© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317

Physiologia Plantarum 2014

Knockout of AtDjB1, a J-domain protein from Arabidopsis thaliana, alters plant responses to osmotic stress and abscisic acid Xingxing Wang‡ , Ning Jia‡ , Chunlan Zhao‡ , Yulu Fang, Tingting Lv, Wei Zhou† , Yongzhen Sun and Bing Li∗ Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Science, Hebei Normal University, Shijiazhuang 050024, PR China

Correspondence *Corresponding author, e-mail: [email protected] Received 9 October 2013; revised 18 January 2014 doi:10.1111/ppl.12169

AtDjB1 is a member of the Arabidopsis thaliana J-protein family. AtDjB1 is targeted to the mitochondria and plays a crucial role in A. thaliana heat and oxidative stress resistance. Herein, the role of AtDjB1 in adapting to saline and drought stress was studied in A. thaliana. AtDjB1 expression was induced through salinity, dehydration and abscisic acid (ABA) in young seedlings. Reverse genetic analyses indicate that AtDjB1 is a negative regulator in plant osmotic stress tolerance. Further, AtDjB1 knockout mutant plants (atj1-1) exhibited greater ABA sensitivity compared with the wild-type (WT) plants and the mutant lines with a rescued AtDjB1 gene. AtDjB1 gene knockout also altered the expression of several ABA-responsive genes, which suggests that AtDjB1 is involved in osmotic stress tolerance through its effects on ABA signaling pathways. Moreover, atj1-1 plants exhibited higher glucose levels and greater glucose sensitivity in the post-germination development stage. Applying glucose promoted an ABA response in seedlings, and the promotion was more evident in atj1-1 than WT seedlings. Taken together, higher glucose levels in atj1-1 plants are likely responsible for the greater ABA sensitivity and increased osmotic stress tolerance.

Introduction Salinity and drought are critical environmental stresses that can dramatically impact agricultural productivity and farm income. Research over the last 10 years has provided valuable insight into the molecular and cellular mechanisms plants use to adapt to salt or drought stress (Zhu 2002, Chinnusamy et al. 2004, Jung et al. 2008,

Magnan et al. 2008, Chinnusamy and Zhu 2009, Covarrubias and Reyes 2010, Miller et al. 2010, Golldack et al. 2011, Castro et al. 2012, Huang et al. 2012, Roelfsema et al. 2012, Ding et al. 2013, Huda et al. 2013, Hwang et al. 2013, Pan 2013, Xie et al. 2013, Li et al. 2014). Phytohormone abscisic acid (ABA) plays an important role in adapting to salinity and drought (Xiong et al. 2002, Verslues and Zhu 2005, Christmann

Abbreviations – ABI1, ABA-Insensitive 1; ABI2, ABA-Insensitive 2; ATPase, adenosine triphosphatase; DAG, diacylglycerol; DNP, 2, 4-dinitrophenol; ETC, mitochondrial electron transport chain; GUS, ß-glucuronidase; Hsp40s, heat-shock protein 40s; HSC70, cognate protein of Hsp70; MES, 2 - (N - morpholine) ethanesulfonic acid; MS, Murashige and Skoog; OST1, open stomata 1; Q-PCR, real-time quantitative PCR; RAB18, response to ABA 18; RD29A, response to desiccation 29A; RD29B, response to desiccation 29B; T-DNA, transferred DNA; WT, wild-type. † Present

address: College of Biology and Engineering, Hebei University of Economics and Business,Shijiazhuang 050061, PR China ‡ These authors contributed equally.

Physiol. Plant. 2014

et al. 2006, Peters et al. 2010, Fujita et al. 2011, Lee and Luan 2012, Fujita et al. 2013). Salinity and drought stress induce ABA production, which changes multiple physiological processes, such as a rapid stomata closure to decrease water loss and gene expression to withstand salinity and drought stress. Multiple ABA-signaling components have been identified, including ABA receptors, PLDα1, PA, G proteins, NADPH oxidases, second messengers [Ca2+ , H2 O2 , NO and diacylglycerol (DAG)], protein kinases, protein phosphatases and anion and cation channels (Pei et al. 2000, Merlot et al. 2001, Mustilli et al. 2002, Marten et al. 2007, Sutter et al. 2007, Pandey et al. 2009, Geiger et al. 2010, Klingler et al. 2010, Guo et al. 2011, Wang et al. 2011, Zhang et al. 2011, Distefano et al. 2012, Aliniaeifard and Meeteren 2013, Wang et al. 2013). An updated model for the ABA-signaling pathway has been proposed. A combination of ABA receptors, PP2Cs/ABI1/ABI2 (as negative modulators) and SnRK2/OST1 (as positive modulators), determines downstream ABA signaling activation or inactivation (Cutler et al. 2010, Hubbard et al. 2010, Umezawa et al. 2010). J-proteins [also referred to as heat shock protein 40s (Hsp40s)] are defined by a J-domain that comprises approximately 75 conserved amino-acid residues. Many J-proteins have been characterized from multiple organisms. In silico localization analyses have shown that J-proteins are distributed in different subcellular compartments. They function as molecular chaperones, alone or in association with HSP70s. HSP70/J-protein molecular chaperone machinery is involved in diverse cellular processes, including neonatal protein folding, assembly and translocation as well as misfolded protein degradation. They play a crucial role in maintaining protein homeostasis by re-establishing the functional native conformation under environmental stress conditions, which protects the cell (Hennessy et al. 2005, Mayer and Bukau 2005, Craig et al. 2006, Rajan and D’Silva 2009, Pukszta et al. 2010, Gillies et al. 2012). A strong indication that chaperones are more than mere molecular ‘sponges’ that passively prevent protein aggregation is that all major chaperone families are adenosine triphosphatases (ATPases), except the small HSPs (Veinger et al. 1998, Priya et al. 2013). HSP70s have a low-level basal ATPase activity. J-protein docking stimulates ATPase activity in their HSP70 partner many-fold and confers functional specificity to HSP70 by recruiting it to specific substrate proteins (Mayer and Bukau 2005, Li et al. 2009, Kampinga and Craig 2010, Ohta et al. 2013). One hundred twenty J-proteins have been identified in the Arabidopsis thaliana genome (Miernyk 2001, Rajan and D’Silva 2009). Emerging evidence indicates that J-proteins are involved in

