- Email: [email protected]
Overexpression of a tobacco J-domain protein enhances drought tolerance in transgenic Arabidopsis
Plant Physiology and Biochemistry 83 (2014) 100e106
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Overexpression of a tobacco J-domain protein enhances drought tolerance in transgenic Arabidopsis Zongliang Xia a, *, 1, Xiaoquan Zhang a, 1, Junqi Li a, Xinhong Su b, Jianjun Liu c a
Henan Agricultural University, Zhengzhou 450002, PR China Henan Tobacco Company, Zhengzhou 450008, PR China c Zhengzhou Branch, Henan Tobacco Company, Zhengzhou 450001, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2014 Accepted 27 July 2014 Available online 6 August 2014
DnaJ proteins constitute a DnaJ/Hsp40 family and are important regulators involved in diverse cellular functions. To date, the molecular mechanisms of DnaJ proteins involved in response to drought stress in plants are largely unknown. In this study, a putative DnaJ ortholog from Nicotiana tabacum (NtDnaJ1), which encodes a putative type-I J-protein, was isolated. The transcript levels of NtDnaJ1 were higher in aerial tissues and were markedly up-regulated by drought stress. Over-expression of NtDnaJ1 in Arabidopsis plants enhanced their tolerance to osmotic or drought stress. Quantitative determination of H2O2 accumulation has shown that H2O2 content increased in wild-type and transgenic seedlings under osmotic stress, but was significantly lower in both transgenic lines compared with the wild-type. Expression analysis of stress-responsive genes in NtDnaJ1-transgenic Arabidopsis revealed that there was significantly increased expression of genes involved in the ABA-dependent signaling pathway (AtRD20, AtRD22 and AtAREB2) and antioxidant genes (AtSOD1, AtSOD2, and AtCAT1). Collectively, these data demonstrate that NtDnaJ1 could be involved in drought stress response and its over-expression enhances drought tolerance possibly through regulating expression of stress-responsive genes. This study may facilitate our understandings of the biological roles of DnaJ protein-mediated abiotic stress in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Drought Gene expression J-domain protein Tobacco
1. Introduction J-domain proteins (also called DnaJ proteins) constitute a DnaJ/ Hsp40 family and are conserved co-chaperones for HSP70s (Caplan et al., 1993; Silver and Way, 1993; Qiu et al., 2006). DnaJ proteins are involved in a variety of essential cellular processes including protein folding, assembly, translocation, degradation, stabilization and refolding (Wang et al., 2004; Mayer and Bukau, 2005; Craig et al., 2006; Rajan and D’Silva, 2009). Besides their coechaperone activity, DnaJ proteins function as protein disulfide isomerases to catalyze protein disulfide formation, reduction, and isomerization (de Crouy-Chanel et al., 1995).
Abbreviations: DnaJ, J-protein; Hsp40, 40 kDa heat shock protein; HPD motif, Histidine, proline, aspartate motif; MS, Murashige and skoog; qPCR, Quantitative PCR. * Corresponding author. College of Life Science, Henan Agricultural University, Zhengzhou 450002, PR China. Tel.: þ86 371 63579676; fax: þ86 371 63555790. E-mail address: [email protected] (Z. Xia). 1 Contributed equally. http://dx.doi.org/10.1016/j.plaphy.2014.07.023 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
DnaJ was originally identified in Escherichia coli as a 41 kDa heat shock protein (Goffin and Georgopoulos, 1998). Subsequently, members of the J-protein family were found to function as molecular chaperones by binding Hsp70 to stimulate ATP hydrolysis, and stabilizing the Hsp70 interaction with substrate proteins (Szyperski et al., 1994; Szabo et al., 1996; Cheetham and Caplan, 1998). The J domain is a highly conserved ~70 amino acid a-helical region in DnaJ proteins (Hennessy et al., 2005). Plant J-domain proteins have been classified into four types (I, II, III, and IV) based on the presence of other conserved domains (Rajan and D’Silva, 2009; Miernyk, 2001; Walsh et al., 2004). Traditional type-I Jdomain proteins contain four domains including a J domain, a Gly/ Phe (G/F) domain, a CXXCXGXG zinc-finger domain and a less conserved C-terminal domain. Type-II J proteins lack the zincfinger domain, whereas type-III J- proteins contain only the J domain. Type-IV J proteins have been recently described and classified as ‘J-like proteins’, with significant sequence and structural similarities with the J domain, but they lack the HPD motif (Walsh et al., 2004). The well-characterized Arabidopsis genome is known to harbor 120 predicted J-domain proteins (Rajan and D’Silva, 2009).
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
Increasing evidence has shown that J-domain proteins play important roles in growth and development (Kneissl et al., 2009; Yang et al., 2009; Shen et al., 2011; Bekh-Ochir et al., 2013), disease resistance (Bekh-Ochir et al., 2013; Liu and Whitham, 2013; Du et al., 2013), and abiotic stress responses (Yang et al., 2010; Zhou et al., 2012). For example, the Arabidopsis J-domain protein OWL1 is involved in the regulation of germination, cotyledon opening, hypocotyl elongation, and perception of very low light influences (Kneissl et al., 2009). TMS1, a DnaJ protein with disulfide isomerase activity, is required for thermotolerance of pollen tubes in Arabidopsis, possibly by functioning as a co-molecular chaperone (Yang et al., 2009). The Arabidopsis Type-I J-domain protein J3 mediates the integration of flowering signals during the floral transition and regulates the plasma membrane Hþ-ATPase through interacting with the PKS5 kinase under high salinity conditions (Yang et al., 2009; Shen et al., 2011). The J-protein AtDjB1 facilitates thermotolerance by protecting cells against heat-induced oxidative damage in Arabidopsis (Zhou et al., 2012). Recently, it has been reported that a type-III J domain protein GmHSP40.1 causes HR-like cell death in tobacco (Liu and Whitham, 2013). More interestingly, a tobacco type-I J-domain protein NbMIP1 has been shown to be required for both tobacco mosaic virus infection and plant innate immunity through functioning as co-chaperones (Du et al., 2013). In spite of progress made in understanding function of DnaJ proteins, few reports have been concerned the role of J-domain proteins in drought stress in plants (Wang et al., 2014). Drought stress often adversely affects plant growth and productivity, thus it is still a serious problem in agriculture worldwide. Tobacco is an important crop as well as a model plant system, and its productivity is vulnerable to drought. To identify genes important in drought stress response in tobacco, we previously identified mRNAs upregulated by drought stress through microarray analysis (unpublished data). One highly induced mRNA encoding a J-domain protein (NtDnaJ1) was characterized in detail. We further characterized the putative NtDnaJ1 in transgenic Arabidopsis to investigate drought tolerance and possible function mechanisms. 2. Materials and methods 2.1. Plant materials and stress treatment Tobacco (Nicotiana tabacum cv. Xanthi) was used throughout this study. Arabidopsis thaliana ecotype Col-0 was used for gene transformation. Plants were grown in a growth room as described previously (Xia et al., 2012a). Drought stress in four-week-old plants was realized by replacing the water with 15% PEG 6000 and leaves were sampled at 0, 6, 12, 24, or 48 h for expression analysis as described by us (Xia et al., 2013). 2.2. Cloning of NtDnaJ1 and sequence analysis The drought-induced EST encoding a putative DnaJ/Hsp40 protein was used to do BLAST (http://www.ncbi.nlm.nih.gov/) and mRNA sequences containing such an EST were downloaded for gene prediction. The gene is highly homologous to DnaJ/Hsp40 family member, and thus is named NtDnaJ1. Two primers NtDnaJ1F and NtDnaJ1-R (Table S1) were designed for amplifying the open reading frame (ORF) of NtDnaJ1. The 1257 bp PCR product was verified by sequencing. The primary structural analysis was performed using InterProScan (http://www.ebi.ac.uk/InterProScan). The alignment of the deduced protein sequences and phylogenetic tree analyses were done by DNASTAR and MEGA 5.1, respectively, using standard parameters (Tamura et al., 2011).
