsp70) (37, 38). The Arabidopsis genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs (5, 12). Like their mammalian counterparts, plant BAG members are multifunctional and regulate PCD processes, including those induced by pathogen attack, abiotic stress, and development (5). In this study we used a combination of reverse and chemical genetics to demonstrate that AtBAG7, an Arabidopsis BAG member, is an important component in the mediation of ER–PCD pathways, including the UPR, during abiotic stress adaptation.

The first aim of this study was to localize AtBAG7 within the cell. Using E-YFP as a marker, an YFP-AtBAG7 fusion was used to trans-form transiently onion epidermal cells and Nicotiana benthamiana leaf tissue where it colocalized with the ER-specific stain, ER-Tracker, and molecular marker AtWAK2 (26). This localization was consistent with proteomic studies that suggested an ER localization for the gene product of the At562390 locus which we have identified as AtBAG7 (39).

The ER has become a major focal point in PCD research in animals and plants, particularly in relation to stress responses through the induction of the UPR (reviewed in ref. 40). A principal component of this research was to determine whether and to what extent AtBAG7 is involved in ER stress pathways. Using BiFC, we demonstrated direct interaction of AtBAG7 with the ER-localized Hsp70 paralog, AtBiP (32). As a marker of ER stress, BiP is well documented as an integral player of the UPR, performing roles including protein translocation, folding, and assembly of ER-modified proteins (32). Although the mechanistic consequences of AtBAG7/BiP binding are unknown, it is reasonable to hypothesize that this interaction is part of a complex required for proper maintenance of the UPR, especially because AtBAG7 knockouts are sensitive to ER stress. Alternatively, and consistent with our real-time data, AtBAG7 may serve a role in the UPR via indirect regulation of BiP3 expression.

Mammalian BAGs are known cochaperones that are involved in PCD pathways during abiotic and biotic stress responses (41–43). Although the demonstration of AtBAG7 and BiP interaction via BiFC assays suggests a role for AtBAG7 as a cochaperone for the mainten-ance of the UPR, further studies were required to confirm this inter-action. Tm, a specific inducer of ER stress, has been used to identify key regulators of ER stress pathways including BiP, Bi-1, and the membrane-tethered transcription factors bZIP28 and bZIP60 (22, 28, 30, 31, 36). Consistent with these studies, AtBAG7 knockouts were significantly more sensitive than wild-type plants to Tm treatment. During Tm treatment unfolded proteins accumulate in the ER; cytoprotective AtBAG7 may delay ER-induced cell death as part of the UPR.

BiP is a chaperone that directs heat-shock responses (22, 32). To confirm that AtBAG7 aids BiP in the protection of cells during heat-induced ER stress via cochaperone activity, we tested whether pretreatment with the chemical chaperone TUDCA could protect heat-stressed AtBAG7 mutants. Treatment with TUDCA compensated for the lack of AtBAG7 and protected AtBAG7 knockouts undergoing heat stress. Collectively, the Tm and TUDCA results confirm a role for AtBAG7 as a cochaperone for the coordination of the UPR and delay of ER-induced cell death.

As mentioned, AtBAG7 also may play a role within the UPR indirectly by regulating BiP expression. Our gene-expression analysis using real-time PCR indicates that BiP3 is significantly induced in the AtBAG7-mutant background during heat and cold stress. In the knockouts, the plant may compensate for the lack of AtBAG7 activity by inducing the expression of another ER chaperone, BiP3. Importantly, no other chaperones were specifically induced in the AtBAG7 knockouts. This finding may indicate that the other chaperones are not involved with AtBAG7 or that their regulation does not occur at the transcriptional level. AtBAG7 transcript levels also were not altered upon heat and cold treatment, thus suggesting that AtBAG7 is regulated at the posttranscriptional level. These observations are in accordance with our bioinformatic studies that failed to identify ER stress elements in the AtBAG7 promoter. The increased level of BiP3 in AtBAG7 knockouts raises the possibility that removal of AtBAG7 from the ER activates a transcription factor required for BiP3 expression. This possibility is consistent with studies involving bZIP60, a transcription factor that regulates BiP3 expression but makes only minor contributions to BiP1/2 (31). Our results show that BAG7 may be required for the optimal regulation of BiP3 under heat and cold stress.

The ER has been associated with biotic responses, and previous studies have shown that AtBAG6, another BAG that contains a calmodulin-binding domain, may be involved in basal defense (5). Therefore we assessed whether AtBAG7 also is involved in biotic stress responses. AtBAG7 knockouts were challenged with the bacterial pathogen Pseudomonas syringae DC3000, and the fungal necrotrophs Botrytis cinerea, Sclerotinia sclerotiorum, and Alternaria alternata. In contrast to AtBAG6, no significant differences were observed between the AtBAG7 knockouts and the wild-type controls. This finding indicates that, unlike AtBAG6, AtBAG7 is involved primarily within abiotic stress pathways.

