The Arabidopsis J-Protein AtDjC5 Facilitates Thermotolerance Likely by Aiding in the ER Stress Response

AtDjC5 belongs to the J-protein family in Arabidopsis thaliana. Its biological functions remain unclear. In this study, we examined the roles of AtDjC5 in resisting heat stress using reverse genetic analysis. After the seedlings were exposed directly to 44 °C for 90 min, AtDjC5 knockout seedlings displayed decreases in the survival rate, membrane system stability, and cell vitality compared to WT seedlings, indicating that AtDjC5 is involved in plant basal thermotolerance. The AtDjC5 knockout seedlings pre-exposed to 37 °C for 30 min exhibited decreases in the survival rate and total chlorophyll contents and increased cell death when they were subsequently exposed to 45 °C compared to the WT seedlings, indicating that AtDjC5 plays an important role in plant acquired thermotolerance. AtDjC5 was found to localize to the endoplasmic reticulum. The expression of the AtDjC5 gene was induced by heat and TM (an ER stress inducer) treatment. Furthermore, we found that the knockout of AtDjC5 inhibited ER stress-induced autophagy and the expression of ER stress-related genes. Taken together, these results suggest that AtDjC5 facilitates thermotolerance, likely by aiding in the ER stress response.


Introduction
A higher growing season temperature has great impacts on agricultural production, farm income, and food security [1][2][3]. High-temperature stress disturbs cellular protein homeostasis, leading to protein denaturation or aggregation [4][5][6]. Thermotolerance is an important component of organisms to adapt to high-temperature stress and is divided into basal and acquired thermotolerance [7,8]. Plants pre-exposed to a nonlethal hightemperature condition for a certain period of time exhibit an evidently increased tolerance to lethal high temperature. This is called acquired thermotolerance. The best-characterized aspect of acquired thermotolerance is the production of heat-shock proteins (HSPs), which are regulated by heat shock transcription factors [9][10][11][12]. HSPs function as molecular chaperones to allow cellular proteins to avoid and/or recover from stress-induced protein aggregation [13][14][15][16].
The accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) generates a condition called ER stress, which may activate the unfolded protein response (UPR), also called the ER stress response. The UPR is a stress mitigation mechanism that monitors protein folding in the cellular secretory pathway [17][18][19]. To alleviate ER stress, the UPR is activated to produce molecular chaperones or HSPs to aid in protein folding [20,21]. The molecular mechanism of the UPR in plant cells is much less well-understood than those in yeast and animal cells. In Arabidopsis thaliana, bZIP28 and bZIP60 of the basic domain/leucine zipper (bZIP) family have been identified as ER stress-activated membrane-related transcription factors [22,23]. bZIP60 mRNA is spliced