101
2.3. Real-time PCR analysis Real-time PCR was used to determine the expression patterns of NtDnaJ1 in different organs and under drought condition. The qRTPCR was performed in triplicate with an IQ5 light cycler system (Bio-Rad) using SYBR Premix ExTaq II (Takara, Japan) with genespecific primers NtDnaJ1-F1 and NtDnaJ1-R1 (Table S1), which produces a 195-bp product. The tobacco NtActin transcript was used as an internal control to quantify the relative transcript levels as described by us previously (Xia et al., 2013). The relative level of gene expression was detected using the 2DDCT method (Livak and Schmittgen, 2001). To examine the relative expression of NtDnaJ1 in transgenic Arabidopsis plants, the AtActin2 transcript (gene-specific primers AtActin2-F1 and AtActin2-R1; Table S1) was used as an internal control to quantify the expression levels, and the lowest expression level among transgenic lines was regarded as standard and the relative level of gene expression was computed as described above. To assay the expression of stress-responsive genes (AtSOD1, AtSOD2, AtCAT1, AtRD20, AtRD22 and AtAREB2) in transgenic Arabidopsis plants, qRT-PCR analysis was also performed with the RNA samples isolated from four-week-old transgenic plants harvested in soil under normal conditions. Total RNA isolation and reverse transcription were performed as described above. PCR amplification was performed with gene specific primers (Table S1). Criteria for designing primers were a primer size between 22 and 25, an optimal Tm at 60 C, and a product size ranging from 150 bp to 250 bp. Amplification of AtActin2 was used as an internal control, and qRT-PCR experimental procedures were performed as described above. For the entire qRT-PCR assay, three technical replicates were performed for each experiment and the expression of each gene was investigated in three biological replicates. 2.4. Construction of plant expression vectors and development of transgenic Arabidopsis lines The NtDnaJ1 coding sequence was amplified and introduced into the pART7 plasmid (Xia et al., 2012b) using primers NtDnaJ1-F2 with BamHI restriction site (underlined) and NtDnaJ1-R2 with XbaI restriction site (underlined) (Table S1) and was subsequently inserted downstream of the 35S promoter in the plasmid vector pART7. The resulting expression cassette containing the 35S promoter and NtDnaJ1 coding sequence was cut and inserted into the binary vector pART27, producing the transformation construct pART27-35S-NtDnaJ1. The binary construct was introduced into Agrobacterium tumefaciens (strain GV3101) and then transformed into Arabidopsis (Col0) via the floral dip method (Clough and Bent, 1998). Transgenic lines were selected by germinating seeds on medium containing Murashige and Skoog (MS) basal salt mixture (SigmaeAldrich, USA) and 50 mg/L kanamycin. After two weeks on selection medium, green seedlings (T1 plants) were transferred to soil pots and grown to maturity in a growth room. The presence of the transgene in each plant was checked by PCR with genomic DNA from leaves of individual plant using primers 35SP-F and NtDnaJ1-R3 (the forward primer 35SP-F is from CaMV 35S promoter sequence) (Table S1). The PCR-positive plants as transgenes were grown to maturity and seeds were collected (T2 seed). T2 seeds were germinated on kanamycin selection medium again and the one-copy lines were identified by examining the segregation ratio (3:1) of the kanamycin selectable marker. Each one-copy line was maintained growth to set seeds until T3 generation. Five independent homozygous NtDnaJ1 transgenic lines (named OE-8, OE-14, OE-18, OE-22 and OE-27) were developed. The expression levels of the transgene
102
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
among the five lines were evaluated by qRT-PCR with the primers NtDnaJ1-F1 and NtDnaJ1-R1 (Table S1) as described above. 2.5. Analysis of NtDnaJ1-overexpressing Arabidopsis for osmotic and drought stress tolerance For osmotic stress tolerance analysis, surface-sterilized seeds of both transgenic Arabidopsis lines (OE-18 and OE-22) or wild-type were germinated and cultured on MS medium for 7 days, and then the seedlings (30e40) were transferred to grow on MS medium supplemented with 0 or 250 mM mannitol in the growth chamber. The phenotype of seedlings was photographed after 7 days of growth. The experiment was repeated at least three times. Survival rates (%) under mannitol treatment were determined as the number of visibly green plants after 7 days. For drought tolerance analysis, four-week-old plants were subjected to progressive drought by withholding water until a nearly lethal effect of dehydration (about 3 weeks) was observed. The drought stress experiment was performed at least three times. Seven days after re-watering, survival rates (%) under drought stress were determined as described above.
2.6. Quantitative determination of H2O2 accumulation Seven days old seedlings of WT and both transgenic lines were transferred to MS medium supplemented with 0 or 250 mM mannitol in the growth chamber. After osmotic stress for 3 days, the content of H2O2 in the WT and transgenic seedlings was measured following the method of Xia et al. (2012b). 3. Results 3.1. Molecular characterization of NtDnaJ1 At the start of this work, total RNAs from the leaves of tobacco plants, which had been treated with 15% PEG for 12 h, were used as samples for microarray experiments. A partial cDNA fragment with about 5-fold induction was obtained (data not shown). Homology search by BLAST analysis showed that the gene is highly homologous to DnaJ, a member of the DnaJ/Hsp40 family, and thus is named NtDnaJ1 (accession no. AJ299254). The ORF of the NtDnaJ1 consists of 1257 nucleotide acids and encodes a protein of 418 amino acids with a predicted molecular mass of about 46 kDa. Like other known
Fig. 1. Sequence alignment and phylogenetic analysis of DnaJ1 proteins from N. tabacum and other plant species. A An alignment is shown for the deduced amino acid sequence of DnaJ1s from N. tabacum, A. thaliana, S. lycopersicum, S. tuberosum, O. sativa and Z. mays. The numbers on the left indicate the amino acid position. Identical residues in all these proteins are shown in a black background. Dashes indicated gaps introduced for optimal alignment. The putative J, G/F, Zinc finger and C-terminal domains are underlined with a thick blue line, a thick red line, a thick purple line and a thick black line, respectively. B Phylogenetic tree based on the six DnaJ1 protein sequences. The bootstrap values shown were calculated based on 500 replications. The tree was constructed using the neighbor-joining method. N. tabacum, AJ299254; A. thaliana, At3g44110; S. lycopersicum, XM_004239689; S. tuberosum, X94301; O.sativa, NM_001058032 and Z. mays, EU960799. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
type-I DnaJ proteins, the deduced amino acid sequence of the NtDnaJ1 exhibits typical structural characteristics with four functional domains, including a J domain (residues 1e72), a Gly/Phe (G/ F) domain (residues 82e115), a CXXCXGXG zinc-finger domain (residues 148e216) and a less conserved C-terminal domain (residues 229e418) (Fig. 1A). Amino acid sequence comparisons have revealed that NtDnaJ1 exhibits high identity to counterpart proteins from A. thaliana (79.1% identity), Solanum lycopersicon (84.3% identity), Symphytum tuberosum (84% identity), Oryza sativa (83.5% identity), and Zea mays (82.4% identity) (Fig. 1A). A phylogenetic tree was established based on DnaJ1 protein sequences available in GenBank from 6 plant species (Arabidopsis, tobacco, tomato, potato, rice, and maize) (Fig. 1B). As shown in Fig. 1B, interestingly, DnaJ from Arabidopsis formed a subgroup distinct from the other DnaJs subgroup, including the solanaceae DnaJs from S. Lycopersicon, S.tuberosum and N. tabacum, and the monocot DnaJs from Oryza sativa and Z.mays. The NtDnaJ1 showed higher identities with DnaJ proteins from tomato, potato, rice, and maize, and thus was clustered into the same isoform subgroup. The solanaceae DnaJs from S. lycopersicon and S. tuberosum were clustered into the same isoform subgroup. These results clearly demonstrated that NtDnaJ1 shares basic structural feature similar to the known DnaJ proteins from Arabidopsis and other crop plants, thus it could be identified as an ortholog of DnaJ protein.
3.2. Transcript levels of NtDnaJ1 in various organs of tobacco and its responses to PEG-induced water stress Transcriptional patterns of NtDnaJ1 were examined in five organs (roots, stems, leaves, flowers, and immature fruits) by qRTPCR. As shown in Fig. 2A, NtDnaJ1 mRNA was detected in roots, stems, leaves, flowers, and immature fruits. The NtDnaJ1 transcript levels were significantly high in leaves, flowers and stems. In contrast, NtDnaJ1 transcripts were low in roots and immature fruits
103
(Fig. 2A). The highest relative expression occurred in the leaves, with about 6 times as high as that of the roots (Fig. 2A). Time-course analysis of NtDnaJ1 transcript levels in tobacco plants under PEG-induced water stress was performed by qRT-PCR (Fig. 2B). The transcript level of NtDnaJ1 was increased rapidly after 6 h, and then remained higher levels during 48 h period of drought stress with a peak at 12 h (about 5-fold increase in transcripts) (Fig. 2B), suggesting that NtDnaJ1 is involved in drought response in tobacco. 3.3. Response of NtDnaJ1-overexpressing Arabidopsis seedlings to osmotic stress To explore the physiological function of NtDnaJ1, a CaMV 35S promoter-driven binary expression construct harboring NtDnaJ1 was developed and transformed into Arabidopsis by Agrobacteriummediated floral dip transformation. To this end, five homozygous transgenic lines (named OE-8, OE-14, OE-18, OE-22, and OE-27) were developed, in which NtDnaJ1 transcript levels were analyzed by qRT-PCR (Fig.3A). Both lines (OE-18 and OE-22) with higher NtDnaJ1 transcripts were chosen for further analysis. Both transgenic and WT seedlings grown on MS medium alone for 7 days were transferred to MS plates supplemented with 0 or 250 mM mannitol for additional 7 days. On MS medium alone, all the transgenic seedlings showed little or no difference in growth compared to WT (Fig. 3B; left panel). On MS medium containing 250 mM mannitol, WT seedlings showed more chlorosis and were severely affected in growth, and nearly 40% of WT seedlings were dead, whereas about 90% of transgenic seedlings were still alive (Fig. 3 B, C). H2O2 accumulation was quantitatively determined in the WT and OE seedlings exposed to 250 mM mannitol for 3 days. As shown in Fig. 3D, the content of H2O2 increased in both WT and OE seedlings under mannitol-induced osmotic stress, but was significantly lower in both transgenic lines compared with the WT (185% increase for WT, and 75% increase for both OE lines averagely) (Fig. 3D). In contrast, under normal condition (control), no significant difference was observed in H2O2 accumulation between WT and both transgenic seedlings (Fig. 3D). These results demonstrate that overexpression of NtDnaJ1 in transgenic Arabidopsis enhances tolerance to osmotic stress possibly by reducing the accumulation of H2O2. 3.4. Response of NtDnaJ1-overexpressing Arabidopsis plants to drought stress
Fig. 2. Transcript profiles of NtDnaJ1 in major organs of N. tabacum and its response to drought stress. A The transcriptional pattern of NtDnaJ1 in N. tabacum root (R), stem (St), leaf (L), flower (F), and immature fruit (If) samples evaluated by real-time PCR. The transcript levels of N. tabacum internal control gene Actin were also evaluated in various samples. For each assay, the expression level in roots was defined as 1.0, and data represented means ± SE of three technical replicates. **t-test, with P < 0.01; *ttest, with P < 0.05. B Time-course analysis of NtDnaJ1 transcript levels under PEGinduced water stress by real-time PCR. Four-week-old tobacco seedlings were exposed to 15% PEG6000 for indicated time points (0, 6, 12, 24 and 48 h), and leaf samples were used for real-time PCR analysis. Actin was used as an internal control. For each treatment, the expression level at time point 0 was defined as 1.0, and data represented means ± SE of three technical replicates. *t-test, with P < 0.05.