Plants must adapt continually to the changing environment. An in-crease in freezing tolerance known as “cold acclimation” typically is observed after plants are exposed to low nonfreezing temperatures. In this study we subjected plants to severe cold without prior acclimation. During freezing temperatures, cell death occurs because of irreversible damage to cellular membranes, particularly during the thawing process. To circumvent this damage, plant membranes, particularly the plasma membrane, undergo structural changes including the accumu-lation of mono- or diunsaturated species of phosphatidylcholine which increase cryostability (44, 45). Plasma membrane proteins may play an important role during cold acclimation (46–48). A recent study by Kawamura and Uemura (33) identified substantial changes in the protein content for range of plasma membrane proteins during the early stages of cold acclimation. These changes included increased levels of the gene product of locus At5g62390 which we identified as AtBAG7. In light of these findings, future studies involving the translocation of AtBAG7 during cold acclimation are of interest.

In summary, this paper details the investigation of an Arabidopsis BAG protein, AtBAG7. In contrast to the mammalian BAGs, the Arabidopsis BAGs appear to have diverse specificities and are localized to a variety of subcellular organelles for a range of cellular functions. Here we show that AtBAG7 is an ER-localized chaperone that is involved in the maintenance of ER stress pathways, a unique role for BAG proteins. Specifically, AtBAG7 is integral for the survival of heat- and cold-stressed cells by delaying PCD pathways of cells undergoing ER stress. This finding is consistent with our previous data that linked AtBAG4 with inhibition of PCD, including DNA laddering and TUNEL-positive nuclei, during cold stress. Taken together, these results further demonstrate the importance of the BAG family in plant cell-death pathways and cytoprotection.

Materials and Methods

Materials and Growth Conditions.

Arabidopsis transfer-DNA insertion mutants of BAG7 (Salk_065883, 058247) and BiP (Salk_073202) in the col-1 background were acquired from the Arabidopsis Biological Resource Center (ABRC). Unless stated otherwise, all plants and cells were grown at 22 °C using a 16-h photoperiod. Plasmids comprising RFP localized to the ER (ER-rk CD3-959), the YFP-fusion construct pEarleygate104 (CD3-686), and BiFC constructs pSAT4A-DEST-n(1-174)EYFP-N1 (CD3-1080) and pSAT5A-DEST-c(175-end)EYFP-N1 (CD3-1096) also were acquired from the ABRC.

Microprojectile Bombardment of Onion Cells.

Onion inner epidermal peels were plated onto MS medium without vitamins [MS salts, 2% (wt/vol) sucrose, 2% agar] no more than 1 h before bombardment. Gold particles (1 μm) (Biorad) were coated with the respective plasmid DNAs in an equal molar ratio and were introduced transiently into epidermal cells by microprojectile bombardment using a Bio-Rad PDS/1000 helium-driven particle accelerator. Bombardment parameters were essentially as outlined in ref. 49 and included the following: 1,100 psi bombardment pressure, helium vacuum of 27 in. Hg, and a distance of 12 cm from the microcarriers to the sample. All samples were incubated for ∼24–48 h under dark conditions at 22 °C before microscopy analysis.

BiFC Analysis.

The AtBAG7-containing fragment was subcloned using LR Clonase II (Invitrogen) from pENTRBAG7 into the destination vector pSAT5A-DEST-c(175-end)EYFP-N1 to generate pBAG7097. Simultaneously, the AtBiP2-containing fragment was cloned from pENTRBiP2 into pSAT4A-DEST-n(1-174)EYFP-N1 to generate pBiP2089. Once generated, pBAG7096 and pBiP2080 were cobombarded into onion epidermal cells. Three days after bombardment, fluorescent cells were viewed by confocal microscopy as described above using the appropriate GFP filter (excitation 474 nm /emission 510 nm).

Abiotic Stress Assays.

Wild-type, AtBAG7-knockout (Salk_065883 and 058247), and AtBiP2-knockout (Salk_073202) seeds were surface sterilized, vernalized at 4 °C for 48 h, germinated on 1/2 MS containing 2% sucrose, and grown for 5 days. All plantlets were subjected to either heat stress (50 °C for 30 min) or cold stress (−20 °C/90 min) and allowed to recover for 4 days before phenotypic analysis. Each treatment was performed in duplicate, and each experiment was repeated twice.

Chemical Chaperone Protection Assay.

Wild-type, AtBAG-knockout, and AtBiP2-knockout seeds were germinated and grown for 5 days as described above. One day before heat treatment (50 °C for 30 min), five plantlets of each genotype were transferred to fresh MS medium or to MS medium containing 0.5 mM TUDCA. After treatment plants were allowed to recover for 4 days before phenotypic analysis.

Tunicamycin Assays.

Wild-type and Arabidopsis BAG7-knockout seeds were prepared and grown as described in the abiotic stress assays. One week after germination, five plantlets of each genotype were submerged in 1/2 MS liquid containing, 0.5 μg mL⁻¹ Tm for 6 h, washed twice in 1/2 MS containing no antibiotic, and grown for a further 5 days on 1/2 MS agar. Control (MS only) plants and Tm-treated plants were analyzed for phenotypic differences and photographed.