Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis

GENE-39944; No. of pages: 8; 4C: Gene xxx (2014) xxx–xxx

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Gene journal homepage: www.elsevier.com/locate/gene

Methods paper

Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis Hyun Yong Cho a,1, Chanhui Lee b,1, Sun-Goo Hwang a, Yong Chan Park a, Hye Lee Lim a, Cheol Seong Jang a,⁎ a b

Plant Genomics Lab., Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-713, Republic of Korea Department of Plant and Environmental New Resources, Kyung Hee University, Yongin 446-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 10 February 2014 Received in revised form 2 September 2014 Accepted 9 September 2014 Available online xxxx Keywords: Chilling inducible gene Copper protein Abiotic stress Subcellular localization Heterogeneous expression WGCNA Coexpression network

a b s t r a c t In a previous study, we identified a number of genes induced by chilling using a microarray approach. In order to investigate the molecular mechanism underlying chilling tolerance and possible crosstalk with other abiotic stresses, we selected a rice gene, OsChI1 (Os01g61160), for further analysis. The OsChI1 gene encodes a putative laccase precursor protein. In accordance with our previous results, its transcript is highly accumulated during a 12-day period of chilling treatment. Higher expression of the OsChI1 gene was also detected in roots and tissues at the vegetative and productive stages. In addition, we also observed increased transcript levels of the OsChI1 gene during dehydration and high salinity conditions. Transient expression of OsChI1 proteins tagged with fluorescence protein in rice protoplasts revealed that OsChI1 is localized in the plasma membrane. The Arabidopsis transgenic plants overexpressing OsChI1-EGFP resulted in an increased tolerance to drought and salinity stress. In silico analysis of OsChI1 suggests that several genes coexpressed with OsChI1 in the root during various abiotic stresses, such as chilling, drought and salt stress, may play an important role in the ROS signaling pathway. Potential roles of OsChI1 in response to abiotic stresses are discussed. © 2014 Published by Elsevier B.V.

1. Introduction Unfavorable environmental conditions such as water deficit, high salinity, and temperature fluctuation lead to a serious reduction in crop yields. In particular, exposure to abiotic stresses at the early seedling stage of crop plants can cause a range of serious developmental defects including retarded seedling development, reduced tillering, and dwarfism (Mittler, 2006). Thus, plants have evolved intricate molecular mechanisms to minimize damages and to ensure cellular homeostasis. Transcriptional profiling of abiotic stress-inducible genes followed by mutagenesis and/or transgenic approaches has contributed to our current understanding of the complexity and crosstalk of the molecular mechanisms of stress tolerance (Sharma et al., 2013). Physiological studies have demonstrated that rice seedlings are very sensitive to chilling in early spring (Su et al., 2010). Low temperature responses such as chilling (sub-optimal temperature), cold (non-optimal temperature) and freezing in higher plants is a complex process that includes Abbreviations: OsCHIs, Oryza sativa chilling inducible genes; SOD, superoxide dismutase; ROS, reactive oxygen species; PC, plastocyanin; ER, endoplasmic reticulum; ARACNE, algorithm for the reconstruction of accurate cellular networks; WGCNA, weighted correlation network analysis. ⁎ Corresponding author. E-mail address: [email protected] (C.S. Jang). 1 These authors contributed equally to this work.

the increased generation of secondary metabolites such as antioxidants and phenolic compounds, alteration of lipid membrane compositions, and the regulation of gene expression (Campos et al., 2003; Weidner et al., 2009). For example, chilling stress leads to a significant overproduction of reactive oxygen species (ROS) by aggravating the imbalance between light absorption and utilization through the inhibition of the Calvin–Benson cycle (Logan et al., 2006). In addition, the decline of photosynthesis under chilling stress might cause photooxidative damage such as lipid peroxidation increment and chlorophyll degradation in chloroplasts (Fryer et al., 1998). Laccases, p-diphenol:dioxygen oxidoreductases, are divided into two different subgroups of multicopper oxidases (MCOs) and laccaselike multicopper oxidases (LMCOs) (McCaig et al., 2005). It is believed that plant laccases function mainly as an oxidase for lignin biosynthesis (Dean and Eriksson, 1994). In particular, a couple of Arabidopsis LMCO genes are known to also be associated with lignin deposition (McCaig et al., 2005). In Arabidopsis, a double mutant of two stem-specific laccase genes, LAC4 and LAC17, resulted in a substantial reduction in the lignin content, providing evidence that both genes are crucial in the lignification of Arabidopsis stems (Berthet et al., 2011). Considering the expression patterns of laccase genes in non-lignified tissues and compositional changes of lignin monomers upon biotic and abiotic stresses in several species, it has been speculated that laccase genes also play an important role in defense response (Turlapati et al., 2011). An increase in lignin

http://dx.doi.org/10.1016/j.gene.2014.09.018 0378-1119/© 2014 Published by Elsevier B.V.