Knockout of the AtDjC5 Gene Decreased Basal Thermotolerance
To understand the role of AtDjC5 in adapting to heat stress, we identified a homozygous T-DNA insertion mutant for the AtDjC5 gene, djc5-1. Sequence analysis of the T-DNA flanking regions revealed that djc5-1 contained a T-DNA insertion in intron 2 of AtDjC5 ( Figure 1A). RT-PCR showed no detectable full AtDjC5 transcript in the djc5-1 plants ( Figure 1B). To assess the effect of AtDjC5 knockout on plant basal thermotolerance, the survival rates were compared between the WT and djc5-1 plants after the seedlings were exposed directly to 44 • C for 90 min. The survival rate of the djc5-1 seedlings was not different from that of the WT seedlings under normal growth conditions. However, the djc5-1 seedlings exhibited hypersensitivity after heat shock (HS). While 49% of the WT seedlings were alive after HS, the survival rate after HS was 31% for the djc5-1 seedlings ( Figure 1C,D).
As an additional test of heat sensitivity, the electrolyte leakage level was compared between the WT and djc5-1 leaves. Under non-HS conditions, there were no significant differences in the ion conductivity of the incubation medium between the WT and djc5-1 leaves ( Figure 1E). After HS at 45 • C for 120 min, the ion conductivity of the incubation medium was markedly increased in WT and djc5-1, and the ion conductivity in djc5-1 was distinctly higher than that in WT ( Figure 1E). The results indicated that AtDjC5 is important for the stability of the membrane system during HS. (D) Survival rates of WT and atdj5c-1 seedlings. Each value is the m SD of ten biological replicates. (E) Electrolyte leakage assay for the WT and djc5-1 leaves. Th are the means ± SDs of three independent experiments. (F) 2,3,5-triphenyltetrazolium ch (TTC) reduction activity of roots for WT and djc5-1. The data are the means ± SDs of four bio replicates, with a total of 30 roots per sample. Asterisks indicate significant differences from t of the same treatment (t test, * p < 0.05, ** p < 0.01).
As an additional test of heat sensitivity, the electrolyte leakage level was com between the WT and djc5-1 leaves. Under non-HS conditions, there were no signi differences in the ion conductivity of the incubation medium between the WT and leaves ( Figure 1E). After HS at 45 °C for 120 min, the ion conductivity of the incub medium was markedly increased in WT and djc5-1, and the ion conductivity in djc5distinctly higher than that in WT ( Figure 1E). The results indicated that AtDjC5 portant for the stability of the membrane system during HS.
To further confirm the role of AtDjC5 in plant thermotolerance, we compare difference in cell vitality between the WT and djc5-1 roots. Under non-HS condition TTC reduction activity in the djc5-1 roots was not obviously different from that in th roots; however, after HS at 45 °C for 1 h, the TTC reduction activity in the WT and roots was markedly decreased, and the TTC reduction activity in djc5-1 was mar lower than that in WT ( Figure 1F). HS prevented the reduction in TTC much m atdjc5-1 than in WT. Taken together, our data suggest that AtDjC5 knockout accele HS-induced cell death and improved the basal thermotolerance of plants. (D) Survival rates of WT and atdj5c-1 seedlings. Each value is the mean ± SD of ten biological replicates. (E) Electrolyte leakage assay for the WT and djc5-1 leaves. The data are the means ± SDs of three independent experiments. (F) 2,3,5-triphenyltetrazolium chloride (TTC) reduction activity of roots for WT and djc5-1. The data are the means ± SDs of four biological replicates, with a total of 30 roots per sample. Asterisks indicate significant differences from the WT of the same treatment (t test, * p < 0.05, ** p < 0.01).
To further confirm the role of AtDjC5 in plant thermotolerance, we compared the difference in cell vitality between the WT and djc5-1 roots. Under non-HS conditions, the TTC reduction activity in the djc5-1 roots was not obviously different from that in the WT roots; however, after HS at 45 • C for 1 h, the TTC reduction activity in the WT and djc5-1 roots was markedly decreased, and the TTC reduction activity in djc5-1 was markedly lower than that in WT ( Figure 1F). HS prevented the reduction in TTC much more in atdjc5-1 than in WT. Taken together, our data suggest that AtDjC5 knockout accelerated HS-induced cell death and improved the basal thermotolerance of plants.

AtDjC5 Is Involved in the Regulation of Acquired Thermotolerance
To determine the role of AtDjC5 in plant acquired thermotolerance, we generated two other AtDjC5 mutant lines (djc5-2 and djc5-3) through the artificial microRNA method. RT-PCR showed no detectable full AtDjC5 transcript in the djc5-2 and djc5-3 seedlings (Figure 2A). The 11-day-old seedlings grown at 22 • C were acclimated at 37 • C for 30 min and then returned to 22 • C for 120 min before challenge at 45 • C for 120 min. The survival rate of seedlings after HS was compared among WT, djc5-1, djc5-2, and djc5-3. The results showed that these three AtDjC5 mutant seedlings were hypersensitive to HS. While 68% of WT seedlings were alive after HS, the survival rates after HS were 56% for djc5-1, 46% for djc5-2, and 51% for djc5-3 ( Figure 2B,C). The decrease in acquired thermotolerance in the AtDjC5 mutants was confirmed by testing the total chlorophyll contents of seedlings. Before HS, the total chlorophyll contents in the three AtDjC5 mutant lines were not obviously different from those in WT; however, the total chlorophyll contents in the three AtDjC5 mutant lines were markedly lower than those in WT after HS ( Figure 2D). As an additional test of heat sensitivity, heat-induced cell death was assayed in WT, djc5-1, djc5-2, and djc5-3 by trypan blue staining. The three AtDjC5 mutant lines exhibited increased cell death after HS compared to WT ( Figure 2E).