development and signal transduction processes (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, Chen et al. 2010, 2011, Shen et al. 2011, Pulido et al. 2013). The role of J-proteins in adapting to environmental stress has been documented. Li et al. (2007) reported that AtDjA2 and AtDjA3, two Arabidopsis J-proteins, improve A. thaliana thermotolerance. Yang et al. (2009) demonstrated that TMS1, which is a thermosensitive male sterile J-protein, plays an important role in pollen tube thermotolerance. Yang et al. (2010) showed that plants lacking AtDjA3 are more sensitive to salt at a high external pH compared with wild-type (WT) plants. AtDjB1 is a member of the Arabidopsis J-protein family. Its amino acid sequence contains a potential N-terminal mitochondrial-targeting sequence (Miernyk 2001). Molecular and genetic evidence has demonstrated that it is localized to the mitochondria (Zhou et al. 2012). Recombinant AtDjB1 stimulates ATPase activity in both Escherichia coli DnaK and maize endosperm HSP70 in vitro (Kroczynska et al. 1996). AtDjB1 directly interacts with an A. thaliana mitochondrial HSP70 (AtmtHSC70-1) and stimulates its ATPase activity. Thus, AtDjB1 knockout leads to cellular ATP accumulation, which inhibits seedling respiration through feedback inhibition. The lower respiration downregulates cellular ascorbate (ASC) levels, which reduces the hydrogen peroxide (H2 O2 ) detoxification rate, suggesting that AtDjB1 plays a crucial role in maintaining redox homeostasis and facilitates thermotolerance by protecting cells against heat-induced oxidative damage (Zhou et al. 2012). In this work, the role of AtDjB1 in A. thaliana adaptation to salinity and drought stress was studied using reverse genetic analyses.

Materials and methods Plant material and growth conditions

Arabidopsis thaliana (L.) Heynh. (ecotype Columbia0) seeds were surface-sterilized, plated on Murashige and Skoog (MS) medium containing 1.0% (w/v) sucrose and 0.8% (w/v) agar and maintained at 4◦ C for 3 days. Next, the plants were grown in a growth chamber at 22◦ C under long-day conditions (16 h light/8 h dark) with a light intensity of approximately 60 μmol m−2 s−1 . Two-week-old seedlings were transplanted to soil and cultured under the original growth conditions. The plants were irrigated with 1× Hoagland nutrient solution once a week. To avoid variation in seed quality, the plants were cultured under identical conditions over the same time period, and the seeds were harvested and stored in the same manner. Physiol. Plant. 2014

Seeds of a putative transferred DNA (T-DNA) insertion mutant for AtDjB1 (At1g28210), SALK_049553, were obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH). The homozygous AtDjB1 mutant (atj1-1) was identified, and the AtDjB1 transcript abundance was determined as described by Zhou et al. (2012). The atj1-1 lines rescued with the AtDjB1 gene (R1 and R6) were obtained and identified as described by Zhou et al. (2012). Stress treatments

Stress-induced AtDjB1 gene expression Salinity or dehydration treatments were performed by transferring 10-day-old seedlings onto a new agar plate supplemented with 200 mM NaCl or 400 mOsm kg−1 sorbitol for the indicated time under the original growth conditions. For the ABA treatment, 10-day-old seedlings were incubated in a 100 μM ABA (Sigma-Aldrich, St Louis, MO) solution for the indicated time, and water was used as a control.

Expression for ABA-responsive genes For the ABA treatment, roots of 4-week-old plants grown in soil in a growth chamber (22 ± 2◦ C, 16-h light:8-h dark) were dipped in a 50 μM ABA solution or deionized water (as control) for 24 h. The plant materials were collected and used for real-time quantitative PCR (Q-PCR). Real-time quantitative PCR The total RNA was isolated from the treated plants with various genotypes using TRIzol® reagent (Invitrogen, Carlsbad, CA) and used for first-strand cDNA synthesis using the ExScriptRT reagent kit (Takara, Otsu, Japan) in accordance with the manufacturer’s instructions. Q-PCR was performed as described by Liu et al. (2007). The reaction was performed using an ABI prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Actin was used as the internal control. The primer pairs for Q-PCR were designed using Primer Express (Applied Biosystems).