Please cite this article as: Cho, H.Y., et al., Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.09.018

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content was observed when poplar seedlings were grown at 10 °C (Moura et al., 2010). In addition, other studies showed that genes involved in the biosynthesis of lignin monolignols were highly upregulated by cold treatment (Christie et al., 1994). In maize roots, ZmLAC1 expression is positively associated with NaCl treatment in a dose dependent manner (Liang et al., 2006). Although little is known about the effects of diverse abiotic stresses on lignin biosynthesis, these previous data raise the possibility that laccase genes as well as other lignin biosynthetic genes play a role in plant defense responses. Plant laccases are also known to be involved in flavonoid oxidation that is an essential biochemical process for cytotoxicity and antioxidant abilities required for plant growth and defense (Pourcel et al., 2007). In accordance with this context, the enzymatic activities of laccase are highly induced by biotic and abiotic stresses as well as developmental signals. Transcriptome analysis based on integrated microarray datasets can provide an effective tool for predicting the gene function relationships and will facilitate a genome-wide scale network-based analysis. The various methods such as weighted correlation network analysis (WGCNA) and rank based methods have been reported to construct the coexpression network (Stuart et al., 2003; Langfelder and Horvath, 2008). For example, weighted correlation network analysis (WGCNA) has been generally used for identifying the modules as highly correlated clusters and this method has successfully provided a clue to understanding the functional association between distinct modules in plants (Ficklin et al., 2010; Ficklin and Feltus, 2011). We have previously reported a comparative transcriptome analysis with a coexpression network with the simple Pearson's Correlation Coefficient (PCC) method and identified twenty chilling-inducible genes in rice (Cho et al., 2012); however, the molecular functions of these genes have not yet been determined. Here, we report that one chilling inducible gene, OsChI1 (Oryza sativa Chilling Induced 1) might participate in various stress-response mechanisms, such as responses to drought and salinity in rice. 2. Materials and methods 2.1. Plant materials and stress treatments Rice seedlings (O. sativa spp. japonica cv. ‘Donganbyeo’) were grown on meshes supported in plastic containers with 1/2 Murashige and Skoog (MS) solution in a growth chamber (16/8-h light/dark photoperiod at 25 °C with 70% relative humidity). For abiotic stress and phytohormone treatments, fourteen day-old seedlings were treated with dehydration, 200 mM NaCl, and 0.1 mM ABA, as described by Jung et al. (2012) with some modifications. Root tissues were harvested at 0, 3, 6, 12, and 24 h after stress or phytohormone treatments, respectively. For chilling stress, fourteen day old seedlings were grown in soil pot (200-hole-type plug tray; 3 × 3 × 4 cm), transferred into a chamber at (9 °C) and then cold-exposed root tissues were harvested at different time points (0, 1, 3, 7, and 12 days). For tissue-specific expression analysis, the plant materials were harvested from root, stem, and leaf tissues at vegetative stage (14 days old) and at reproductive stage (80 days old). Panicles were sampled at the reproductive stage (80 days old). All samples were ground using liquid nitrogen and immediately stored at −80 °C. 2.2. Isolation and cloning of the OsChI1 gene The coding sequence for OsChI1 (Os01g61160) was retrieved from the Rice Genome Annotation Project database (http://rice.plantbiology.msu. edu/). The designed primer pair sequences harboring restriction enzyme sites were listed in Supplementary Table 1. Total RNA was extracted from 2-week-old rice roots using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and first-strand cDNA was synthesized using MMLV reverse transcriptase (Takara Bio, Kyoto, Japan). The full-length cDNA was amplified using high-fidelity Pfu Turbo DNA polymerase

(Stratagene, La Jolla, CA, USA). The amplified polymerase chain reaction (PCR) products were digested by XbaI/KpnI enzymes and introduced into pBIN35S-GFP plasmids (Lim et al., 2013). The PCR products were verified at a DNA sequencing facility (Macrogen, Seoul, Korea). 2.3. Reverse transcription polymerase chain reaction (RT-PCR) Total RNAs were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. First-strand cDNA synthesis from total RNA (1 μg) was conducted using the PrimeScript™ RT-PCR kit (Takara-Bio, Ohtsu, Japan). For tissue-specific expression analysis, semi-quantitative RT-PCR was performed as described by Lim et al. (2010). To normalize each sample for cDNA quality and quantity, the rice Os18S-rRNA gene (Os09g00999) was employed as an internal control with specific primers (for sequences see Supplementary Table 1). Gene specific primers were designed by using the NCBI Primer BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primerbalst/). The PCR program was run as follows: denaturing for 5 min at 95 °C, followed by 30 cycles of 30 s of denaturation at 95 °C, 30 s of annealing at 57 °C, and 30 s of extension at 72 °C, with a final extension step at 72 °C. 2.4. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) Synthesized cDNA using the PrimeScript™ RT-PCR kit (Takara-Bio, Ohtsu, Japan) was finally diluted into 50 μL, and then each 2 μL was used as template for quantitative real-time RT-PCR (qRT-PCR). qRTPCR was performed using a Rotor-Gene-Q real time PCR cycler (Qiagen, Venlo, Netherland) with 20 μL reaction volume using SYBR® Green TOPreal qPCR 2× PreMIX (Enzynomics™, Daejeon, Korea) with the gene specific primers (for sequences see Supplementary Table 1). A housekeeping gene encoding Os18S-rRNA (Os09g00999) was used as a control and was amplified with the primer 5′-ATG ATA ACT CGA CGG ATC GC-3′ (forward) and 5′-CTT GGA TGT GGT AGC CGT TT-3′ (reverse). The thermal cycler conditions recommended by the manufacturer were used: 15 min at 95 °C, followed by 50 cycles at 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s. The fluorescent product was detected during the final step of each cycle. Amplification, detection, and data analysis were carried out on a Rotor-Gene Q real-time PCR cycler. To determine the relative fold-differences in template abundance for each sample, the Ct value for OsChI1 was normalized to the Ct value for Os18S-rRNA and calculated relative to a calibrator using the formula 2 − ΔΔCT. Three independent experiments were performed, and the primer efficiencies were determined according to the previously reported method (Livak and Schmittgen, 2001) to validate the ΔΔCT method used in our experiment. 2.5. Protoplast isolation Protoplasts were isolated from two-week-old rice seedlings. Rice seedlings were grown on 1/2 MS nutrient solution in a growth chamber (16/8-h light/dark photoperiod at 25/23 °C with 70% relative humidity). Young leaves and sheaths were chopped and dipped in an enzyme solution [1% cellulose R-10 (Yakult Honsa Co,. Ltd, Tokyo, Japan), 0.25% macerozyme R-10 (Yakult Honsa), 0.1% BSA, 10 mM MES, 500 mM Mannitol, 1 mM CaCl2] for 6 h with gentle shaking then filtered through Miracloth. Protoplasts were pelleted by centrifugation and resuspended in an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, 1.5 mM MES) and incubated on ice for 5 h. Protoplasts were centrifuged and resuspended in the MMg solution (0.4 M mannitol, 15 mM MgCl2, 4.7 mM MES). 2.6. Subcellular localization In order to evaluate subcellular localization of OsChI1, the gene was amplified using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA)