AtDjC5 Is Involved in the Regulation of Acquired Thermotolerance
To determine the role of AtDjC5 in plant acquired thermotolerance, we gen two other AtDjC5 mutant lines (djc5-2 and djc5-3) through the artificial micr method. RT-PCR showed no detectable full AtDjC5 transcript in the djc5-2 and seedlings ( Figure 2A). The 11-day-old seedlings grown at 22 °C were acclimated at for 30 min and then returned to 22 °C for 120 min before challenge at 45 °C for 12 The survival rate of seedlings after HS was compared among WT, djc5-1, djc5-djc5-3. The results showed that these three AtDjC5 mutant seedlings were hypersen to HS. While 68% of WT seedlings were alive after HS, the survival rates after HS 56% for djc5-1, 46% for djc5-2, and 51% for djc5-3 ( Figure 2B,C). The decrease in acq thermotolerance in the AtDjC5 mutants was confirmed by testing the total chlor contents of seedlings. Before HS, the total chlorophyll contents in the three AtDjC tant lines were not obviously different from those in WT; however, the total chlor contents in the three AtDjC5 mutant lines were markedly lower than those in WT HS ( Figure 2D). As an additional test of heat sensitivity, heat-induced cell death w sayed in WT, djc5-1, djc5-2, and djc5-3 by trypan blue staining. The three AtDjC5 m lines exhibited increased cell death after HS compared to WT ( Figure 2E). (C) Survival rates of WT and AtDjC5 mutant seedlings. Each value is the mean ± SD of ten biological replicates. (D) Total chlorophyll contents in WT, djc5-1, djc5-2, and djc5-3 leaves. The data are the means ± SDs of four biological replicates. (E) Analysis of cell death in the WT and three AtDjC5 mutant lines before and after heat stress by trypan blue staining. Asterisks indicate significant differences from the WT of the same treatment (t test, * p < 0.05, ** p < 0.01). Furthermore, we examined the effect of AtDjC5 overexpression on plant acquired thermotolerance. A binary vector containing the AtDjC5 coding region (pCAMBIA1300-35S::AtDjC5) or an empty vector (pCAMBIA1300-35S) was transformed into Col plants. PCR assays confirmed that the AtDjC5 gene was introduced into all AtDjC5 transgenic lines obtained. Q-PCR assays showed that the levels of AtDjC5 mRNA were strongly increased in the OE1 and OE2 overexpression lines compared to the WT and transgenic lines containing an empty vector (EV) ( Figure 3A). The WT, EV, and AtDjC5 overexpression seedlings grown at 22 • C were acclimated at 37 • C for 30 min and then returned to 22 • C for 120 min before challenge at 45 • C for 120 min. While 50% of the WT seedlings were alive after HS, the survival rates after HS were 44% for EV, 66% for OE1, and 93% for OE2 ( Figure 3B,C), indicating that AtDjC5 overexpression increased the acquired thermotolerance. Taken together, our data suggest a role for AtDjC5 in improving the acquired thermotolerance of A. thaliana seedlings. mutant lines before and after heat stress by trypan blue staining. Asterisks indicate significant differences from the WT of the same treatment (t test, * p < 0.05, ** p < 0.01). Furthermore, we examined the effect of AtDjC5 overexpression on plant acquired thermotolerance. A binary vector containing the AtDjC5 coding region (pCAM-BIA1300-35S::AtDjC5) or an empty vector (pCAMBIA1300-35S) was transformed into Col plants. PCR assays confirmed that the AtDjC5 gene was introduced into all AtDjC5 transgenic lines obtained. Q-PCR assays showed that the levels of AtDjC5 mRNA were strongly increased in the OE1 and OE2 overexpression lines compared to the WT and transgenic lines containing an empty vector (EV) ( Figure 3A). The WT, EV, and AtDjC5 overexpression seedlings grown at 22 °C were acclimated at 37 °C for 30 min and then returned to 22 °C for 120 min before challenge at 45 °C for 120 min. While 50% of the WT seedlings were alive after HS, the survival rates after HS were 44% for EV, 66% for OE1, and 93% for OE2 ( Figure 3B,C), indicating that AtDjC5 overexpression increased the acquired thermotolerance. Taken together, our data suggest a role for AtDjC5 in improving the acquired thermotolerance of A. thaliana seedlings.