To characterize the performance of NtDnaJ1 transgenic lines under watering-stress (drought) in soil, both OE lines were tested. Under well-watered conditions, there was no obvious difference between WT and transgenic lines in leaves size and number of plants (Fig. 4 A). After 20 days without watering, all WT plants displayed severe wilting (all leaves were severely curled and more than 70% leaves were turning dark purple and dead), whereas NtDnaJ1 transgenic lines showed signs of heavy water stress and most leaves of transgenic plants were still fully expanded (Fig. 4 A). Seven days after re-watering, nearly 60% of WT plants survived, whereas almost all transgenic lines survived the stress and started to grow (Fig. 4 A, B). These results provide evidence that overexpression of NtDnaJ1 in transgenic Arabidopsis plants also improves tolerance to drought stress. 3.5. Changes in transcript levels of stress-responsive genes in NtDnaJ1 transgenic Arabidopsis To reveal the possible molecular mechanisms underlying the improvement of drought stress tolerance in transgenic Arabidopsis by NtDnaJ1, we examined the expression patterns of antioxidant genes (AtSOD1, AtSOD2, and AtCAT1) and stress-responsive genes
104
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
Fig. 3. Osmotic tolerance analysis of NtDnaJ1-overexpressing and wild-type Arabidopsis seedlings. A Transcription levels of NtDnaJ1 in wild-type (WT; Col-0) Arabidopsis plants and five homozygous over-expression (OE) lines (named OE-8, OE-14, OE-18, OE-22 and OE-27). NtDnaJ1 transcripts detected by qPCR were present in the OE lines, but not in the wild-type plants. B Representative phenotypes of NtDnaJ1-overexpressing Arabidopsis seedlings under mannitol treatment. Surface-sterilized seeds of both transgenic lines (OE-18 and OE-22) or WT were germinated and cultured on MS medium for 7 days, and then the seedlings (30e40) were transferred to grow on MS medium supplemented with 0 or 250 mM mannitol for 7 days. C Survival rates (%) under mannitol stress in Fig. b were determined as the number of visibly green plants after 7 days. Values are mean ± SE, n ¼ 30e40. *t-test, with P < 0.05. D H2O2 level was quantified in the wild-type and OE (OE-18 and OE-22) seedlings exposed to 250 mM mannitol for 3 days. Error bars indicate SE. **t-test, with P < 0.01.
(AtRD20, AtRD22 and AtAREB2) in both transgenic lines (OE-18 and OE-22) and WT Arabidopsis. The expression of all of the genes was significantly up-regulated in both transgenic plants compared with WT plants. Furthermore, it is noticeable that the antioxidant gene AtSOD2 and the ABA responsive element-binding gene AtAREB2 exhibited much higher levels of up-regulation than other genes (about 10-fold increase averagely for both lines) (Fig. 5). These data demonstrate that NtDnaJ1 overexpression up-regulates transcriptional levels of stress-related genes in plants. 4. Discussion Drought stress has drastic effects on biomass and grain yields of crops worldwide. However, the molecular mechanisms underlying Jdomain proteins-mediated drought tolerance in plants are largely unknown. In this study, we investigated physiological roles of a putative J-domain protein NtDnaJ1 from tobacco and its possible function mechanisms. Our genetic evidence has demonstrated that NtDnaJ1 over-expression enhances drought stress tolerance in transgenic plants. To our knowledge, this is the first J-domain protein from tobacco to be functionally characterized during drought stress. 4.1. DnaJ proteins are conserved, but have diverse function in higher plants Like known plant J-domain proteins, NtDnaJ1 has higher sequence identities (79e84%) and four typical structural domains
(Fig. A). From this, it can be reasonably concluded that the NtDnaJ1 cDNA clone encodes tobacco DnaJ isoform. NtDnaJ1 was found to be highly expressed in aerial tissues of tobacco, and its highest expression level occurred in leaves (Fig. 2A), indicating this gene may be constitutively expressed during both vegetative and reproductive growth. Previous studies on J-domain proteins from Arabidopsis have shown that AtDjB1 was induced by heat stress; AtDjB1 overexpression can facilitate thermotolerance and oxidative stress tolerance in Arabidopsis (Zhou et al., 2012). The Arabidopsis Type-I Jdomain protein J3 knock-out mutation led to sensitive to high salinity (Yang et al., 2010). However, no drought-induced expression of DnaJ gene has been reported. Compared to this expression pattern, NtDnaJ1 is significantly affected by PEG-induced water stress (Fig.2 B). Genetic evidence further proved that NtDnaJ1 OE lines showed enhanced osmotic or drought tolerance in transgenic Arabidopsis (Figs. 3 and 4). These results indicate that function divergence of the DnaJ orthologs may occur in the genomes of Arabidopsis and tobacco, although these genes have high similarities in sequences. 4.2. NtDnaJ1 probably participates in ABA-dependent signaling pathway in transgenic plants during drought stress The molecular mechanisms underlying the response of higher plants to drought stress are complicated. In response to drought stress, various genes are up-regulated, which can mitigate the
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
105
effects of stress and lead to an adjustment of plant stress tolerance. ABA-dependent signaling pathway is involved in the inducible expression of specific genes during drought stress (Shinozak and Yamaguchi-Shinozaki, 2000). RD20 is highly responsive to stress and is often used as a stress marker gene (Aubert et al., 2010). RD22 is induced by drought and ABA, and is involved in an ABAdependent stress signaling pathway (Yamaguchi-Shinozaki and Shinozaki, 1993). AREB2 is an important regulatory gene involved in signal transduction or transcriptional regulation during stress conditions (Shinozaki and Yamaguchi-Shinozaki, 2007; Saibo et al., 2009). The increased expression of RD20, RD22 and AREB2 in NtDnaJ1 transgenic plants suggests that NtDnaJ1 may participate in the ABA-dependent signaling pathway during drought stress. In support of this notion, a type-III J-protein BIL2 from Arabidopsis has been shown to be involved in plant growth, and salt and strong light tolerance in brassinosteroid (BR) signal transduction recently (Bekh-Ochir et al., 2013). 4.3. The antioxidant mechanism might be involved in NtDnaJ1 conferring drought stress tolerance
Fig. 4. Phenotypes of NtDnaJ1-overexpressing and wild-type Arabidopsis plants in response to drought stress. A Drought tolerance of potted plants of wild-type and NtDnaJ1-overexpressing Arabidopsis. Four-week-old WT and transgenic OE (OE-18 and OE-22) plants were grown in soil in pots for 20 days without watering, and then rewatering after 7 days. B Survival rates (%) of drought-stressed wild-type and NtDnaJ1-overexpressing plants after 7 days recovery. Values are mean ± SE, n ¼ 30. *ttest, with P < 0.05.
Drought stress can reduce photosynthesis and result in excess ROS accumulation which leads to cell toxicity, membrane peroxidation and even cell death (Apel and Hirt, 2004). There is a constant need for efficient mechanisms to avoid oxidative damage to cells, and antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) play essential roles in the defense of plants against ROS (Miller et al., 2010). In this study, we have found that the transcripts of several antioxidant genes AtSOD1, AtSOD2, and AtCAT1 exhibited much higher levels in NtDnaJ1 transgenic plants (Fig. 5); moreover, the accumulation of H2O2 was significantly lower in NtDnaJ1-transgenic plants than the WT under drought stress (Fig. 3D); indicating that NtDnaJ1 might protect plants from dehydration stress by functioning as molecular chaperones for
Fig. 5. The expression of stress-responsive genes in NtDnaJ1-overexpressing and wild-type Arabidopsis. Relative expression levels of stress-responsive genes (AtSOD1, AtSOD2, AtCAT1, AtRD20, AtRD22 and AtAREB2) were determined by qRT-PCR using cDNA synthesized from total RNAs isolated from the leaves of 4-week-old Arabidopsis grown in soil under normal conditions. Relative expression levels of these six genes were normalized to the transcripts of Actin2 in the same samples. For each assay, the expression level of WT was taken as 1.0, and data represented means ± SE of three biological replicates. Bar indicates SE. **t-test, with P < 0.01; *t-test, with P < 0.05.