Please cite this article as: Cho, H.Y., et al., Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.09.018

H.Y. Cho et al. / Gene xxx (2014) xxx–xxx

and inserted into the multiple cloning cite of EGFP vector (Lim et al., 2013). Each of plasmid DNA (10 μg) was transfected into 2 × 106 rice protoplasts using a 40% polyethylene glycol (PEG) solution (40% PEG, 400 mM mannitol, 100 mM Ca(NO3)2) for 30 min at room temperature. W5 solution was added for dilution of the PEG solution and then discarded. The protoplasts were resuspended again with W5 and incubated overnight at room temperature. pm-rk-mCherry was used as a plasma membrane marker protein (Nelson et al., 2007). Excitation/ emission wavelengths were 488/500 550 nm for EGFP, 543/565 615 nm for mCherry constructs. Fluorescent images were obtained using a Multiphoton Confocal Laser Scanning Microscope (model LSM 410 META NLO, Carl Zeiss) at the Korea Basic Science Institute Chuncheon Center. 2.7. Heterogeneous overexpression in Arabidopsis Transformants (Arabidopsis thaliana) with 35S:OsChI1-EGFP or 35S: EGFP (control) were generated by the floral dipping method (Zhang et al., 2006). For selection of transgenic lines, seeds harvested from T3 transformants were placed on MS agar plates containing 50 μg/ml kanamycin in a growth chamber (16/8-h light/dark photoperiod at 25 °C with 70% relative humidity). The presence of OsChI1 in the transgenic lines was confirmed by using One-step 5X RT-PCR Master premix (ELPIS Biotech., Inc, Korea). Three-independent 35S:OsChI1-EGFP- and 35S:EGFP-overexpressing Arabidopsis lines were treated under salt and drought stresses as well as ABA treatment. For seed germination assays, seeds of OsChI1-EGFP, and 35S:EGFP were sterilized by chlorine gas (Kuromori et al., 2004), which was produced by mixing 100 ml of chlorine bleach (Yuhan Yanghang, Seoul, Korea) and 3 ml HCl. The seeds in a gas container were exposed for 3 h, and were then rinsed three times with distilled water. Sterilized seeds were sown and monitored on 1/2 MS medium containing different concentrations (0, 100, or 150 mM) of NaCl and (−0.25, −0.7, or −0.12 MPa) of PEG 8000 (Sigma-Aldrich). PEG plates were prepared as described by Verslues and Bray (2004). Briefly, each plate was prepared that 1/2 MS medium of 40 ml containing 1.5% agar were solidified and were then overlaid with 60 ml of a liquid PEG containing 0, 400, and 550 g/L PEG8000, yielding final a MPas of − 0.25, − 0.7, and − 1.2, respectively, according to van der Weele et al. (2000). After the plates were allowed to be kept for 16 h, the liquid PEG was then removed from the plates. Germination percentages were scored at 1-day interval for 7 days. For root elongation assays, transgenic seeds were germinated on 1/2 MS medium for 3 days and transferred into medium harboring NaCl (0, 100, or 150 mM) and PEG (− 0.25,− 0.7, or − 1.2 MPa). Plant growth was then monitored and photographed after 7 days, at which point root length was analyzed using Image J software. For ABA treatment, the sterilized seeds were placed on the 1/2 MS medium with different concentrations of ABA (0.4, 0.7 and 1.0 μM) for 5 days and thus their germination ratios were evaluated at 5 days. 2.8. Measurements of electrolyte leakage To evaluate cold tolerance using electrolyte leakage assays, 35S: OsChI1-EGFP or 35S:EGFP (control) transgenic plants were grown in a chamber with 16/8-h light/dark photoperiod at 25 °C with 70% relative humidity for 4 weeks and then treated at 4 °C for 1 week for cold acclimation. Leaf disks were punched from fully developed rosette leaves, then transferred into 8 ml of water and incubated for 24 h. The tubes containing leaf disks and water were then autoclaved. Each percentage of ion leakage before and after autoclave was calculated and plotted. Three biological replicates were conducted in each treatment. 2.9. In silico analysis of OsChI1 The complete genome sequences for five plant species (Arabidopsis lyrata, A. thaliana, Zea mays, Brachypodium distachyon and O. sativa)