Cotyledon greening assay

ABA level measurements

Seeds from different genotypes were plated on separate regions of the same MS plate containing 1.0% (w/v) sucrose, with or without 100 mM NaCl, 400 mM mannitol or 1 μM ABA. The seeds were incubated for 3 days at 4◦ C in the dark prior to transfer to a growth chamber. Cotyledon greening was scored after growth at 22◦ C for 6–12 days. The experiments were repeated using at least three different batches of seeds, and the results from one representative experiment are shown. The cotyledon greening rates were calculated using results from three independent experiments.

Leaves from 25-day-old Arabidopsis grown in soil with or without 150 mM NaCl or a drought treatment were harvested, rinsed in water, frozen in liquid nitrogen and ground into powder. The ABA was extracted by suspending 0.1 g of tissue in 1.5 ml of an extraction solution (80% methanol, 100 mg l−1 butylated hydroxytoluene and 0.5 g l−1 citric acid monohydrate) and stirring overnight at 4◦ C. The suspension was centrifuged at 4000 g for 20 min, and the supernatant was transferred to a clean tube and dried under vacuum. The dry residue was dissolved using 0.1 ml of modified Trisbuffered saline (45 mM Tris–HCl, pH 7.8, 90 μM MgCl2 , 0.135 M NaCl and 10% methanol) and centrifuged at 10 000 g for 10 min. The supernatant ABA concentration was then determined using a plant hormone ABA ELISA Kit (R & D Systems, Minneapolis, MN).

Stress tolerance assay The salt tolerance assay was performed with 4-week-old plants grown in soil in a growth chamber (22 ± 2◦ C, 16-h light:8-h dark). The plants were irrigated with 175 mM NaCl solution every 4 days and subsequently monitored for stress symptoms over the following 2 weeks. Photographs were collected 2 weeks after the salt treatment, and the fresh weight was recorded. For the drought tolerance assay, water was withheld from 4week-old soil-grown plants. Photographs were collected 4 weeks after the drought treatment, and the fresh weight was recorded. To minimize experimental variation, the genotype plants were placed in the same pot. The experiments were repeated at least three times with more than 20 plants for each experiment. The results from one representative experiment are shown. Physiol. Plant. 2014

Seed germination test For the germination assays, approximately 100 seeds were plated on MS with 1% sucrose and different concentration of ABA. To break the dormancy, fully desiccated seeds were incubated at 4◦ C for 4 days in the dark before germination and were subsequently grown in a growth chamber as described above. Seed germination was observed for 6 days. A seed was considered germinated when the radicle protruded through the seed coat. The germination rate was calculated as a percentage of the total number of seeds plated.

Stomatal aperture measurements

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For the stomatal closing experiments, fully expanded leaves from 3- to 4-week-old plants were excised, and the epidermal pieces were peeled from the abaxial surface. The epidermal peels were floated in a stomatal opening solution [50 mM KCl, 0.1 mM CaCl2 and 10 mM 2 (N - morpholine) ethanesulfonic acid (MES), pH 6.1] for 2.5 h. After incubation in 50 μM ABA for 0.5 h, the stomatal aperture was measured. Control experiments were performed in parallel without ABA. Glucose level measurements Seven-day-old seedlings grown at 22◦ C were dried at 110◦ C for 15 min then 70◦ C overnight in an oven. The dried seedlings were ground into powder using a mortar and pestle, and 1 ml of deionized water was added to 5 mg of a ground sample, which was incubated at 70◦ C for 20 min and decolorized with active carbon for 10 min followed by centrifugation at 10 000 g for 5 min. The supernatant absorbance at 340 nm was measured through spectrophotometry using a D-glucose kit (BioSenTec, Auzeville-Tolosane, France).

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Statistical analysis The statistical analyses were performed using STATISTICA 6.0. The significance of the differences was determined using P < 0.05 and Student’s t -test.

Results AtDjB1 expression is enhanced under different stimuli To evaluate AtDjB1 gene expression in response to osmotic stress or ABA, 10-day-old A. thaliana seedlings were subjected to salinity, dehydration or exogenous ABA treatments. The AtDjB1 transcript levels were analyzed using Q-PCR. The results showed that salinity, dehydration or ABA treatment significantly increased the AtDjB1 expression levels. Under salt stress, AtDjB1 expression began to increase within 4 h of the treatment and reached a 1.9-fold peak level maximum 12 h after the treatment, which then decreased 24 h after the treatment (Fig. 1A). Under dehydration stress, AtDjB1 expression increased 1.85-fold within 6 h of the treatment and remained constant within 24 h (Fig. 1B). One hundred micromolar ABA was applied to seedlings, which rapidly upregulated the AtDjB1 expression level (1.6-fold within 15 min of the treatment, Fig. 1C). The AtDjB1 gene expression pattern suggests that AtDjB1 may be involved in ABA-dependent abiotic stress signaling.