106
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
ROS-scavenging proteins to maintain higher levels of antioxidant activities. Supporting these interesting findings, mutation of the Arabidopsis type-II J-protein AtDjiB1 decreased thermotolerance under heat stress by down-regulating activities of antioxidant enzymes and accumulating ROS levels (Zhou et al., 2012); implying that the antioxidant system may be involved in J-protein-mediated abiotic stress tolerance in plants. In the future, further study will be interesting to clarify the mechanism of activation of the antioxidant system in NtDnaJ1-transgenic plants under drought stress. Acknowledgments This work was financially supported by the science and technology R&D project from Henan Tobacco Company (grant no. HYKJ201010). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.07.023. Authors' contributions ZX designed the study and wrote the manuscript; ZX, XZ, J-Li, XS and J-Liu performed the experiments and analyzed the data; ZX contributed reagents/materials/analysis tools. References Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev. Plant Biol. 55, 373e399. Aubert, Y., Vile, D., Pervent, M., Aldon, D., Ranty, B., Simonneau, T., Vavasseur, A., Jean- Philippe, G., 2010. RD20, A stress-inducible caleosin, participates in stomatal control, transpiration and drought tolerance in Arabidopsis thaliana. Plant Cell. Physiol. 51, 1975e1987. Bekh-Ochir, D., Shimada, S., Yamagami, A., Kanda, S., Ogawa, K., Nakazawa, M., Matsui, M., Sakuta, M., Osada, H., Asami, T., Nakano, T., 2013. A novel mitochondrial DnaJ/Hsp40 family protein BIL2 promotes plant growth and resistance against environmental stress in brassinosteroid signaling. Planta 237,1509e1525. Caplan, A.J., Cyr, D.M., Douglas, M.G., 1993. Eukaryotic homologs of Escherichia coli Dnaj-a diverse protein family that functions with Hsp70 stress proteins. Mol. Biol. Cell. 4, 555e563. Cheetham, M.E., Caplan, A.J., 1998. Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell. Stress Chaperones 3, 28e36. Clough, S.J., Bent, A.F., 1998. Floral dip: a simplified method for Agrobateriummediated transformation of Arabidopsis thaliana. Plant J. 16, 735e743. Craig, E.A., Huang, P., Aron, R., Andrew, A., 2006. The diverse roles of J-proteins, the obligate Hsp70 co-chaperone. Rev. Physiol. Biochem Pharmacol. 156, 1e21. de Crouy-Chanel, A., Kohiyama, M., Richarme, G., 1995. A novel function of Escherichia coli chaperone DnaJ protein-disulfide isomerase. J. Biol. Chem. 270, 22669e22672. Du, Y., Zhao, J., Chen, T., Liu, Q., Zhang, H., Wang, Y., Hong, Y., Xiao, F., Zhang, L., Shen, Q., Liu, Y., 2013. Type I J-domain NbMIP1 proteins are required for both tobacco mosaic virus infection and plant innate immunity. PLoS Pathog. 9, e1003659. Goffin, L., Georgopoulos, C., 1998. Genetic and biochemical characterization of mutations affecting the carboxy-terminal domain of the Escherichia coli molecular chaperone DnaJ. Mol. Microbiol. 30, 329e340. Hennessy, F., Boshoff, A., Blatch, G.L., 2005. Rational mutagenesis of a 40 kDa heat shock protein from Agrobacterium tumefaciens identifies amino acid residues critical to its in vivo function. Int. J. Biochem Cell. Biol. 37, 177e191. Kneissl, J., Wachtler, V., Chua, N.H., Bolle, C., 2009. OWL1: an Arabidopsis J-domain protein involved in perception of very low light fluences. Plant Cell. 21, 3212e3225. Liu, J.Z., Whitham, S.A., 2013. Overexpression of a soybean nuclear localized type III DnaJ domain-containing HSP40 reveals its roles in cell death and disease resistance. Plant J. 74, 110e121.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real- time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods 25, 402e408. Mayer, M.P., Bukau, B., 2005. Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62, 670e684. Miernyk, J.A., 2001. The J-domain proteins of Arabidopsis thaliana: an unexpectedly large and diverse family of chaperones. Cell. Stress Chaperones 6, 209e218. Miller, G., Suzuki, N., Ciftci-Yilmaz, S., Mittler, R., 2010. Reactive oxygen species homeostasis and signaling during drought and salinity stresses. Plant Cell. Environ. 33, 453e457. Qiu, X.B., Shao, Y.M., Miao, S., Wang, L., 2006. The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones. Cell. Mol. Life Sci. 63, 2560e2570. Rajan, V.B., D'Silva, P., 2009. Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors. Funct. Integr. Genomics 9, 433e446. Saibo, N.J.M., Lourenço, T., Oliveira, M.M., 2009. Transcription factors and regulation of photosynthetic and related metabolism under environmental stresses. Ann. Bot. 103, 609e623. Shen, L., Kang, Y.G., Liu, L., Yu, H., 2011. The J-domain protein J3 mediates the integration of flowering signals in Arabidopsis. Plant Cell. 23, 499e514. Shinozak, I.K., Yamaguchi-Shinozaki, K., 2000. Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signal pathways. Curr. Opin. Plant Biol. 3, 217e223. Shinozaki, K., Yamaguchi-Shinozaki, K., 2007. Gene networks involved in drought stress response and tolerance. J. Exp. Bot. 58, 221e227. Silver, P.A., Way, J.C., 1993. Eukaryotic Dnaj homologs and the specificity of Hsp70 activity. Cell 74, 5e6. Szabo, A., Korszun, R., Hartl, F.U., Flanagan, J., 1996. A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates. EMBO J. 15, 408e417. Szyperski, T., Pellecchia, M., Wall, D., Georgopoulos, C., Wuthrich, K., 1994. NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2e108) containing the highly conserved J domain. Proc. Natl. Acad. Sci. U. S. A. 91, 11343e11347. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731e2739. Walsh, P., Bursac, D., Law, Y.C., Cyr, D., Lithgow, T., 2004. The J-protein family: modulating protein assembly, disassembly and translocation. EMBO Rep. 5, 567e571. Wang, W., Vinocur, B., Shoseyov, O., Altman, A., 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9, 244e252. Wang, G., Cai, G., Kong, F., Deng, Y., Ma, N., Meng, Q., 2014. Overexpression of tomato chloroplast-targeted DnaJ protein enhances tolerance to drought stress and resistance to Pseudomonas solanacearum in transgenic tobacco. Plant Physiol. Biochem 82, 95e104. Xia, Z., Su, X., Wu, J., Wu, K., Zhang, H., 2012. Molecular cloning and functional characterization of a putative sulfite oxidase (SO) ortholog from Nicotiana benthamiana. Mol. Biol. Rep. 39, 2429e2437. Xia, Z., Sun, K., Wang, M., Wu, K., Zhang, H., 2012. Overexpression of a maize sulfite oxidase gene in tobacco enhances tolerance to sulfite stress via sulfite oxidation and CAT-mediated H2O2 scavenging. PLoS One 7, e37383. Xia, Z., Wei, Y., Sun, K., Wu, J., Wang, Y., Wu, K., 2013. The maize AAA-type protein SKD1 confers enhanced salt and drought stress tolerance in transgenic tobacco by interacting with Lyst-interacting protein 5. PLoS One 8, e69787. Yamaguchi-Shinozaki, K., Shinozaki, K., 1993. The plant hormone abscisic acid mediates the drought-induced expression but not the seed-specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis thaliana. Mol. Gen. Genet. 238, 17e25. Yang, K., Xia, C., Liu, X., Dou, X., Wang, W., Chen, L., Zhang, X., Xie, L., He, L., Ma, X., Ye, D., 2009. A mutation in Thermosensitive Male Sterile 1, encoding a heat shock protein with DnaJ and PDI domains, leads to thermosensitive gametophytic male sterility in Arabidopsis. Plant J. 57, 870e882. Yang, Y., Qin, Y., Xie, C., Zhao, F., Zhao, J., Liu, D., Chen, S., Fuglsang, A.T., Palmgren, M.G., Schumaker, K.S., Deng, X.W., Guo, Y., 2010. The Arabidopsis chaperone J3 regulates the plasma membrane Hþ-ATPase through interaction with the PKS5 kinase. Plant Cell. 22, 1313e1332. Zhou, W., Zhou, T., Li, M., Zhao, C., Jia, N., Wang, X., Sun, Y., Li, G., Xu, M., Zhou, R., Li, B., 2012. The Arabidopsis J-protein AtDjB1 facilitates thermotolerance by protecting cells against heat-induced oxidative damage. New. Phytol. 194, 364e378.