(B) Comparison of WT and
AtDjC5-overexpressing seedling viability. (C) Survival rates of WT and AtDjC5 overexpression seedlings. Each value is the mean ± SD of ten biological replicates. Asterisks indicate significant differences from WT of the same treatment (t test, ** p < 0.01). AtDjC5 transcript level in the WT sample was used as the calibrator and was set to 1. Data are the means ± SDs of three biological replicates. (B) Comparison of WT and AtDjC5-overexpressing seedling viability. (C) Survival rates of WT and AtDjC5 overexpression seedlings. Each value is the mean ± SD of ten biological replicates. Asterisks indicate significant differences from WT of the same treatment (t test, ** p < 0.01).

Expression Pattern and Subcellular Localization of AtDjC5
Bioinformatics analysis showed that AtDjC5 may exist in the cytosol, endoplasmic reticulum (ER), Golgi apparatus, and other cellular compartments (http://utoronto.ca/ cell_efp/cgi-bin/cell_efp.cgi). We examined the subcellular localization of AtDjC5 using transgenic plants stably expressing 35S::AtDjC5-sGFP. ER-Tracker Red, an ER-specific fluorescent dye, was used to highlight the ER in the cells of root hairs. Merged images of GFP fluorescence (green) and ER-Tracker Red fluorescence (red) showed bright yellow fluorescence, indicating that the AtDjC5-sGFP fusion protein was partially present in the ER ( Figure 4A).

Expression Pattern and Subcellular Localization of AtDjC5
Bioinformatics analysis showed that AtDjC5 may exist in the cytosol, endoplasmic reticulum (ER), Golgi apparatus, and other cellular compartments (http://utoronto.ca/cell_efp/cgi-bin/cell_efp.cgi). We examined the subcellular localization of AtDjC5 using transgenic plants stably expressing 35S::AtDjC5-sGFP. ER-Tracker Red, an ER-specific fluorescent dye, was used to highlight the ER in the cells of root hairs. Merged images of GFP fluorescence (green) and ER-Tracker Red fluorescence (red) showed bright yellow fluorescence, indicating that the AtDjC5-sGFP fusion protein was partially present in the ER ( Figure 4A).  The spatiotemporal expression pattern of the AtDjC5 gene was examined using two approaches. First, the GUS reporter assay was used to monitor the activity of the promoter of the AtDjC5 gene. The results obtained from transgenic plants harboring PAtDjC5::GUS showed that GUS under the AtDjC5 promoter was expressed in cotyledons, roots, stems, cauline leaves, flowers, and young siliques ( Figure 4B). Then, Q-PCR was used to compare the expression levels of AtDjC5 in different tissues. AtDjC5 was expressed to different degrees in all of the tissues analyzed, consistent with the results obtained by the GUS reporter assay ( Figure 4C). Furthermore, we investigated the effect of heat shock treatment on the expression of the AtDjC5 gene using Q-PCR. Ten-day-old seedlings grown at 22 • C were exposed to 37 • C for 0, 15, 30, 60 or 90 min, which induced an increase in the expression level of the AtDjC5 gene. The level of AtDjC5 mRNA began to increase 30 min after HS and continued to rise until 90 min ( Figure 5A). Due to the ER localization of AtDjC5, we evaluated the effect of tunicamycin (TM) treatment, an ER stress inducer, on AtDjC5 gene expression. After the 10-day-old seedlings grown at 22 • C were treated with 5 µg/mL TM for 2 h, the expression of the AtDjC5 gene was markedly increased, reaching 3.5 times that detected before treatment ( Figure 5B). The data above suggest that the expression of the AtDjC5 gene was induced by heat stress and ER stress.
ER-Tracker Red (red) fluorescence in the root hair cells was observed using a confocal laser microscope. In the photomicrographs on the right, a merge of the two images is presented, and yellow fluorescence was visible in the cells. (B) Transgenic plants harboring PAtDjC5::GUS were histochemically stained. GUS staining in 1, 6-day-old seedling; 2, cauline leaf; 3, stem; 4, anthotaxy; 5, young silique; 6, mature silique. (C) Tissue-specific expression of the AtDjC5 gene by Q-PCR. The expression level in roots was set to 1 and used for normalization. Data are the means ± SDs of three biological replicates. R, roots; St, stems; L, leaves; F, flowers; Si, siliques.
The spatiotemporal expression pattern of the AtDjC5 gene was examined using two approaches. First, the GUS reporter assay was used to monitor the activity of the promoter of the AtDjC5 gene. The results obtained from transgenic plants harboring PAtDjC5::GUS showed that GUS under the AtDjC5 promoter was expressed in cotyledons, roots, stems, cauline leaves, flowers, and young siliques ( Figure 4B). Then, Q-PCR was used to compare the expression levels of AtDjC5 in different tissues. AtDjC5 was expressed to different degrees in all of the tissues analyzed, consistent with the results obtained by the GUS reporter assay ( Figure 4C). Furthermore, we investigated the effect of heat shock treatment on the expression of the AtDjC5 gene using Q-PCR. Ten-day-old seedlings grown at 22 °C were exposed to 37 °C for 0, 15, 30, 60 or 90 min, which induced an increase in the expression level of the AtDjC5 gene. The level of AtDjC5 mRNA began to increase 30 min after HS and continued to rise until 90 min ( Figure 5A). Due to the ER localization of AtDjC5, we evaluated the effect of tunicamycin (TM) treatment, an ER stress inducer, on AtDjC5 gene expression. After the 10-day-old seedlings grown at 22 °C were treated with 5 μg/mL TM for 2 h, the expression of the AtDjC5 gene was markedly increased, reaching 3.5 times that detected before treatment ( Figure 5B). The data above suggest that the expression of the AtDjC5 gene was induced by heat stress and ER stress.  The effect of TM treatment on AtDjC5 expression. Ten-day-old seedlings grown at 22 • C were treated with 5 µg/mL TM for 2 h. The level of AtDjC5 in the nontreated seedlings was normalized to 1. Q-PCR was performed with AtDjC5-specific primers (Table S1). Actin was used as an internal control. Data are the mean ± SD from three independent experiments.