Fig. 1. Expression of the AtDjB1 gene in A. thaliana seedlings treated with salinity (A), dehydration (B) or ABA (C). For salinity or dehydration stress, 10-day-old seedlings grown on MS medium at 22◦ C were transferred to MS medium with 200 mM NaCl or 400 mOsm kg−1 sorbitol and cultured for the indicated time period. For the ABA treatment, 10-day-old seedlings grown on MS medium at 22◦ C were treated with 100 μM ABA for the indicated time period. Total RNA was analyzed using Q-PCR with AtDjB1 gene-specific primers (Table S1). The AtDjB1 gene expression level in untreated seedlings was set to 1 and used for normalization. The values are the means ± SD from three independent experiments.

Salt and water stress responses are altered in the atj1-1 mutant To evaluate the importance of AtDjB1 in an abiotic stress response, a homozygous T-DNA insertion mutant (atj1-1) with T-DNA in AtDjB1 and two homozygous rescued mutant lines (R1 and R6) with a single copy of exogenous AtDjB1 were used for the stress Physiol. Plant. 2014

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Fig. 2. AtDjB1 knockout impaired seedling cotyledon greening under osmotic stress. WT, wild-type A. thaliana plants; atj1-1, AtDjB1 mutant; and R1 and R6, two atj1-1/AtDjB1 lines. (A) Intron/exon organization of the AtDjB1 coding region and T-DNA insertion location. Solid boxes, exons; lines, introns; and triangle, T-DNA insertion position. (B, C) Salt stress response of the different seedling genotypes. Photographs were collected 12 days after a 100 mM NaCl treatment. The data are the means ± SD from six individual experiments (n = 300). (D, E) Dehydration stress response of the different seedling genotypes. The photographs were collected 7 days after a 400 mM mannitol treatment. The data are the means ± SD from nine individual experiments (n = 225). The percentage values represent the % of cotyledon greening, which was inhibited by salt or dehydration stress.

response assays. Sequence analyses of the T-DNA flanking regions revealed that atj1-1 contained a T-DNA insertion in AtDjB1 intron 14 (Fig. 2A). RT-PCR showed no detectable full AtDjB1 transcript, and the partial AtDjB1 transcript was significantly downregulated in the atj1-1 seedlings (Zhou et al. 2012). Q-PCR showed that the AtDjB1 expression level was very low in atj1-1 seedlings. Introduction of the AtDjB1 gene into atj1-1 rescued AtDjB1 gene expression, and the R1 and R6 seedling displayed a similar AtDjB1 expression level to WT seedlings (Zhou et al. 2012). Under standard culture conditions, though the atj1-1 seedlings grown on the MS medium were smaller than the WT, R1 and R6 seedlings, their cotyledon greening rates were not clearly different. In the presence of 100 mM NaCl, the cotyledon greening of atj1-1 seedling was severely inhibited, whereas most WT, R1 and R6 seedlings exhibited a green cotyledon (Fig. 2B, C), which indicates that AtDjB1 might be involved in regulating the plant salt stress response during the post-germination development stage. To examine whether AtDjB1 is an osmotic regulator in the salt stress response, cotyledon greening of the seedlings with different genotypes was analyzed upon Physiol. Plant. 2014

mannitol application, which is a strong dehydration substance. With 400 mM mannitol, cotyledon greening rate of atj1-1 seedling was also clearly lower (60%) than for the WT (92%), R1 (81%) and R6 (90%) seedlings (Fig. 2D, E), which suggests that AtDjB1 plays an important role in regulating the plant osmotic stress response during the post-germination development stage. Further, tolerance of mature WT, atj1-1, R1 and R6 plants to salt and drought stress was examined. For the salt tolerance assay, 4-week-old plants were irrigated with either water as a control or a 175 mM NaCl solution every 4 days. After 2 weeks, the atj1-1 plants displayed greater salt stress tolerance than the WT, R1 and R6 plants (Fig. 3A). When the salt stress treatment was complete, the fresh weight of each genotype plant was clearly downregulated compared with their respective controls. However, this downregulation was lower in the atj1-1 plants (35%) compared with the WT (52%), R1 (50%) and R6 (56%) plants (Fig. 3B). For the drought tolerance assay, 4-week-old plants were subjected to drought stress by stopping the irrigation. After 4 weeks, the atj1-1 plants exhibited greater drought stress tolerance compared with the WT, R1 and R6 plants (Fig. 3C).

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Fig. 3. AtDjB1 knockout altered the salt and drought tolerance of plants. WT, wild-type A. thaliana plants; atj1-1, AtDjB1 mutant; and R1 and R6, two atj1-1/AtDjB1 lines. The phenotype (A, C) and fresh weight (B, D) of WT, atj1-1, R1 and R6 plants under salt (A, B) and drought (C, D) stress. For the salt stress, 4-week-old plants grown in soil were irrigated every 4 days for 2 weeks with water (control) or 175 mM NaCl. For the drought stress, 4-week-old plants grown in soil were subjected to a drought treatment for 4 weeks by stopping irrigation. The experiments were repeated three times with similar results. The values are the means ± SD from 30 (for salt stress) or 20 (for drought stress) plants. The percentage values represent the fresh weight, which was inhibited by salt or drought stress.