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were downloaded from the Phytozome v9.1 (http://www.phytozome. net/). In order to employ the rice LMCOs, BLASTP analysis was conducted using the laccase protein (IPR017761) retrieved from the Interpro database with a cutoff b 1e-10. The phylogenetic tree in five plant species using the maximum likelihood (ML) method of the PHYLIP program with 100 bootstrap replicates (http://evolution.genetics.washington. edu/phylip.html), and then the ML tree was visualized by MEGA software (http://www.megasoftware.net/). In order to study functionally related genes with OsChI1 a total of 1000 CEL files of Affymetrix GeneChip genome arrays of O. sativa (GPL2025) were manually downloaded from the Gene Expression Omnibus datasets of the NCBI database (http://www.ncbi.nlm.nih.gov/ geo/). The microarray CEL files were normalized by R statistical software with RMA method (http://www.rproject. org/). In order to retrieve the top 1000 probes with high correlation with the OsCHI1 gene, we evaluated PCC values using the normalized microarray dataset. Subsequently, we identified the module genes of OsChI1 using R package Weighted Correlation Network Analysis (WGCNA) following the procedure described by Zhang and Horvath (2005). Amino acids of coepxressed genes were retrieved from the Phytozome v8.0 (http://www. phytozome.net/) and then the subcellular localization of these genes were predicted by WoLF PSORT (http://www.psort.org/). The expression profiles of coexpressed genes with OsChI1 in developmental stages and various tissues presented as heat maps was generated by the Genevestigator (http://www.genevestigator.ethz.ch) webtool with default parameters. For subcellular network analysis of coexpressed genes with OsChI1 were constructed by R package ARACNE (Margolin et al., 2006) and visualized using Cytoscape software (Shannon et al., 2003). Functional enrichment analyses of coexpressed genes were conducted by agriGO (Du et al., 2010). 3. Results 3.1. Expression patterns of the OsChI1 gene Previously, we reported the identification of a chilling-tolerant mutant line generated by gamma ray irradiation to Donganbyeo. Comparative transcriptome analysis revealed that a host of genes were upregulated in the chilling-tolerant line compared to wild type (Cho et al., 2012). Among them, one gene (Os01g61160), which encodes laccase precursor protein, is known to be a multicopper-containing glycoprotein that functions as an oxidase for lignin polymerization. In our previous microarray data, OsChI1 gene is more than threefold higher expressed in a chilling tolerant mutant line upon chilling stress. We determined the molecular functions of the gene in response to other abiotic stresses as well as chilling stress, and selected it for further study. The gene was named OsChI1 and its expression was examined under chilling, drought and salinity conditions and in tissues of vegetative and reproductive stages. Interestingly, low levels of expression were seen in the reproductive stage, but the OsChI1 gene was highly expressed in roots and stems at both vegetative and reproductive stages (Fig. 1A). To confirm that the accumulation of mRNA is induced by chilling stress, we tested the expression pattern in response to chilling stress (9 °C) in 14-day-old root tissues (Fig. 1B). The transcript level of the OsChI1 gene gradually increased in response to chilling stress at 3 to 7 days and then decreased at 12 days. Since a number of stress-induced genes have been shown to have extensive overlap and crosstalk with different types of abiotic stresses, it is plausible that the OsChI1 gene is also induced by other abiotic stresses. We further analyzed expression patterns of OsChI1 in rice roots under dehydration and high salinity (250 mM NaCl) conditions (Fig. 1C). Interestingly, transcript levels of the OsChI1 gene were significantly increased by drought and high salinity treatments for up to 24 hrs. Meanwhile, the transcript level of OsChI1 was not significantly altered by ABA treatment. We employed the reliable stress-inducible gene, OssalT, as a quality control (Claes et al., 1990) for salinity, dehydration, and ABA treatment. Altogether, OsChI1 gene

Please cite this article as: Cho, H.Y., et al., Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.09.018

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Fig. 1. Expression analysis of OsChI1 gene in rice plants. A. Expression patterns of OsChI1 gene in 4 different tissues (root, stem, leaf and panicle). STAGE I indicates vegetative stages and STAGE II indicates reproductive stages. B. Relative mRNA level of OsChI1 gene in root tissue during chilling stress (9 °C). Fourteen-day-old plants were exposed to chilling stress for up to 12 days. Root tissue was harvested at the time points specified and analyzed by qRT-PCR. Relative level of each transcript was calculated by comparison to the time-matched nonstressed controls. Data represent mean ± se (n = 3). C. qRT-PCR analysis of OsChI1 expression under dehydration, high-salinity and ABA treatment. Twelve-day-old seedlings under different treatments were used for the analysis, and the expression of OsChI1 without treatment was set at 1.0. Biological replicates of the experiments were performed, and the data are presented as averages of three independent experiments. The OssalT gene is marker gene for dehydration, high-salinity, and ABA treatment.