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Overexpression of a tobacco J-domain protein enhances drought tolerance in transgenic Arabidopsis Zongliang Xia a, *, 1, Xiaoquan Zhang a, 1, Junqi Li a, Xinhong Su b, Jianjun Liu c a
Henan Agricultural University, Zhengzhou 450002, PR China Henan Tobacco Company, Zhengzhou 450008, PR China c Zhengzhou Branch, Henan Tobacco Company, Zhengzhou 450001, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2014 Accepted 27 July 2014 Available online 6 August 2014
DnaJ proteins constitute a DnaJ/Hsp40 family and are important regulators involved in diverse cellular functions. To date, the molecular mechanisms of DnaJ proteins involved in response to drought stress in plants are largely unknown. In this study, a putative DnaJ ortholog from Nicotiana tabacum (NtDnaJ1), which encodes a putative type-I J-protein, was isolated. The transcript levels of NtDnaJ1 were higher in aerial tissues and were markedly up-regulated by drought stress. Over-expression of NtDnaJ1 in Arabidopsis plants enhanced their tolerance to osmotic or drought stress. Quantitative determination of H2O2 accumulation has shown that H2O2 content increased in wild-type and transgenic seedlings under osmotic stress, but was significantly lower in both transgenic lines compared with the wild-type. Expression analysis of stress-responsive genes in NtDnaJ1-transgenic Arabidopsis revealed that there was significantly increased expression of genes involved in the ABA-dependent signaling pathway (AtRD20, AtRD22 and AtAREB2) and antioxidant genes (AtSOD1, AtSOD2, and AtCAT1). Collectively, these data demonstrate that NtDnaJ1 could be involved in drought stress response and its over-expression enhances drought tolerance possibly through regulating expression of stress-responsive genes. This study may facilitate our understandings of the biological roles of DnaJ protein-mediated abiotic stress in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Drought Gene expression J-domain protein Tobacco
1. Introduction J-domain proteins (also called DnaJ proteins) constitute a DnaJ/ Hsp40 family and are conserved co-chaperones for HSP70s (Caplan et al., 1993; Silver and Way, 1993; Qiu et al., 2006). DnaJ proteins are involved in a variety of essential cellular processes including protein folding, assembly, translocation, degradation, stabilization and refolding (Wang et al., 2004; Mayer and Bukau, 2005; Craig et al., 2006; Rajan and D’Silva, 2009). Besides their coechaperone activity, DnaJ proteins function as protein disulfide isomerases to catalyze protein disulfide formation, reduction, and isomerization (de Crouy-Chanel et al., 1995).
Abbreviations: DnaJ, J-protein; Hsp40, 40 kDa heat shock protein; HPD motif, Histidine, proline, aspartate motif; MS, Murashige and skoog; qPCR, Quantitative PCR. * Corresponding author. College of Life Science, Henan Agricultural University, Zhengzhou 450002, PR China. Tel.: þ86 371 63579676; fax: þ86 371 63555790. E-mail address: [email protected] (Z. Xia). 1 Contributed equally. http://dx.doi.org/10.1016/j.plaphy.2014.07.023 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
DnaJ was originally identified in Escherichia coli as a 41 kDa heat shock protein (Goffin and Georgopoulos, 1998). Subsequently, members of the J-protein family were found to function as molecular chaperones by binding Hsp70 to stimulate ATP hydrolysis, and stabilizing the Hsp70 interaction with substrate proteins (Szyperski et al., 1994; Szabo et al., 1996; Cheetham and Caplan, 1998). The J domain is a highly conserved ~70 amino acid a-helical region in DnaJ proteins (Hennessy et al., 2005). Plant J-domain proteins have been classified into four types (I, II, III, and IV) based on the presence of other conserved domains (Rajan and D’Silva, 2009; Miernyk, 2001; Walsh et al., 2004). Traditional type-I Jdomain proteins contain four domains including a J domain, a Gly/ Phe (G/F) domain, a CXXCXGXG zinc-finger domain and a less conserved C-terminal domain. Type-II J proteins lack the zincfinger domain, whereas type-III J- proteins contain only the J domain. Type-IV J proteins have been recently described and classified as ‘J-like proteins’, with significant sequence and structural similarities with the J domain, but they lack the HPD motif (Walsh et al., 2004). The well-characterized Arabidopsis genome is known to harbor 120 predicted J-domain proteins (Rajan and D’Silva, 2009).
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
Increasing evidence has shown that J-domain proteins play important roles in growth and development (Kneissl et al., 2009; Yang et al., 2009; Shen et al., 2011; Bekh-Ochir et al., 2013), disease resistance (Bekh-Ochir et al., 2013; Liu and Whitham, 2013; Du et al., 2013), and abiotic stress responses (Yang et al., 2010; Zhou et al., 2012). For example, the Arabidopsis J-domain protein OWL1 is involved in the regulation of germination, cotyledon opening, hypocotyl elongation, and perception of very low light influences (Kneissl et al., 2009). TMS1, a DnaJ protein with disulfide isomerase activity, is required for thermotolerance of pollen tubes in Arabidopsis, possibly by functioning as a co-molecular chaperone (Yang et al., 2009). The Arabidopsis Type-I J-domain protein J3 mediates the integration of flowering signals during the floral transition and regulates the plasma membrane Hþ-ATPase through interacting with the PKS5 kinase under high salinity conditions (Yang et al., 2009; Shen et al., 2011). The J-protein AtDjB1 facilitates thermotolerance by protecting cells against heat-induced oxidative damage in Arabidopsis (Zhou et al., 2012). Recently, it has been reported that a type-III J domain protein GmHSP40.1 causes HR-like cell death in tobacco (Liu and Whitham, 2013). More interestingly, a tobacco type-I J-domain protein NbMIP1 has been shown to be required for both tobacco mosaic virus infection and plant innate immunity through functioning as co-chaperones (Du et al., 2013). In spite of progress made in understanding function of DnaJ proteins, few reports have been concerned the role of J-domain proteins in drought stress in plants (Wang et al., 2014). Drought stress often adversely affects plant growth and productivity, thus it is still a serious problem in agriculture worldwide. Tobacco is an important crop as well as a model plant system, and its productivity is vulnerable to drought. To identify genes important in drought stress response in tobacco, we previously identified mRNAs upregulated by drought stress through microarray analysis (unpublished data). One highly induced mRNA encoding a J-domain protein (NtDnaJ1) was characterized in detail. We further characterized the putative NtDnaJ1 in transgenic Arabidopsis to investigate drought tolerance and possible function mechanisms. 2. Materials and methods 2.1. Plant materials and stress treatment Tobacco (Nicotiana tabacum cv. Xanthi) was used throughout this study. Arabidopsis thaliana ecotype Col-0 was used for gene transformation. Plants were grown in a growth room as described previously (Xia et al., 2012a). Drought stress in four-week-old plants was realized by replacing the water with 15% PEG 6000 and leaves were sampled at 0, 6, 12, 24, or 48 h for expression analysis as described by us (Xia et al., 2013). 2.2. Cloning of NtDnaJ1 and sequence analysis The drought-induced EST encoding a putative DnaJ/Hsp40 protein was used to do BLAST (http://www.ncbi.nlm.nih.gov/) and mRNA sequences containing such an EST were downloaded for gene prediction. The gene is highly homologous to DnaJ/Hsp40 family member, and thus is named NtDnaJ1. Two primers NtDnaJ1F and NtDnaJ1-R (Table S1) were designed for amplifying the open reading frame (ORF) of NtDnaJ1. The 1257 bp PCR product was verified by sequencing. The primary structural analysis was performed using InterProScan (http://www.ebi.ac.uk/InterProScan). The alignment of the deduced protein sequences and phylogenetic tree analyses were done by DNASTAR and MEGA 5.1, respectively, using standard parameters (Tamura et al., 2011).