When the drought stress was complete, the fresh weight of each genotype plant was clearly lower compared with their respective controls. However, this decrease was lower in the atj1-1 plants (29%) compared with the WT (42%), R1 (42%) and R6 (45%) plants (Fig. 3D). These results suggest an important role of AtDjB1 as a negative regulator in plant osmotic stress tolerance. AtDjB1 mutants are more sensitive to exogenous ABA To determine whether the atj1-1 response to osmotic stress results from an ABA-dependent process, ABA levels in WT, atj1-1 and R6 plants were first evaluated under salinity and water deficit conditions. Under salt stress, the mature atj1-1 plants did not display a marked difference in ABA levels compared with the WT and R6 plants (Fig. 4A). Under drought stress, the ABA levels in the mature atj1-1 plants were slightly greater than that in the WT plants and were similar to the R6 plants (Fig. 4B). ABA sensitivity for the WT, atj1-1, R1 and R6 plants was then examined. One of the best-known ABA effects is seed germination inhibition. In the absence of ABA, 45, 24 and 40% of the WT, R1 and R6 seeds germinated

1 day after transfer to 22◦ C, respectively, whereas only 13% of the atj1-1 seeds germinated. The seed germination rate for each genotype was more than 96% 4 days after transfer to 22◦ C (Fig. 5A), which indicates that atj1-1 seeds broke dormancy after the WT, R1 and R6 seeds. In the presence of 1 μM ABA, seed germination for each genotype was inhibited to a certain extent within 6 days. After 2 days of the ABA treatment, the seed germination rate was 88% for the WT, 64% for R1, and 75% for R6. In contrast, the seed germination rate in atj11 was merely 37% (Fig. 5B). In the presence of increasing ABA concentrations, seeds from each genotype exhibited a lower germination rate, and the atj1-1 seed germination rates were more severely impaired by ABA compared with the WT, R1 and R6 seeds. After 3 days with 5 μM ABA, the seed germination rate was 88% for WT, 55% for atj1-1, 68% for R1 and 75% for R6. At 10 μM ABA, the seed germination remained more inhibited in atj1-1 than WT, R1 and R6 (Fig. 5C), which indicates that the atj1-1 seeds were more sensitive to ABA than the WT, R1 and R6 seeds. Further, seedling cotyledon greening was analyzed with or without ABA. Under control conditions, most WT, atj1-1 and R6 seedlings exhibited fully expanded and green cotyledons. On the MS medium Physiol. Plant. 2014

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increased stress tolerance of atj1-1 plants might be caused by enhanced ABA sensitivity, not ABA levels. AtDjB1 regulates the expression of ABA-responsive genes

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To evaluate the role of AtDjB1 in plant responses to ABA, expression of several known ABA-responsive genes was analyzed in WT, atj1-1 and R6 plants following exogenous ABA treatment. The Q-PCR analyses were performed using specific primers from the Response to Desiccation 29A (RD29A), Response to Desiccation 29B (RD29B), ABA-Insensitive 1 (ABI1), ABA-Insensitive 2 (ABI2), Open Stomata 1 (OST1) and Response to ABA 18 (RAB18) genes. When the plants were treated with 50 μM ABA, the expression level of RD29A, RD29B, ABI2 and RAB18 genes was clearly lower in atj1-1 than WT and R6 (Fig. 6A, B, D, F). However, the expression level of ABI1 and OST1 genes did not exhibit significant difference among WT, atj1-1 and R6 (Fig. 6C, E). These data indicate that AtDjB1 affects plant sensitivity to ABA likely through regulating expression of ABA-responsive genes. Higher glucose levels in atj1-1 mutants results in greater ABA sensitivity

Fig. 4. The ABA levels in mature plants exposed to salinity (A) or drought (B) stress. WT, wild-type A. thaliana plants; atj1-1, AtDjB1 mutant; and R6, an atj1-1/AtDjB1 line. For the salt stress, 4-week-old plants grown in soil were irrigated with water (control) or 150 mM NaCl for 24 h. For the drought stress, 6-week-old plants grown in soil were subjected to a drought treatment by stopping irrigation for 9 days. The ABA levels were analyzed by enzyme-linked immunosorbent assay (ELISA). The values are the means ± SD from three independent experiments. The asterisks indicate significant differences compared with WT using the same treatment (t-test, * P < 0.05).

with 1 μM ABA, cotyledon greening for each seedling genotype was inhibited, and the inhibition rate was 33% for WT, 86% for atj1-1 and 22% for R6 (Fig. 5D, E), which indicates that the atj1-1 seedlings were more sensitive to ABA than the WT and R6 seedlings. An additional well-documented ABA function is promoting stomatal closure. To test whether atj1-1 was altered in ABAmediated stomatal movement, the AtDjB1 expression in guard cells and guard cell sensitivity were measured for the different genotypes with ABA. ß-glucuronidase (GUS) expression driven by the AtDjB1 promoter (1144 bp) (Zhou et al. 2012) showed that AtDjB1 was clearly expressed in guard cells from transgenic plants with PAtDjB1: GUS (Fig. 5F). In addition, guard cells from atj1-1 plants were more sensitive to ABA compared with guard cells from the WT, R1 and R6 plants (Fig. 5G, H). Taken together, the above results suggest that the Physiol. Plant. 2014