AtDjC5 Is Required for ER Stress-Induced Autophagy and the ER Stress Response
To clarify whether AtDjC5 is related to ER stress-induced autophagy, the induction of autophagy was examined in WT and djc5-1 mutant plants. Seven-day-old WT and djc5-1 seedlings were treated with 5 µg/mL TM for 8 h, followed by MDC staining; nontreated seedlings were used as a control. Both WT and djc5-1 seedlings showed very few autophagosomes in the control group ( Figure 6A). The WT seedlings showed markedly elevated autophagy after TM treatment; however, autophagy induction was not observed in response to TM treatment in djc5-1 seedlings ( Figure 6A). To quantify these results, autophagosome numbers were analyzed per root section for WT and djc5-1. Autophagosome numbers in the WT roots strongly increased after TM treatment; however, the djc5-1 roots did not show obvious differences from WT roots in autophagosome numbers after TM treatment ( Figure 6B), indicating that AtDjC5 is likely to be involved in the regulation of ER stress-induced autophagy.
22 °C were treated with 5 μg/mL TM for 2 h. The level of AtDjC5 in the nontreated seedlings was normalized to 1. Q-PCR was performed with AtDjC5-specific primers (Table S1). Actin was used as an internal control. Data are the mean ± SD from three independent experiments.