101
2.3. Real-time PCR analysis Real-time PCR was used to determine the expression patterns of NtDnaJ1 in different organs and under drought condition. The qRTPCR was performed in triplicate with an IQ5 light cycler system (Bio-Rad) using SYBR Premix ExTaq II (Takara, Japan) with genespecific primers NtDnaJ1-F1 and NtDnaJ1-R1 (Table S1), which produces a 195-bp product. The tobacco NtActin transcript was used as an internal control to quantify the relative transcript levels as described by us previously (Xia et al., 2013). The relative level of gene expression was detected using the 2DDCT method (Livak and Schmittgen, 2001). To examine the relative expression of NtDnaJ1 in transgenic Arabidopsis plants, the AtActin2 transcript (gene-specific primers AtActin2-F1 and AtActin2-R1; Table S1) was used as an internal control to quantify the expression levels, and the lowest expression level among transgenic lines was regarded as standard and the relative level of gene expression was computed as described above. To assay the expression of stress-responsive genes (AtSOD1, AtSOD2, AtCAT1, AtRD20, AtRD22 and AtAREB2) in transgenic Arabidopsis plants, qRT-PCR analysis was also performed with the RNA samples isolated from four-week-old transgenic plants harvested in soil under normal conditions. Total RNA isolation and reverse transcription were performed as described above. PCR amplification was performed with gene specific primers (Table S1). Criteria for designing primers were a primer size between 22 and 25, an optimal Tm at 60 C, and a product size ranging from 150 bp to 250 bp. Amplification of AtActin2 was used as an internal control, and qRT-PCR experimental procedures were performed as described above. For the entire qRT-PCR assay, three technical replicates were performed for each experiment and the expression of each gene was investigated in three biological replicates. 2.4. Construction of plant expression vectors and development of transgenic Arabidopsis lines The NtDnaJ1 coding sequence was amplified and introduced into the pART7 plasmid (Xia et al., 2012b) using primers NtDnaJ1-F2 with BamHI restriction site (underlined) and NtDnaJ1-R2 with XbaI restriction site (underlined) (Table S1) and was subsequently inserted downstream of the 35S promoter in the plasmid vector pART7. The resulting expression cassette containing the 35S promoter and NtDnaJ1 coding sequence was cut and inserted into the binary vector pART27, producing the transformation construct pART27-35S-NtDnaJ1. The binary construct was introduced into Agrobacterium tumefaciens (strain GV3101) and then transformed into Arabidopsis (Col0) via the floral dip method (Clough and Bent, 1998). Transgenic lines were selected by germinating seeds on medium containing Murashige and Skoog (MS) basal salt mixture (SigmaeAldrich, USA) and 50 mg/L kanamycin. After two weeks on selection medium, green seedlings (T1 plants) were transferred to soil pots and grown to maturity in a growth room. The presence of the transgene in each plant was checked by PCR with genomic DNA from leaves of individual plant using primers 35SP-F and NtDnaJ1-R3 (the forward primer 35SP-F is from CaMV 35S promoter sequence) (Table S1). The PCR-positive plants as transgenes were grown to maturity and seeds were collected (T2 seed). T2 seeds were germinated on kanamycin selection medium again and the one-copy lines were identified by examining the segregation ratio (3:1) of the kanamycin selectable marker. Each one-copy line was maintained growth to set seeds until T3 generation. Five independent homozygous NtDnaJ1 transgenic lines (named OE-8, OE-14, OE-18, OE-22 and OE-27) were developed. The expression levels of the transgene
102
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
among the five lines were evaluated by qRT-PCR with the primers NtDnaJ1-F1 and NtDnaJ1-R1 (Table S1) as described above. 2.5. Analysis of NtDnaJ1-overexpressing Arabidopsis for osmotic and drought stress tolerance For osmotic stress tolerance analysis, surface-sterilized seeds of both transgenic Arabidopsis lines (OE-18 and OE-22) or wild-type were germinated and cultured on MS medium for 7 days, and then the seedlings (30e40) were transferred to grow on MS medium supplemented with 0 or 250 mM mannitol in the growth chamber. The phenotype of seedlings was photographed after 7 days of growth. The experiment was repeated at least three times. Survival rates (%) under mannitol treatment were determined as the number of visibly green plants after 7 days. For drought tolerance analysis, four-week-old plants were subjected to progressive drought by withholding water until a nearly lethal effect of dehydration (about 3 weeks) was observed. The drought stress experiment was performed at least three times. Seven days after re-watering, survival rates (%) under drought stress were determined as described above.
2.6. Quantitative determination of H2O2 accumulation Seven days old seedlings of WT and both transgenic lines were transferred to MS medium supplemented with 0 or 250 mM mannitol in the growth chamber. After osmotic stress for 3 days, the content of H2O2 in the WT and transgenic seedlings was measured following the method of Xia et al. (2012b). 3. Results 3.1. Molecular characterization of NtDnaJ1 At the start of this work, total RNAs from the leaves of tobacco plants, which had been treated with 15% PEG for 12 h, were used as samples for microarray experiments. A partial cDNA fragment with about 5-fold induction was obtained (data not shown). Homology search by BLAST analysis showed that the gene is highly homologous to DnaJ, a member of the DnaJ/Hsp40 family, and thus is named NtDnaJ1 (accession no. AJ299254). The ORF of the NtDnaJ1 consists of 1257 nucleotide acids and encodes a protein of 418 amino acids with a predicted molecular mass of about 46 kDa. Like other known
Fig. 1. Sequence alignment and phylogenetic analysis of DnaJ1 proteins from N. tabacum and other plant species. A An alignment is shown for the deduced amino acid sequence of DnaJ1s from N. tabacum, A. thaliana, S. lycopersicum, S. tuberosum, O. sativa and Z. mays. The numbers on the left indicate the amino acid position. Identical residues in all these proteins are shown in a black background. Dashes indicated gaps introduced for optimal alignment. The putative J, G/F, Zinc finger and C-terminal domains are underlined with a thick blue line, a thick red line, a thick purple line and a thick black line, respectively. B Phylogenetic tree based on the six DnaJ1 protein sequences. The bootstrap values shown were calculated based on 500 replications. The tree was constructed using the neighbor-joining method. N. tabacum, AJ299254; A. thaliana, At3g44110; S. lycopersicum, XM_004239689; S. tuberosum, X94301; O.sativa, NM_001058032 and Z. mays, EU960799. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
type-I DnaJ proteins, the deduced amino acid sequence of the NtDnaJ1 exhibits typical structural characteristics with four functional domains, including a J domain (residues 1e72), a Gly/Phe (G/ F) domain (residues 82e115), a CXXCXGXG zinc-finger domain (residues 148e216) and a less conserved C-terminal domain (residues 229e418) (Fig. 1A). Amino acid sequence comparisons have revealed that NtDnaJ1 exhibits high identity to counterpart proteins from A. thaliana (79.1% identity), Solanum lycopersicon (84.3% identity), Symphytum tuberosum (84% identity), Oryza sativa (83.5% identity), and Zea mays (82.4% identity) (Fig. 1A). A phylogenetic tree was established based on DnaJ1 protein sequences available in GenBank from 6 plant species (Arabidopsis, tobacco, tomato, potato, rice, and maize) (Fig. 1B). As shown in Fig. 1B, interestingly, DnaJ from Arabidopsis formed a subgroup distinct from the other DnaJs subgroup, including the solanaceae DnaJs from S. Lycopersicon, S.tuberosum and N. tabacum, and the monocot DnaJs from Oryza sativa and Z.mays. The NtDnaJ1 showed higher identities with DnaJ proteins from tomato, potato, rice, and maize, and thus was clustered into the same isoform subgroup. The solanaceae DnaJs from S. lycopersicon and S. tuberosum were clustered into the same isoform subgroup. These results clearly demonstrated that NtDnaJ1 shares basic structural feature similar to the known DnaJ proteins from Arabidopsis and other crop plants, thus it could be identified as an ortholog of DnaJ protein.
3.2. Transcript levels of NtDnaJ1 in various organs of tobacco and its responses to PEG-induced water stress Transcriptional patterns of NtDnaJ1 were examined in five organs (roots, stems, leaves, flowers, and immature fruits) by qRTPCR. As shown in Fig. 2A, NtDnaJ1 mRNA was detected in roots, stems, leaves, flowers, and immature fruits. The NtDnaJ1 transcript levels were significantly high in leaves, flowers and stems. In contrast, NtDnaJ1 transcripts were low in roots and immature fruits
103
(Fig. 2A). The highest relative expression occurred in the leaves, with about 6 times as high as that of the roots (Fig. 2A). Time-course analysis of NtDnaJ1 transcript levels in tobacco plants under PEG-induced water stress was performed by qRT-PCR (Fig. 2B). The transcript level of NtDnaJ1 was increased rapidly after 6 h, and then remained higher levels during 48 h period of drought stress with a peak at 12 h (about 5-fold increase in transcripts) (Fig. 2B), suggesting that NtDnaJ1 is involved in drought response in tobacco. 3.3. Response of NtDnaJ1-overexpressing Arabidopsis seedlings to osmotic stress To explore the physiological function of NtDnaJ1, a CaMV 35S promoter-driven binary expression construct harboring NtDnaJ1 was developed and transformed into Arabidopsis by Agrobacteriummediated floral dip transformation. To this end, five homozygous transgenic lines (named OE-8, OE-14, OE-18, OE-22, and OE-27) were developed, in which NtDnaJ1 transcript levels were analyzed by qRT-PCR (Fig.3A). Both lines (OE-18 and OE-22) with higher NtDnaJ1 transcripts were chosen for further analysis. Both transgenic and WT seedlings grown on MS medium alone for 7 days were transferred to MS plates supplemented with 0 or 250 mM mannitol for additional 7 days. On MS medium alone, all the transgenic seedlings showed little or no difference in growth compared to WT (Fig. 3B; left panel). On MS medium containing 250 mM mannitol, WT seedlings showed more chlorosis and were severely affected in growth, and nearly 40% of WT seedlings were dead, whereas about 90% of transgenic seedlings were still alive (Fig. 3 B, C). H2O2 accumulation was quantitatively determined in the WT and OE seedlings exposed to 250 mM mannitol for 3 days. As shown in Fig. 3D, the content of H2O2 increased in both WT and OE seedlings under mannitol-induced osmotic stress, but was significantly lower in both transgenic lines compared with the WT (185% increase for WT, and 75% increase for both OE lines averagely) (Fig. 3D). In contrast, under normal condition (control), no significant difference was observed in H2O2 accumulation between WT and both transgenic seedlings (Fig. 3D). These results demonstrate that overexpression of NtDnaJ1 in transgenic Arabidopsis enhances tolerance to osmotic stress possibly by reducing the accumulation of H2O2. 3.4. Response of NtDnaJ1-overexpressing Arabidopsis plants to drought stress
Fig. 2. Transcript profiles of NtDnaJ1 in major organs of N. tabacum and its response to drought stress. A The transcriptional pattern of NtDnaJ1 in N. tabacum root (R), stem (St), leaf (L), flower (F), and immature fruit (If) samples evaluated by real-time PCR. The transcript levels of N. tabacum internal control gene Actin were also evaluated in various samples. For each assay, the expression level in roots was defined as 1.0, and data represented means ± SE of three technical replicates. **t-test, with P < 0.01; *ttest, with P < 0.05. B Time-course analysis of NtDnaJ1 transcript levels under PEGinduced water stress by real-time PCR. Four-week-old tobacco seedlings were exposed to 15% PEG6000 for indicated time points (0, 6, 12, 24 and 48 h), and leaf samples were used for real-time PCR analysis. Actin was used as an internal control. For each treatment, the expression level at time point 0 was defined as 1.0, and data represented means ± SE of three technical replicates. *t-test, with P < 0.05.