35S:OsChI1-EGFP

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Fig. 2. Subcellular localization of OsChI1-EGFP fusion proteins. Agrobacterium strain (GV3101) harboring 35S:OsChI1-EGFP construct was transfected in rice protoplasts. Its subcellular location was examined with a laser confocal microscope. Images were captured and merged by z-series optical sections. The 35S:EGFP construct was used as a control. pm-rk-mCherry was used as a plasma membrane marker protein. EGFP, EGFP fluorescence; visible, visible light images; merged, merged images of GFP and visible light images. Bars = 2 μm. A and B. Confocal images of 35S:OsChI1-EGFP and 35S:EGFP. Chloroplast location was shown for each image.

Please cite this article as: Cho, H.Y., et al., Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.09.018

H.Y. Cho et al. / Gene xxx (2014) xxx–xxx

was highly induced by all of the stress treatments and hormone treatment. 3.2. Subcellular localization of the OsChI1 protein Sequence analysis using the TMHMM2.0 program (http://www.cbs. dtu.dk/services/TMHMM-2.0/) predicted that OsChI1 does not contain a transmembrane helix (data not shown). To determine the actual subcellular location of OsChI1, we fused the full-length OsChI1 gene with the enhanced green fluorescent protein (EGFP) at its C terminal region under the control of the CaMV35S promoter and transfected it into rice protoplasts. Examination of the rice protoplasts transfected with 35S:OsChI1-EGFP or a control (35S:EGFP) showed that the 35S: OsChI1-EGFP signal was clearly colocalized with a plasma membrane marker protein (pm-rk-mCherry) (Fig. 2A). The control protoplasts expressing 35S:EGFP alone had fluorescent signals throughout the cytosol and nucleus (Fig. 2B). This result indicates that OsChI1 is a plasma membrane-localized protein. 3.3. Functional analysis of OsChI1 in Arabidopsis transgenic plants We next investigated the functional role of OsChI1 by overexpressing it in Arabidopsis. Transgenic Arabidopsis plants expressing OsChI1-EGFP

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were generated under the control of the CaMV 35S promoter (35S: OsChI1-EGFP). We selected three independent lines (T3) with the highest expression levels of the OsChI1 gene (Fig. 3A). To evaluate the effect of OsChI1 overexpression on cold stress tolerance, the electrolyte leakage test was conducted in the 35S:OsChI1-EGFP and 35S:EGFP plants under cold stress (Fig. 3B). After the low temperature treatment, the 35S:EGFP control plants exhibited less electrolyte leakage compared to the 35S:OsChI1-EGFP plants, indicating that there was no substantial increase in chilling tolerance in 35S:OsChI1-EGFP overexpression lines. Next, we evaluated the drought and high salinity stress tolerance of 35S:OsChI1-EGFP plants. For germination rate analysis, three transgenic plants as well as a 35S:EGFP plant as a control were placed in 1/2 Murashige and Skoog (MS) medium containing a different concentration of PEG (0, − 0.7 or − 1.2 MPa) and NaCl (0, 100 or 150 mM), respectively. Under the normal treatment, germination rates of transgenic lines and control seeds were not significantly different in 1/2 MS medium (Fig. 3C). By contrast, the transgenic seeds of 35S:OsChI1EGFP showed a much higher germination rate than the control seeds when seeds were plated in 1/2 MS medium supplemented with PEG (− 0.7 or −1.2 MPa). For example, less than 60% of the control seeds germinated within 7 days under PEG treatment (− 0.7 MPa), while the three independent transgenic lines exhibited more than 80% germination ratios (Fig. 3C). We subsequently evaluated the root lengths of

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Fig. 3. Effects of OsChI1 overexpression under osmotic stress insensitivity in Arabidopsis. Each of plants was treated with different concentrations of PEG and NaCl during seed germinations and seedling stage. A. RT-PCR analysis of three independent Col-0/35S:OsChI1 T3 transgenic plants and control wild-type (E.V.). B. Electrolyte leakage analysis in 35S:OsChI1-EGFP and 35S:EGFP plants under cold condition. Data represent means ± SD of three independent experiments (n = 3). C. Germination rates of wild-type and three independent transgenic plants on control medium and increasing PEG or NaCl concentrations. D. Root growth assay on different concentration of PEG or NaCl for 7 days. Seedlings were germinated on 1/2 MS medium for 3 days prior to transfer to PEG or NaCl-supplemented or control plates and analyzed using Image J software. Data represent means ± SD of three independent experiments (n = 10). ‘**’, P values b 0.01, and ‘*’, P values b 0.05, indicating significant difference from corresponding controls, t-test of independence.