AtDjC5 Is Required for ER Stress-Induced Autophagy and the ER Stress Response
To clarify whether AtDjC5 is related to ER stress-induced autophagy, the induction of autophagy was examined in WT and djc5-1 mutant plants. Seven-day-old WT and djc5-1 seedlings were treated with 5 μg/mL TM for 8 h, followed by MDC staining; nontreated seedlings were used as a control. Both WT and djc5-1 seedlings showed very few autophagosomes in the control group ( Figure 6A). The WT seedlings showed markedly elevated autophagy after TM treatment; however, autophagy induction was not observed in response to TM treatment in djc5-1 seedlings ( Figure 6A). To quantify these results, autophagosome numbers were analyzed per root section for WT and djc5-1. Autophagosome numbers in the WT roots strongly increased after TM treatment; however, the djc5-1 roots did not show obvious differences from WT roots in autophagosome numbers after TM treatment ( Figure 6B), indicating that AtDjC5 is likely to be involved in the regulation of ER stress-induced autophagy.  Furthermore, we explored the possibility of AtDjC5 participating in UPR regulation. We examined the effects of TM treatment on the expression levels of the BiP1, BiP2, BiP3, bZIP17, bZIP28, and bZIP60 genes. Except for bZIP17, the expression levels of the other five genes were upregulated to different degrees in the WT and djc5-1 seedlings after treatment with 5 µg/mL TM for 30 min (Figure 7A-F). Meanwhile, we found that AtDjC5 knockout significantly suppressed the TM-induced upregulation of BiP3 and bZIP28 expression ( Figure 7G). The above results indicate that AtDjC5 is required for the ER stress response.
Furthermore, we explored the possibility of AtDjC5 participating in UPR regulation. We examined the effects of TM treatment on the expression levels of the BiP1, BiP2, BiP3, bZIP17, bZIP28, and bZIP60 genes. Except for bZIP17, the expression levels of the other five genes were upregulated to different degrees in the WT and djc5-1 seedlings after treatment with 5 μg/mL TM for 30 min (Figure 7A-F). Meanwhile, we found that AtDjC5 knockout significantly suppressed the TM-induced upregulation of BiP3 and bZIP28 expression ( Figure 7G). The above results indicate that AtDjC5 is required for the ER stress response.  BiP2, BiP3, bZIP17, bZIP28, and bZIP60 genes in WT and djc5-1. Q-PCR was performed with gene-specific primers (Table S1). Actin was used as an internal control. The expression levels of these genes in the nontreated WT seedlings Figure 7. The effects of AtDjC5 knockout on the expression levels of the three BiP and three bZIP genes before and after TM treatment. WT, wild-type plants; djc5-1, a T-DNA insertion mutant line for the AtDjC5 gene; Control, nontreated seedlings; TM, seedlings treated with 5 µg/mL TM for 30 min. (A-F) Analysis of the expression levels of the BiP1, BiP2, BiP3, bZIP17, bZIP28, and bZIP60 genes in WT and djc5-1. Q-PCR was performed with gene-specific primers (Table S1). Actin was used as an internal control. The expression levels of these genes in the nontreated WT seedlings were set to 1 and used for normalization. Data are the mean ± SD from three independent experiments. Asterisks indicate significant differences from the control group. (G) Comparison of the relative mRNA amount ratios (TM treatment/control) of BiP1, BiP2, BiP3, bZIP17, bZIP28, or bZIP60 between WT and the djc5-1 seedlings. (* p < 0.05, ** p < 0.01).

Discussion
In plants, J-domain proteins have been reported to localize in different subcellular compartments and participate in various biological processes. A previous study showed that AtDjC5 (also named AtPam18-3) mainly exists in mitochondria [55]. However, in this study, we found that AtDjC5 showed a partial overlap with the ER maker ( Figure 4A), indicating that AtDjC5 possibly has multiple subcellular localization.
Eukaryotic have two evolutionarily highly conserved systems to adapt to environmental stress conditions: the heat shock response (HSR) and the UPR. Various environmental stresses may lead to the accumulation of unfolded proteins or denatured proteins in plant cells. HSPs/chaperones respond to environmental stress, while cells respond to the accumulation of unfolded proteins in the ER by activating the UPR to generate HSPs/chaperones. It is likely that there is crosstalk between the HSR and the UPR. Heat stress has been shown to transiently induce X-box binding protein 1 (XBP1) splicing and to lead to increases in the mRNA expression levels of HSPA5 and DNAJB9 (ERdj4) [56][57][58][59], two typical UPR genes. The IRE1-bZIP60 pathway in plants is activated by heat stress [56]. Gao reported that bZIP28 is an important component of the plant response to heat stress and is involved in the regulation of chaperone expression [60]. Further work has confirmed the role of bZIP28 in the plant response to heat stress and connected it to the maintenance of fertility under heat stress [61]. In this study, we showed that AtDjC5 was induced by heat and TM treatment, and the knockout of AtDjC5 led to decreased thermotolerance and inhibited the ER stress-induced upregulation of BiP3 and bZIP28 expression ( Figures 5 and 7), suggesting that AtDjC5 promotes plant thermotolerance, likely by assisting the BiP3-bZIP28 pathway. The dissociation of BiP3 from bZIP28 is a major switch that activates the UPR signaling pathway in plants. In response to heat stress, BiP3 competes with unfolded proteins in the ER to release bZIP28, allowing it to be mobilized from the ER to the Golgi apparatus and then enter the nucleus to activate the UPR. AtDjC5, as a chaperone, is likely to play an important role in assisting the dissociation of BiP3 from the C-terminal tail of bZIP28. Loss of AtDjC5 function might block bZIP28 exit from the ER and inhibit the UPR, which downregulates the expression of chaperone proteins, leading to decreased thermotolerance. Whether AtDjC5 plays a role as a partner of BiP3 and how they regulate UPR still need answers.