Previously, authors have reported that AtDjB1 is targeted to the mitochondria and directly interacts with mitochondrial cognate protein of Hsp70 (HSC70)-1 (Fig. S1, Supporting information) to activate its ATPase activity (Fig. S2A) and that an AtDjB1 knockout leads to the upregulation of cellular ATP levels (Fig. S2B) and inhibition of respiration (Fig. S2C) (Zhou et al. 2012). The data presented here show that the glucose levels in atj1-1 seedlings were clearly higher than in WT and R6 seedlings, and 2, 4-dinitrophenol (DNP) treatment, which reduces ATP production, decreased the glucose levels in atj1-1 seedlings (Fig. 7A). These results suggest that ATP accumulation (as a respiration product) caused by AtDjB1 knockout inhibits the mitochondrial electron transport chain (ETC) through feedback inhibition, which leads to cellular glucose accumulation (as a respiration substrate). To discern the link between higher glucose concentrations and greater ABA sensitivity in atj1-1, the glucose response was first compared among WT, atj1-1 and R6 plants. The results showed that the elevated glucose concentration in an MS medium inhibited hypocotyl, root elongation and cotyledon opening/greening processes in each seedling genotype (Fig. 7B–D). Further, the inhibiting role of glucose was more evident in atj1-1 seedlings compared with the WT and R6 seedlings (Fig. 7E–G), which indicates that the atj1-1 plants are more sensitive to glucose in

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Fig. 5. AtDjB1 knockout generated ABA oversensitivity in seed germination, cotyledon greening and stomatal closure. WT, wild-type A. thaliana plants; atj1-1, AtDjB1 mutant; and R1 and R6, two atj1-1/AtDjB1 lines. (A–C) Seed germination rate of the different genotypes without ABA (A), with 1 μM ABA (B) or with different concentrations of ABA (C). Germination was scored every day (A, B) or the third day (C) after the seeds were transferred to 22◦ C. One hundred seeds per genotype were measured in each experiment. The values are the means ± SD from three independent experiments (n = 300). (D, E) Cotyledon greening of different genotypes with or without 1 μM ABA. The photographs were collected 6 days after the 1 μM ABA treatment. Twenty-five seeds per genotype were measured in each experiment. The data are the means ± SD from nine individual experiments (n = 225). The percentage values represent % of cotyledon greening, which were inhibited by 1 μM ABA. (F) Leaf epidermis histochemical GUS staining of transgenic plants transformed with a PAtDjB1::GUS construct. (G) The photographs show the stomatal apertures from ABA-induced stomata closure assays with 0 (top row) and 50 μM ABA (bottom row) after incubating for 0.5 h. (H) Stomatal aperture measurements of WT, atj1-1, R1 and R6 plants with 50 μM ABA. The data are the means ± SD with 50 stomata per data point. Three independent experiments were performed, and similar results were generated. The percentage values represent the stomatal aperture, which was decreased with 50 μM ABA.

the post-germination development stage. In addition, the effects of sucrose on post-germination development were also examined in the WT, atj1-1 and R6 plants (Fig. S3). The results were consistent with the glucose response results. The above results show that the glucose-arrested atj1-1 seedlings mimic ABA-arrested atj1-1 seedlings, which implies a link between glucose and the ABA response. Furthermore, the WT and atj1-1 seedlings were co-treated with both glucose and ABA. The WT seedling results showed that the inhibition rate from 1 μM ABA

on cotyledon opening/greening was 23% in 1% glucose, 52% in 3% glucose and 96% in 5% glucose (Fig. 8A), which indicates that the higher glucose levels promote the ABA response. In atj1-1 seedlings that exhibited a higher level of endogenous glucose, the inhibition rate from 1 μM ABA on cotyledon opening/greening was 89% even with the lower glucose levels (1%) (Fig. 8A), which confirms the regulatory role of glucose in the ABA response and suggests that the higher endogenous glucose levels in atj1-1 are likely responsible for the Physiol. Plant. 2014

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Fig. 6. AtDjB1 knockout altered the expression of ABA-responsive genes. WT, wild-type A. thaliana plants; atj1-1, AtDjB1 mutant; and R6, an atj1-1/AtDjB1 line. The gene expression levels were measured using Q-PCR with the gene-specific primers (Table S1). Four-week-old plants were treated with 50 μM ABA or deionized water (as control) for 24 h. The gene expression levels in water-treated WT plants were set to 1 and used for normalization. The values are the means ± SD from three independent experiments. The asterisks indicate significant differences from WT using the same treatment (t-test, ** P < 0.01).

increased ABA sensitivity. Moreover, the effects of mannitol on the ABA response were also examined in WT seedlings. The results show that higher mannitol levels also promote seedling sensitivity to ABA. However, the effects of glucose and mannitol on ABA responses were distinguishable (Fig. 8B), which suggests that glucose plays a regulatory role in the ABA response not only as an osmotic substance, maybe also as a signaling molecular.