Please cite this article as: Cho, H.Y., et al., Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.09.018

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the three 35S:OsChI1-EGFP plants as well as the control plants under nontreatment and PEG treatments (Fig. 3D). Under normal conditions, the root lengths of the three 35S:OsChI1-EGFP plants were not significantly different from the control plants, whereas the transgenic plants' roots were significantly longer than the control plants' in 1/2 MS medium with PEG (−0.7 or −1.2 MPa) (Fig. 3D). While significant differences in the germination ratios were not found between the overexpressing plants and the control plants under normal conditions, 54 to 36% of the control seeds were germinated within 7 days in NaCl (100 or 150 mM) but the OsChI1-overexpression transgenic seeds showed much higher germination rates with a range of 81 to 100% (Fig. 3D). For root growth assays, three independent lines of 35S:OsChI1-EGFP and 35S:EGFP were plated in 1/2 MS medium for 7 days with or without salt treatment. We found no significant differences in root lengths between the control and the transgenic plants under normal conditions. By contrast, root lengths were longer in the transgenic plants than in the control plants under high salt treatments (100 mM or 150 mM) (Fig. 3D). Under ABA treatment, no significant difference in germination ratios was found between the transgenic and control plants at different concentrations of ABA up to 1.0 μM (Fig. S1A and B).

Arabiodpopsis lyrata Arabiodpopsis thaliana Zea mays Brachypodium distachyon Oryza sativa

100 100 51

78 66

68

100 100 81

59 100 100 100

100 100 100 100 100 100

62 100

52 34

100 100

100 73

58

Os11g01730 Os12g01730

Bradi4g44810 Os03g18640

GRMZM5G814718 Bradi1g65100

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487334 AT5G05390 903327 AT2G40370 325328 AT5G07130 481932 AT2G30210 AT3G09220 478237 AT5G01040 486975 472269 AT5G01050 Os01g63200

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Bradi2g55060 GRMZM5G842071 Bradi2g55050 Os01g63190

92 100

GRMZM5G800488 64 95

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86 87

100 100 78 100 81

3.4. Phylogenetic analysis of LMCO genes and coexpression network of OsChI1

90 100 87 89

100 100 93

67 97

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91 93

98

55

100 70

100 100 100

45

43 30 84

100 100

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86

100

In order to study the evolutionary relationship of the LMCO gene family among rice gene family members and other plants, a phylogenetic tree was constructed using multiple sequence alignment from various plant species (A. lyrata, A.thaliana, Z. mays, and B. distachyon). There exist 17 laccase members in the rice genome. In accordance with previous phylogenetic studies (McCaig et al., 2005; Turlapati et al., 2011), laccase members found in A. lyrata and A. thaliana cluster into six groups. Interestingly, laccase members found in monocot species studied were grouped into the five groups and no monocot laccases belong to group VI (Fig. 4). While group V contains 11 rice genes, the OsChI1 gene was clustered into group III. Also, a total of 14 genes in Arabidopsis (7 genes) and rice (7 genes) were generated by W/SGDs and OsChI1 gene was duplicated with Os12g01730 in group III. In order to study the functional gene interactions with OsChI1, we retrieved the top 1000 probes of OsChI1 by using PCC values and constructed their coexpression network. In addition, scale-free topology for 10 soft threshold values and a model fitting index R2 value of 0.8 and regression line slope value of − 1.49 were used to determine the highly coexpressed genes (Fig. S2A), resulting in six modules exhibited in the coexpression network; therefore, the module of yellow color was composed of 86 genes including OsChI1 (Fig. S2B). To study the molecular dissection of the highly coexpressed module genes including OsChI1, we subsequently constructed the OsChI1functional interaction network based on their protein localization prediction (Fig. S3). The predicted subcellular localizations were mainly associated with chloroplasts (43 genes) and extracellular (23 genes). However, the five genes including OsChI1 were predicted to be mainly associated with the cytosol. Functional enrichment analysis was conducted to detect enriched functions of OsChI1-module genes. These genes exhibited the overrepresented GO functions, such as lipid metabolic processes (genes), lipid transport and macromolecule localization, response to oxidative stress, oxidoreductase activity, antioxidant activity, response to stress, electron carrier activity, copper ion binding, and tetrapyrrole binding. In particular, the copper ion binding function was composed of five genes coding for plastocyanin-like domain containing proteins (Os02g43660, Os04g46120, Os03g59280 and Os03g63390) and OsChI1. Also, oxidative stress related functions were composed of five genes, Os07g48060, Os03g25330, Os03g22020, Os01g19020, and Os06g16350, and these genes are commonly associated with the differentially overrepresented functions such as response to stress, electron carrier activity and tetrapyrrole binding. OsChI1 was

GRMZM2G336337 GRMZM2G132169 Bradi2g53800 OsChI1 (Os01g61160) GRMZM2G388587

94 100

Os05g38410 Os05g38420

GRMZM2G447271 GRMZM2G072780 GRMZM2G164467 Bradi1g24880 Bradi1g24910 Bradi2g54680 Os01g62480 497195 901874 AT2G29130 496140 AT5G60020 AT5G58910 496014 482815 AT2G38080 AT5G01190 860582