To characterize the performance of NtDnaJ1 transgenic lines under watering-stress (drought) in soil, both OE lines were tested. Under well-watered conditions, there was no obvious difference between WT and transgenic lines in leaves size and number of plants (Fig. 4 A). After 20 days without watering, all WT plants displayed severe wilting (all leaves were severely curled and more than 70% leaves were turning dark purple and dead), whereas NtDnaJ1 transgenic lines showed signs of heavy water stress and most leaves of transgenic plants were still fully expanded (Fig. 4 A). Seven days after re-watering, nearly 60% of WT plants survived, whereas almost all transgenic lines survived the stress and started to grow (Fig. 4 A, B). These results provide evidence that overexpression of NtDnaJ1 in transgenic Arabidopsis plants also improves tolerance to drought stress. 3.5. Changes in transcript levels of stress-responsive genes in NtDnaJ1 transgenic Arabidopsis To reveal the possible molecular mechanisms underlying the improvement of drought stress tolerance in transgenic Arabidopsis by NtDnaJ1, we examined the expression patterns of antioxidant genes (AtSOD1, AtSOD2, and AtCAT1) and stress-responsive genes
104
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
Fig. 3. Osmotic tolerance analysis of NtDnaJ1-overexpressing and wild-type Arabidopsis seedlings. A Transcription levels of NtDnaJ1 in wild-type (WT; Col-0) Arabidopsis plants and five homozygous over-expression (OE) lines (named OE-8, OE-14, OE-18, OE-22 and OE-27). NtDnaJ1 transcripts detected by qPCR were present in the OE lines, but not in the wild-type plants. B Representative phenotypes of NtDnaJ1-overexpressing Arabidopsis seedlings under mannitol treatment. Surface-sterilized seeds of both transgenic lines (OE-18 and OE-22) or WT were germinated and cultured on MS medium for 7 days, and then the seedlings (30e40) were transferred to grow on MS medium supplemented with 0 or 250 mM mannitol for 7 days. C Survival rates (%) under mannitol stress in Fig. b were determined as the number of visibly green plants after 7 days. Values are mean ± SE, n ¼ 30e40. *t-test, with P < 0.05. D H2O2 level was quantified in the wild-type and OE (OE-18 and OE-22) seedlings exposed to 250 mM mannitol for 3 days. Error bars indicate SE. **t-test, with P < 0.01.
(AtRD20, AtRD22 and AtAREB2) in both transgenic lines (OE-18 and OE-22) and WT Arabidopsis. The expression of all of the genes was significantly up-regulated in both transgenic plants compared with WT plants. Furthermore, it is noticeable that the antioxidant gene AtSOD2 and the ABA responsive element-binding gene AtAREB2 exhibited much higher levels of up-regulation than other genes (about 10-fold increase averagely for both lines) (Fig. 5). These data demonstrate that NtDnaJ1 overexpression up-regulates transcriptional levels of stress-related genes in plants. 4. Discussion Drought stress has drastic effects on biomass and grain yields of crops worldwide. However, the molecular mechanisms underlying Jdomain proteins-mediated drought tolerance in plants are largely unknown. In this study, we investigated physiological roles of a putative J-domain protein NtDnaJ1 from tobacco and its possible function mechanisms. Our genetic evidence has demonstrated that NtDnaJ1 over-expression enhances drought stress tolerance in transgenic plants. To our knowledge, this is the first J-domain protein from tobacco to be functionally characterized during drought stress. 4.1. DnaJ proteins are conserved, but have diverse function in higher plants Like known plant J-domain proteins, NtDnaJ1 has higher sequence identities (79e84%) and four typical structural domains
(Fig. A). From this, it can be reasonably concluded that the NtDnaJ1 cDNA clone encodes tobacco DnaJ isoform. NtDnaJ1 was found to be highly expressed in aerial tissues of tobacco, and its highest expression level occurred in leaves (Fig. 2A), indicating this gene may be constitutively expressed during both vegetative and reproductive growth. Previous studies on J-domain proteins from Arabidopsis have shown that AtDjB1 was induced by heat stress; AtDjB1 overexpression can facilitate thermotolerance and oxidative stress tolerance in Arabidopsis (Zhou et al., 2012). The Arabidopsis Type-I Jdomain protein J3 knock-out mutation led to sensitive to high salinity (Yang et al., 2010). However, no drought-induced expression of DnaJ gene has been reported. Compared to this expression pattern, NtDnaJ1 is significantly affected by PEG-induced water stress (Fig.2 B). Genetic evidence further proved that NtDnaJ1 OE lines showed enhanced osmotic or drought tolerance in transgenic Arabidopsis (Figs. 3 and 4). These results indicate that function divergence of the DnaJ orthologs may occur in the genomes of Arabidopsis and tobacco, although these genes have high similarities in sequences. 4.2. NtDnaJ1 probably participates in ABA-dependent signaling pathway in transgenic plants during drought stress The molecular mechanisms underlying the response of higher plants to drought stress are complicated. In response to drought stress, various genes are up-regulated, which can mitigate the
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
105
effects of stress and lead to an adjustment of plant stress tolerance. ABA-dependent signaling pathway is involved in the inducible expression of specific genes during drought stress (Shinozak and Yamaguchi-Shinozaki, 2000). RD20 is highly responsive to stress and is often used as a stress marker gene (Aubert et al., 2010). RD22 is induced by drought and ABA, and is involved in an ABAdependent stress signaling pathway (Yamaguchi-Shinozaki and Shinozaki, 1993). AREB2 is an important regulatory gene involved in signal transduction or transcriptional regulation during stress conditions (Shinozaki and Yamaguchi-Shinozaki, 2007; Saibo et al., 2009). The increased expression of RD20, RD22 and AREB2 in NtDnaJ1 transgenic plants suggests that NtDnaJ1 may participate in the ABA-dependent signaling pathway during drought stress. In support of this notion, a type-III J-protein BIL2 from Arabidopsis has been shown to be involved in plant growth, and salt and strong light tolerance in brassinosteroid (BR) signal transduction recently (Bekh-Ochir et al., 2013). 4.3. The antioxidant mechanism might be involved in NtDnaJ1 conferring drought stress tolerance
Fig. 4. Phenotypes of NtDnaJ1-overexpressing and wild-type Arabidopsis plants in response to drought stress. A Drought tolerance of potted plants of wild-type and NtDnaJ1-overexpressing Arabidopsis. Four-week-old WT and transgenic OE (OE-18 and OE-22) plants were grown in soil in pots for 20 days without watering, and then rewatering after 7 days. B Survival rates (%) of drought-stressed wild-type and NtDnaJ1-overexpressing plants after 7 days recovery. Values are mean ± SE, n ¼ 30. *ttest, with P < 0.05.
Drought stress can reduce photosynthesis and result in excess ROS accumulation which leads to cell toxicity, membrane peroxidation and even cell death (Apel and Hirt, 2004). There is a constant need for efficient mechanisms to avoid oxidative damage to cells, and antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) play essential roles in the defense of plants against ROS (Miller et al., 2010). In this study, we have found that the transcripts of several antioxidant genes AtSOD1, AtSOD2, and AtCAT1 exhibited much higher levels in NtDnaJ1 transgenic plants (Fig. 5); moreover, the accumulation of H2O2 was significantly lower in NtDnaJ1-transgenic plants than the WT under drought stress (Fig. 3D); indicating that NtDnaJ1 might protect plants from dehydration stress by functioning as molecular chaperones for
Fig. 5. The expression of stress-responsive genes in NtDnaJ1-overexpressing and wild-type Arabidopsis. Relative expression levels of stress-responsive genes (AtSOD1, AtSOD2, AtCAT1, AtRD20, AtRD22 and AtAREB2) were determined by qRT-PCR using cDNA synthesized from total RNAs isolated from the leaves of 4-week-old Arabidopsis grown in soil under normal conditions. Relative expression levels of these six genes were normalized to the transcripts of Actin2 in the same samples. For each assay, the expression level of WT was taken as 1.0, and data represented means ± SE of three biological replicates. Bar indicates SE. **t-test, with P < 0.01; *t-test, with P < 0.05.