Discussion Salt and water stress induces ABA biosynthesis and triggers ABA-dependent signaling pathways (Verslues and Zhu 2005, Huang et al. 2012). To evaluate the role of AtDjB1 in abiotic stress, the authors first detected the AtDjB1 gene expression pattern under osmotic stress and upon ABA application. The results show that salinity and dehydration stress increases the AtDjB1 expression level by less than twofold in WT seedlings (Fig. 1A, B). This result is consistent with the results from the public Arabidopsis microarray database (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) Physiol. Plant. 2014

(Fig. S4A–D). The data presented here also show that a 100 μM ABA application rapidly upregulated the AtDjB1 expression level (1.6-fold within 15 min, Fig. 1C). However, in the public Arabidopsis microarray databases, the AtDjB1 expression level does not clearly increase after 30 min of a 10 μM ABA treatment (Fig. S4E). The authors assert that the inconsistency between the two results was likely caused by the different ABA treatment concentrations and times. The AtDjB1 gene expression pattern implies that AtDjB1 is likely involved in ABA-dependent abiotic stress signaling. Further, the effects of AtDjB1 knockout and overexpression on the seedling osmotic stress response during postgermination development were examined. The results show that salinity or dehydration treatment inhibited cotyledon greening for each seedling genotype and atj11 seedlings then displayed greater inhibition than WT. Introducing the AtDjB1 gene into atj1-1 restored the WT phenotype (Fig. 2), which confirms the role of AtDjB1 in modulating the osmotic stress response. However, AtDjB1 gene overexpression did not clearly affect the salt and dehydration stress responses (Fig. S5) likely because the endogenous AtDjB1 gene expression level in WT is sufficient. In addition, the atj1-1 plants exhibited increased sensitivity to ABA through seed germination and cotyledon greening compared with WT and the rescued mutant plants (Fig. 5A–E). Further, the ABA levels in the atj1-1 plants did not markedly differ from the WT plants after the salt stress and was slightly higher than the WT plants after the drought stress (Fig. 4). These results suggest that the greater growth inhibition observed in atj1-1 seedling after a salinity or dehydration treatment might result from increased ABA sensitivity. ABA plays a primary role in decreasing plant water loss under drought conditions by regulating stomatal closure and opening (Nilson and Assmann 2007, Cutler et al. 2010, Aliniaeifard and Meeteren 2013). The data presented here show that the AtDjB1 gene was clearly expressed in guard cells, and guard cells in isolated epidermal peels from atj1-1 plants were more sensitive to a 50 μM ABA application compared with guard cells from the WT, R1 and R6 plants (Fig. 5F–H), which suggests a role for AtDjB1 in stomatal control and transpiration under stress conditions. ABA is involved in plant tolerance under salinity and drought stress, and the plants that display greater ABA sensitivity are more resistant to salinity and drought stress (Magnan et al. 2008, Kim et al. 2009, Peters et al. 2010). The results presented here show that mature atj1-1 plants display a higher tolerance to salt and water deficits than the WT, R1 and R6 plants (Fig. 3), which is consistent with the greater ABA sensitivity observed for the atj1-1 plants. Taken together, the data presented here suggest that

A

B

C

D

E

F

G

Fig. 7. The effects of AtDjB1 knockout on glucose responses in WT, atj1-1 and R6 seedlings. WT, wild-type A. thaliana plants; atj1-1, AtDjB1 mutant; R6, an atj1-1/AtDjB1 line; and Glc, glucose. (A) Comparison of Glc levels among WT, atj1-1 and R6 seedlings. (B–D) The effects of exogenous Glc on hypocotyl (B), root (C) elongation and cotyledon opening/greening (D) for the different genotypes. The data are the means ± SD from three biological replicates. (E, F) The hypocotyl (E) or root (F) length ratio of 3% Glc-treated seedlings to 1% Glc-treated seedlings. (G) The cotyledon greening ratio of 5% Glc-treated seedlings to 1% Glc-treated seedlings.

AtDjB1 is a negative regulator of ABA signaling and osmotic stress tolerance in A. thaliana. Among the ABA mutants described, npx1-1 and cml9 display similar ABA responses to atj1-1; these mutants are more sensitive to ABA in seed germination, cotyledon opening/greening or stomatal closure (Magnan et al. 2008, Kim et al. 2009). ABF3, ABF4, AtMYB44 and NPC4 overexpression also yields greater ABA sensitivity (Kang et al. 2002, Jung et al. 2008, Peters et al. 2010). The ABA-oversensitive mutants and transgenic plants exhibit enhanced salt or drought tolerance, as observed for the atj1-1 mutant plants. ABA acts through complex signaling networks to induce changes in expression of many genes that participate in the onset of adaptive responses. Microarray data analyses indicate that over 2900 genes respond to ABA in A. thaliana (Nemhauser et al. 2006), including the RD29A, RD29B, ABI1, ABI2, OST1 and RAB18 genes. These genes are convenient markers for monitoring the stress and/or ABA response pathways in plants (Kim et al. 2009, Peters et al. 2010). To determine how AtDjB1 deletion provokes ABA oversensitivity, the authors examined the effect of AtDjB1 knockout on expression of the

aforementioned ABA marker genes by Q-PCR. When the plants were treated with 50 μM ABA, the expression level of RD29A, RD29B, ABI2 and RAB18 genes was clearly lower in atj1-1 than WT and R6 (Fig. 6). This result suggests that the salt and water stress tolerance exhibited by atj1-1 mutants is not conferred by the proteins encoded by these genes, as was recently reported by Magnan et al. (2008) for mutant cml9 and Jung et al. (2008) in transgenic Arabidopsis plants that overexpressed the transcription factor AtMYB44. The mutant analyses showed that knockout lines for RD29A and RD29B, which are two NaCl-, drought- or ABA-inducible genes, maintain greater root growth, photosynthesis and water-use efficiency under salt stress relative to a control (Msanne et al. 2011), which suggests that the RD29A and RD29B proteins are likely not directly protective molecules. ABI1 and ABI2, which are two A-type protein phosphatase 2C (PP2C) members, are negative regulators of ABA signaling. ABA binding to PYR/PYL/RCAR proteins (ABA receptors) initiates PP2C physical interaction and inhibition. PP2C deactivation releases SnRK2 kinases from PP2C–SnRK2 complexes, which activates kinases that target downstream proteins (Merlot et al. Physiol. Plant. 2014