Bradi1g74320 Os11g48060 487097 AT5G03260 Os01g44330 889226 AT1G18140 871194 AT2G46570 487747 AT5G09360 917686 AT5G48100

Bradi3g59180 Os02g51440 Os10g30120 Os01g27700 Os10g30140 Os11g47390

I

II VI

IV

Bradi3g59187 Bradi3g59210 GRMZM2G169033 GRMZM2G320786

Fig. 4. Phylogenetic analysis of LMCOs in different five plant species (Arabidopsis lyrata, A.thaliana, Zea mays, Brachypodium distachyon and Oryza sativa) using the maximum likelihood (ML) method of the PHYLIP program with 100 bootstrap replicates (http:// evolution.genetics.washington.edu/phylip.html). Circle colors represent the different plant species. The amino acid substitutions were evaluated by Jones–Taylor–Thornton (JTT) model. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

closely connected with the copper ion binding (Os03g59280) and oxidative stress-related genes (Os03g22020 and Os06g16360). To evaluate the coexpression of OsChI1-module genes, we identified their expression patterns in different developmental stages and various plant tissues using Genevestigator (http://www.genevestigator.ethz. ch). Many of the OsChI1-module genes were relatively induced during stage I of the vegetative stage, for example during germination, seedling, the tillering stage and the stem elongation stage (Fig. S4). Also, these genes generally exhibited the strong transcript levels in root-related tissues and exhibited stronger expression in the culm (stem) and crown than in other tissues (Fig. S5). 4. Discussion In this report, we showed that OsChI1, a putative rice laccase encoding gene, appears to play an important role in abiotic stress responses. Higher expression of the transcript level of OsChI1 in response to abiotic stresses indicates its important role in stress tolerance (Fig. 1).

Please cite this article as: Cho, H.Y., et al., Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.09.018

H.Y. Cho et al. / Gene xxx (2014) xxx–xxx

Transgenic Arabidopsis plants overexpressing OsChI1 gene exhibited enhanced tolerance to salt and drought stresses, indicating its involvement in stress tolerance (Fig. 3). Although many members of laccase genes, which are specifically expressed in lignified cell types, are responsible for lignin monolignol polymerization in cell walls, several studies have also suggested that plant laccase genes with the expression in vegetative tissues have an additional role, particularly in defense responses (Cai et al., 2006; Liang et al., 2006; Pourcel et al., 2007). For example, flavonoid oxidation catalyzed by laccases protects the plants from ultraviolet radiation and herbivores (Marles et al., 2003; Scalbert, 1991). The transcript levels of plant laccase genes are enhanced in the nonlignifying cell types with biotic and abiotic stresses (Pourcel et al., 2007). Also, it has been shown that mutations of three laccase genes in Arabidopsis resulted in defective root growth under PEG-induced dehydration condition (Cai et al., 2006). In addition, the expression level of ZmLAC1, a putative maize laccase gene, is highly enhanced in the primary root under high concentration of NaCl (Liang et al., 2006). Although detailed mechanism on how laccases perform such different physiological functions has not been elucidated, all these observations suggest that oxidation of a broad range of substrates by laccases is an indispensible enzymatic reaction to maintain cellular homeostasis against abiotic stresses. Our subcellular localization analysis using rice protoplasts clearly showed that OsChI1 is targeted to the plasma membrane (Fig. 2). Most of the laccase proteins have often been predicted to be localized in the apoplast owing to the presence of an N-terminal signal peptide, which directs the protein into the secretory pathway. Apoplastlocalized laccases are implicated to catalyze lignin polymerization. However, a laccase of ryegrass (LpLAC3) does not have a signal peptide and it has been proposed that it remains inside the cell (Gavnholt and Larsen, 2002). AtSKU5 protein involved in directional root growth encodes laccase and is localized in both the plasma membrane and cell walls (Sedbrook et al., 2002). Because rice protoplast system was used for protein localization experiment, we were unable to observe apoplast localization of OsChI1. Thus, we cannot rule out the possibility of dual localization of OsChI1 in the plasma membrane and cell walls. Given the fact that OsChI1 protein lacks a transmembrane domain, it can be predicted that OsChI1 protein is glycosyl phosphatidylinositol (GPI) anchored and targeted to the plasma membrane (Alexandersson et al., 2004). It is our ongoing project to find out the functional role of plasma membrane-localized OsChI1 by identifying target phenolic compounds. Although OsChI1 gene is strongly induced by chilling, dehydration, and high salinity conditions, its overexpression in Arabidopsis resulted in enhanced tolerance to drought and salinity stresses, but not to chilling stress (Fig. 3). One possible explanation for this discrepancy is due to the difference of target substrates of OsChI1 between Arabidopsis and rice. Our working hypothesis is that oxidation of unknown substrates by OsChI1 renders such tolerance to rice and those substrates are absent or function differently in Arabidopsis. Definite proof that OsChI1 is involved in chilling tolerance would depend on demonstration of phenotypic analysis of OsChI1-overexpressing plants in rice. The laccase enzymes are associated with monolignol activation to transfer between monolignols and electron receptors (Frei, 2013). The signaling function of apoplastic enzymes, including laccases, peroxidases and polyphenol oxidases, is modulated by ROS, such as hydrogen peroxide or superoxide (Suzuki et al., 2012). Lang et al. (2000) have reported that the activities of laccase and peroxidase highly increased in Pleurotus sp. and Dichomitus squalens under low temperature condition. Also, OsChI1-module genes were mainly located in the chloroplasts (43 genes, 50%) and extracellularly (23 genes, 27%). The plant's apoplastic copper (Cu) proteins such as laccases (OsChI1) and phytocyanins (Os03g59280) function as electron carriers in the chloroplast and these proteins also act as cofactors of superoxide and a member of extracellular enzymes. Also, module genes of OsChI1 exhibited obvious coherence of their expression patterns in the vegetative stage and