Plant Materials and Growth Conditions
Seeds of Arabidopsis thaliana (ecotype Columbia-0) were surface-sterilized, plated on Murashige and Skoog (MS) medium containing 1.0% (w/v) sucrose and 0.8% (w/v) agar, and kept at 4 • C for 3 d. Then, plants were cultured in a growth chamber under longday conditions (16/8 h photoperiod) at approximately 100 µmol photons m −2 s −1 and 22 • C. Two-week-old seedlings were transplanted to soil and cultured under the original growth conditions.

Identification and Isolation of AtDjC5 T-DNA Insertional Mutants
Seeds of a putative T-DNA insertional mutant for AtDjC5 (At5g03030) and SALK_091892 (djc5-1) were obtained from the Arabidopsis Biological Resource Center (Columbus, OH, USA). The homozygous AtDjC5 mutant was identified by PCR as described by the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu/tdna_FAQs.html). PCR was conducted with genomic DNA from T2 generation seedlings using AtDjC5-specific primers (LP, 5 -GCGTCTGTAATCGACGGTAAG-3 ; RP, 5 -CTGGCTATACACAGGCTACGC-3 ) and a T-DNA left border primer LBb1.3 (5 -ATTTTGCCGATTTCGGAAC-3 ). The position of the T-DNA insertion was determined by PCR product sequencing.
The transcript levels of AtDjC5 in WT and djc5-1 were detected by RT-PCR using a RT-PCR Kit (TaKaRa, Otsu, Japan). Total RNA was isolated from 10-day-old seedlings with TRIzol reagent (Life Technologies, Carlsbad, CA, USA). The AtDjC5 coding region was amplified using a forward primer (FP) 5 -ATGGCTACGCCAATGATTGC-3 and a reverse primer (RP) 5 -TCAAAAGGCAGAACCGCTGT-3 . Actin was used as a loading control and amplified using FP 5 -AGGCACCTCTTAACCCTAAAGC-3 and RP 5 -GGACAACGGAATCTCTCAGC-3 . PCR products were analyzed by agarose gel electrophoresis.

Heat Stress Treatment and Chlorophyll Measurement
Seeds of different genotypes were planted on separate regions of the same MS plate, with 30 seeds per genotype and experiment. For the basal thermotolerance assay, 11-dayold seedlings grown at 22 • C were exposed to 44 • C for 90 min and recovered at 22 • C for 3 to 7 d prior to calculating the survival rate. For the acquired thermotolerance assay, 11-d-old seedlings grown at 22 • C were acclimated at 37 • C for 30 min and returned to 22 • C for 120 min before challenge at 45 • C for 120 min. Then, the seedlings recovered at 22 • C for 3 to 7 d prior to calculating the survival rate or analyzing the total chlorophyll content. Plants that were still green and produced new leaves were scored as surviving. The total chlorophyll content was determined as described by Porra [62].

Electrolyte Leakage Measurement
Membrane system stability was evaluated by an electrolyte leakage assay before and after heat treatment. Green leaves from 11-day-old seedlings grown at 22 • C were washed three times with ion-free water and then incubated in 5 mL of ion-free water at 45 • C for 120 min. The conductivity of the incubation medium was measured using a Leici conductivity meter (DDS-IIA, Shanghai, China).

Assay of Root Vitality
The vitality of the root tissue was detected with 2,3,5-triphenyltetrazolium chloride (TTC) according to Gong et al. [63] with modifications. Eleven-day-old seedlings grown at 22 • C were exposed to 45 • C for 1 h. Heat-treated seedlings were transferred to 0.6% (w/v) TTC solution and cultured at 22 • C for 20 h. After washing three times, 1.0-cm tips of primary roots (total 30 per sample) were homogenized in 95% (v/v) ethanol and centrifuged at 4 000× g for 5 min. The absorbance of the supernatant at 530 nm was measured using a spectrophotometer (TU-1900, PERSEE, Beijing, China).