A

B

Fig. 8. The effects of glucose or mannitol on ABA response. WT, wildtype A. thaliana plants; atj1-1, AtDjB1 mutant; Glc, glucose; and Man, mannitol. (A) The effects of Glc on ABA response in WT and atj1-1 seedlings. (B) The effects of mannitol on ABA response in WT seedlings. The percentage values represent the % of cotyledon greening, which was inhibited by 1 μM ABA.

2001, Cutler et al. 2010, Yu et al. 2012, Aliniaeifard and Meeteren 2013). These data support the authors’ conclusion that the lower ABA-induced RD29A, RD29B, ABI2 and RAB18 gene levels may have increased the ABA sensitivity and osmotic stress tolerance in atj1-1 plants. AtDjB1 is targeted to the mitochondria (Zhou et al. 2012), which raises the question of how AtDjB1 causes the altered ABA response in plants. The authors’ previous work indicates that AtDjB1 directly associates with mitochondrial HSC70-1 and stimulates its ATPase activity and that cellular ATP accumulates upon AtDjB1 knockout, which inhibits respiration through feedback inhibition (Zhou et al. 2012). In atj1-1 mutants, cellular glucose accumulates due to lower respiration (Fig. 7A, Fig. S2). Glucose has a hormone-like function and controls many vital life processes, including seed germination, early seedling growth and development, flowering and senescence (Gibson 2000, 2005, Smeekens 2000, Rolland et al. 2006, Yuan et al. 2013). Exogenous glucose feeding altered the expression of many genes in A. thaliana seedlings (Price et al. 2004, Villadsen and Smith 2004, Li et al. 2006). Genetic analyses indicate that glucose signaling in plants is closely associated with plant Physiol. Plant. 2014

Fig. 9. A working model for the function of AtDjB1 in mediating the plant response to ABA and hyperosmotic stress. Deleting the AtDjB1, which associates with mtHSC70-1 and stimulates its ATPase activity, leads to cellular ATP accumulation, which inhibits the mitochondrial electron transport chain (ETC) through feedback inhibition. Cellular glucose accumulates due to a lower respiration rate. Higher glucose level in atj1-1 likely inhibits the expression of ABA-responsive genes. The lower expression of these genes in atj1-1 may contribute to the elevated ABA sensitivity, which increases osmotic stress resistance. The arrows indicate positive effects; the bars show blockage effects.

hormone biosynthesis and signaling, particularly for ABA (Finkelstein and Gibson 2001, Gazzarrini and ´ and Sheen McCourt 2001, Cheng et al. 2002, Leon 2003, Zhu et al. 2009, Yuan et al. 2013, Hsu et al. 2014). Increasing evidence shows that glucose and ABA act synergistically to control the expression of many genes and seedling development. For example, era12 and abi2-1, two ABA response mutants, displayed an altered glucose response and arrested early seedling development (Dekkers et al. 2008). The data presented here show that exogenous glucose promoted WT seedling sensitivity to ABA (Fig. 8A), which implies a regulatory role of glucose in the ABA response. This supposition is supported by observations that atj11 seedlings exhibit higher endogenous glucose levels (Fig. 7A), and the inhibitory role of exogenous glucose in the ABA response was more apparent in atj1-1 than WT seedlings (Fig. 8A). This result also suggests that higher glucose levels in atj1-1 plants might alter ABA sensitivity in plants. In conclusion, authors’ previous and present data suggest a working model for the function of AtDjB1 in mediating the plant response to ABA and hyperosmotic stress, as summarized in Fig. 9. AtDjB1 indirectly modulates plant adaptation to osmotic stress likely by affecting mitochondrial function. This work supports the roles of molecular chaperones and mitochondria in modulating ABA signaling and osmotic stress tolerance in plants.

Acknowledgements – We thank ABRC for distributing the AtDjB1 insertion mutant seeds. This research was supported by the National Natural Science Foundation of China (Grant No.31070257 and 31270308).

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Edited by B. Huang

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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. AtDjB1 interacts with mtHSC70-1. Fig. S2. AtDjB1 stimulates ATP hydrolysis and regulates the cellular ATP level and respiration rate of Arabidopsis thaliana seedlings. Fig. S3. The effects of AtDjB1 knockout on the sucrose response in WT, atj1-1 and R6 seedlings. Fig. S4. Microarray analyses of AtDjB1 gene expression in roots (A, C), shoots (B, D) or seedlings (E) of Arabidopsis thaliana plants treated with salinity (A, B), dehydration (C, D) or ABA (E). Fig. S5. The effects of AtDjB1 overexpression on seedling cotyledon greening under osmotic stress. Table S1. Primers used for the real-time PCR amplifications.