106
Z. Xia et al. / Plant Physiology and Biochemistry 83 (2014) 100e106
ROS-scavenging proteins to maintain higher levels of antioxidant activities. Supporting these interesting findings, mutation of the Arabidopsis type-II J-protein AtDjiB1 decreased thermotolerance under heat stress by down-regulating activities of antioxidant enzymes and accumulating ROS levels (Zhou et al., 2012); implying that the antioxidant system may be involved in J-protein-mediated abiotic stress tolerance in plants. In the future, further study will be interesting to clarify the mechanism of activation of the antioxidant system in NtDnaJ1-transgenic plants under drought stress. Acknowledgments This work was financially supported by the science and technology R&D project from Henan Tobacco Company (grant no. HYKJ201010). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.07.023. Authors' contributions ZX designed the study and wrote the manuscript; ZX, XZ, J-Li, XS and J-Liu performed the experiments and analyzed the data; ZX contributed reagents/materials/analysis tools. References Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev. Plant Biol. 55, 373e399. Aubert, Y., Vile, D., Pervent, M., Aldon, D., Ranty, B., Simonneau, T., Vavasseur, A., Jean- Philippe, G., 2010. RD20, A stress-inducible caleosin, participates in stomatal control, transpiration and drought tolerance in Arabidopsis thaliana. Plant Cell. Physiol. 51, 1975e1987. Bekh-Ochir, D., Shimada, S., Yamagami, A., Kanda, S., Ogawa, K., Nakazawa, M., Matsui, M., Sakuta, M., Osada, H., Asami, T., Nakano, T., 2013. A novel mitochondrial DnaJ/Hsp40 family protein BIL2 promotes plant growth and resistance against environmental stress in brassinosteroid signaling. Planta 237,1509e1525. Caplan, A.J., Cyr, D.M., Douglas, M.G., 1993. Eukaryotic homologs of Escherichia coli Dnaj-a diverse protein family that functions with Hsp70 stress proteins. Mol. Biol. Cell. 4, 555e563. Cheetham, M.E., Caplan, A.J., 1998. Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell. Stress Chaperones 3, 28e36. Clough, S.J., Bent, A.F., 1998. Floral dip: a simplified method for Agrobateriummediated transformation of Arabidopsis thaliana. Plant J. 16, 735e743. Craig, E.A., Huang, P., Aron, R., Andrew, A., 2006. The diverse roles of J-proteins, the obligate Hsp70 co-chaperone. Rev. Physiol. Biochem Pharmacol. 156, 1e21. de Crouy-Chanel, A., Kohiyama, M., Richarme, G., 1995. A novel function of Escherichia coli chaperone DnaJ protein-disulfide isomerase. J. Biol. Chem. 270, 22669e22672. Du, Y., Zhao, J., Chen, T., Liu, Q., Zhang, H., Wang, Y., Hong, Y., Xiao, F., Zhang, L., Shen, Q., Liu, Y., 2013. Type I J-domain NbMIP1 proteins are required for both tobacco mosaic virus infection and plant innate immunity. PLoS Pathog. 9, e1003659. Goffin, L., Georgopoulos, C., 1998. Genetic and biochemical characterization of mutations affecting the carboxy-terminal domain of the Escherichia coli molecular chaperone DnaJ. Mol. Microbiol. 30, 329e340. Hennessy, F., Boshoff, A., Blatch, G.L., 2005. Rational mutagenesis of a 40 kDa heat shock protein from Agrobacterium tumefaciens identifies amino acid residues critical to its in vivo function. Int. J. Biochem Cell. Biol. 37, 177e191. Kneissl, J., Wachtler, V., Chua, N.H., Bolle, C., 2009. OWL1: an Arabidopsis J-domain protein involved in perception of very low light fluences. Plant Cell. 21, 3212e3225. Liu, J.Z., Whitham, S.A., 2013. Overexpression of a soybean nuclear localized type III DnaJ domain-containing HSP40 reveals its roles in cell death and disease resistance. Plant J. 74, 110e121.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real- time quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods 25, 402e408. Mayer, M.P., Bukau, B., 2005. Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62, 670e684. Miernyk, J.A., 2001. The J-domain proteins of Arabidopsis thaliana: an unexpectedly large and diverse family of chaperones. Cell. Stress Chaperones 6, 209e218. Miller, G., Suzuki, N., Ciftci-Yilmaz, S., Mittler, R., 2010. Reactive oxygen species homeostasis and signaling during drought and salinity stresses. Plant Cell. Environ. 33, 453e457. Qiu, X.B., Shao, Y.M., Miao, S., Wang, L., 2006. The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones. Cell. Mol. Life Sci. 63, 2560e2570. Rajan, V.B., D'Silva, P., 2009. Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors. Funct. Integr. Genomics 9, 433e446. Saibo, N.J.M., Lourenço, T., Oliveira, M.M., 2009. Transcription factors and regulation of photosynthetic and related metabolism under environmental stresses. Ann. Bot. 103, 609e623. Shen, L., Kang, Y.G., Liu, L., Yu, H., 2011. The J-domain protein J3 mediates the integration of flowering signals in Arabidopsis. Plant Cell. 23, 499e514. Shinozak, I.K., Yamaguchi-Shinozaki, K., 2000. Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signal pathways. Curr. Opin. Plant Biol. 3, 217e223. Shinozaki, K., Yamaguchi-Shinozaki, K., 2007. Gene networks involved in drought stress response and tolerance. J. Exp. Bot. 58, 221e227. Silver, P.A., Way, J.C., 1993. Eukaryotic Dnaj homologs and the specificity of Hsp70 activity. Cell 74, 5e6. Szabo, A., Korszun, R., Hartl, F.U., Flanagan, J., 1996. A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates. EMBO J. 15, 408e417. Szyperski, T., Pellecchia, M., Wall, D., Georgopoulos, C., Wuthrich, K., 1994. NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2e108) containing the highly conserved J domain. Proc. Natl. Acad. Sci. U. S. A. 91, 11343e11347. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731e2739. Walsh, P., Bursac, D., Law, Y.C., Cyr, D., Lithgow, T., 2004. The J-protein family: modulating protein assembly, disassembly and translocation. EMBO Rep. 5, 567e571. Wang, W., Vinocur, B., Shoseyov, O., Altman, A., 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 9, 244e252. Wang, G., Cai, G., Kong, F., Deng, Y., Ma, N., Meng, Q., 2014. Overexpression of tomato chloroplast-targeted DnaJ protein enhances tolerance to drought stress and resistance to Pseudomonas solanacearum in transgenic tobacco. Plant Physiol. Biochem 82, 95e104. Xia, Z., Su, X., Wu, J., Wu, K., Zhang, H., 2012. Molecular cloning and functional characterization of a putative sulfite oxidase (SO) ortholog from Nicotiana benthamiana. Mol. Biol. Rep. 39, 2429e2437. Xia, Z., Sun, K., Wang, M., Wu, K., Zhang, H., 2012. Overexpression of a maize sulfite oxidase gene in tobacco enhances tolerance to sulfite stress via sulfite oxidation and CAT-mediated H2O2 scavenging. PLoS One 7, e37383. Xia, Z., Wei, Y., Sun, K., Wu, J., Wang, Y., Wu, K., 2013. The maize AAA-type protein SKD1 confers enhanced salt and drought stress tolerance in transgenic tobacco by interacting with Lyst-interacting protein 5. PLoS One 8, e69787. Yamaguchi-Shinozaki, K., Shinozaki, K., 1993. The plant hormone abscisic acid mediates the drought-induced expression but not the seed-specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis thaliana. Mol. Gen. Genet. 238, 17e25. Yang, K., Xia, C., Liu, X., Dou, X., Wang, W., Chen, L., Zhang, X., Xie, L., He, L., Ma, X., Ye, D., 2009. A mutation in Thermosensitive Male Sterile 1, encoding a heat shock protein with DnaJ and PDI domains, leads to thermosensitive gametophytic male sterility in Arabidopsis. Plant J. 57, 870e882. Yang, Y., Qin, Y., Xie, C., Zhao, F., Zhao, J., Liu, D., Chen, S., Fuglsang, A.T., Palmgren, M.G., Schumaker, K.S., Deng, X.W., Guo, Y., 2010. The Arabidopsis chaperone J3 regulates the plasma membrane Hþ-ATPase through interaction with the PKS5 kinase. Plant Cell. 22, 1313e1332. Zhou, W., Zhou, T., Li, M., Zhao, C., Jia, N., Wang, X., Sun, Y., Li, G., Xu, M., Zhou, R., Li, B., 2012. The Arabidopsis J-protein AtDjB1 facilitates thermotolerance by protecting cells against heat-induced oxidative damage. New. Phytol. 194, 364e378.