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root-related tissues (Fig. S4 and S5). In Arabidopsis, 16 out of 17 genes analyzed were expressed in root (Cai et al., 2006). These results of the coexpression network suggest that the module genes that are expressed with OsChI1 in the root during various abiotic stresses, such as chilling, drought and salt stress, may play a role in the ROS signaling pathway. A number of studies have demonstrated that stress-response signaling pathways converge, indicating extensive overlap and crosstalk in response to different abiotic stresses (Bostock, 2005; Fujita et al., 2006; Mittler, 2006; Sharma et al., 2013). For example, a transcriptomic study of sunflower subjected to diverse abiotic stresses demonstrated that a large number of genes are upregulated by the treatment of low temperature and salinity (Fernandez et al., 2008). Changes in cytosolic calcium levels are commonly observed in plants under abiotic stresses (Xiong et al., 2002). Transient elevation of calcium levels is instantly perceived by calmodulin proteins, and then common downstream targets are activated. Also, physiological studies have shown that plants acclimated to one type of stress displayed enhanced tolerance to other types of stresses (Sanghera et al., 2011). Our finding that overexpression of the OsChI1 gene in Arabidopsis resulted in enhanced tolerance to salt and dehydration may indicate that the OsChI1-mediated response modulates salt and drought tolerance in Arabidopsis presumably in an ABAindependent manner, and that a newly produced phenolic polymer or an enhanced level of flavonoid oxidation by the plasma membranelocalized laccase plays an important role in plant tolerance to salt and dehydration. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.09.018. Acknowledgments This research was supported by grants from iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea and from 2013 Research Grant from Kangwon National University (No. 120131456). This work was also supported by a grant from the Kyung Hee University in 2012 (KHU-20121642). References Alexandersson, E., Saalbach, G., Larsson, C., Kjellbom, P., 2004. Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking. Plant Cell Physiol. 45, 1543–1556. Berthet, S., Demont-Caulet, N., Pollet, B., Bidzinski, P., Cezard, L., Le Bris, P., Borrega, N., Herve, J., Blondet, E., Balzergue, S., Lapierre, C., Jouanin, L., 2011. Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems. Plant Cell 23, 1124–1137. Bostock, R.M., 2005. Signal crosstalk and induced resistance: straddling the line between cost and benefit. Annu. Rev. Phytopathol. 43, 545–580. Cai, X., Davis, E.J., Ballif, J., Liang, M., Bushman, E., Haroldsen, V., Torabinejad, J., Wu, Y., 2006. Mutant identification and characterization of the laccase gene family in Arabidopsis. J. Exp. Bot. 57, 2563–2569. Campos, P.S., Quartin, V., Ramalho, J.C., Nunes, M.A., 2003. Electrolyte leakage and lipid degradation account for cold sensitivity in leaves of Coffea sp. plants. J. Plant Physiol. 160, 283–292. Cho, H.Y., Hwang, S.G., Kim, D.S., Jang, C.S., 2012. Genome-wide transcriptome analysis of rice genes responsive to chilling stress. Can. J. Plant Sci. 92, 447–460. Christie, P.J., Alfenito, M.R., Walbot, V., 1994. Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194, 541–549. Claes, B., Dekeyser, R., Villarroel, R., Van den Bulcke, M., Bauw, G., Van Montagu, M., Caplan, A., 1990. Characterization of a rice gene showing organ-specific expression in response to salt stress and drought. Plant Cell 2, 19–27. Dean, J.F.D., Eriksson, K.E.L., 1994. Laccase and the deposition of lignin in vascular plants. Holzforschung 48, 21–38. Du, Z., Zhou, X., Ling, Y., Zhang, Z., Su, Z., 2010. agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res. 38, W64–W70. Fernandez, P., Rienzo, J.D., Fernandez, L., Hoppl, E.H., Paniego, N., Heinz, R.A., 2008. Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis. BMC Plant Biol. 8, 11. Ficklin, S.P., Feltus, F.A., 2011. Gene coexpression network alignment and conservation of gene modules between two grass species: maize and rice. Plant Physiol. 156, 1244–1256. Ficklin, S.P., Luo, F., Feltus, F.A., 2010. The association of multiple interacting genes with specific phenotypes in rice using gene coexpression networks. Plant Physiol. 154, 13–24.

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Please cite this article as: Cho, H.Y., et al., Overexpression of the OsChI1 gene, encoding a putative laccase precursor, increases tolerance to drought and salinity stress in transgenic Arabidopsis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.09.018