Plant Transformation
To generate the P35S::AtDjC5 construct, the AtDjC5 coding region was PCR-amplified with cDNA from A. thaliana (Columbia-0) seedlings using FP 5 -TCTAGAATGGCTACGCCA ATGATT-3 and RP -GAGCTCTCAAAAGGCAGAACCGCT-3 . The PCR product was cloned into the binary vector pCAMBIA1300-35S digested with Xba I/Sac I.
To generate the PAtDjC5::β-glucuronidase (GUS) construct, a 1239-bp DNA fragment upstream of the AtDjC5 translational start codon was PCR-amplified with genomic DNA from A. thaliana seedlings using FP 5 -CTGCAGAACTCCCTCAAGGCTAAACC-3 and RP 5 -TCTAGATATTCAGCTAAGTAGTTGTTCGG-3 . The PCR fragment was ligated into the binary vector pCAMBIA1300-GUS digested with Pst I/Xba I.
To generate the P35S::AtDjC5-sGFP construct, the AtDjC5 coding region was obtained from a plasmid containing the AtDjC5 coding region by digestion with Xba I/Sac I. The AtDjC5 coding region was fused to the N terminus of sGFP in the binary vector pCAMBIA1300-35S::sGFP digested with XbaI/SacI.
The resulting plasmids were introduced into Agrobacterium tumefaciens (GV3101). Transformation of A. thaliana was performed by the floral-dipping method [64]. Transgenic lines were selected on MS medium containing 25 µg mL −1 hygromycin.

Real-Time Quantitative RT-PCR
Total RNA was isolated from different tissues or different genotypic seedlings using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Real-time quantitative RT-PCR (Q-PCR) was performed as described by Zhang et al. [65]. Primer pairs were designed using Primer Express software (Applied Biosystems) (Table S1). ACTIN was used as the internal control.

Obtaining AtDjC5 amiRNA Mutants
Using WMD3-Web MicroRNA Designer (http://wmd3.weigelworld.org/cgi-bin/ webapp.cgi, accessed on 10 February 2016), we obtained two AtDjC5-specific artificial microRNAs (amiRNAs) ("TAAGCAAGATAGTTACTGCCT" and "TTCTGGGTGATTTA-CAACCAT") and four oligonucleotide sequences (I to IV) (Table S2), which were used to engineer amiRNA into the endogenous miR319a precursor by site-directed mutagenesis. The detailed protocol for amiRNA cloning is as described by Schwab [66]. The resulting two amiRNAs were cloned into the binary vector pCAMBIA1300 via the BamH I/EcoR I site. The resulting plasmids were introduced into Agrobacterium tumefaciens (GV3101). Transformation of A. thaliana was performed by the floral-dipping method [64]. Transgenic lines were selected on MS medium containing 25 µg mL −1 hygromycin. The transgenic plants introduced into the above two amiRNAs were defined as djc5-2 and djc5-3, respectively.

Subcellular Localization of AtDjC5
Roots of 5-day-old transgenic seedlings harboring P35S::AtDjC5-sGFP were incubated in 1 µM ER-Tracker Red (Molecular Probes, Carlsbad, CA) solution for 30 min in the dark. Green fluorescent protein (GFP) fluorescence and ER-Tracker Red fluorescence were observed using a laser scanning confocal microscope (FV3000; OLYMPUS, Tokyo Prefecture, Japan) with excitation/emission wavelengths of 488/510 nm for GFP and 587/615 nm for ER-Tracker Red.

Histochemical Staining
Trypan blue staining was conducted as described by Choi et al. [67]. Histochemical staining for GUS expression was performed according to Jefferson et al. [68].

MDC Staining and Microscopy
Seven-day-old WT or djc5-1 seedlings were incubated in MS liquid medium with or without 5 µg/mL TM for 8 h, subjected to MDC staining with 0.05 mM monodansylcadaverine (MDC) for 10 min, and washed three times with PBS. MDC fluorescence was visualized using a laser scanning confocal microscope (LSM 710; Zeiss, Oberkochen, Germany) with an excitation/emission of 335/508 nm.

Statistical Analysis
Statistical analyses were carried out with STATISTICA 6.0 (StatSoft, Inc., Tulsa, OK, USA). The significance of differences was determined at p < 0.05 using ANOVA with Tukey's HSD test.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms232113134/s1, Additional supporting information may be found in the online version of this article. Table S1 Primers for real-time PCR amplification. Table S2 Primers for cloning of AtDjC5-specific amiRNAs.