JIPB

Journal of Integrative Plant Biology

OsSIDP366, a DUF1644 gene, positively regulates responses to drought and salt stresses in rice Chiming Guo1†, Chengke Luo1†,§, Lijia Guo1§, Min Li1, Xiaoling Guo2, Yuxia Zhang1, Liangjiang Wang3 and Liang Chen1*

Research Article

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Xiamen Key Laboratory for Plant Genetics, School of Life Sciences, Xiamen University, Xiamen 361102, China, 2College of the Environment and Ecology, Xiamen University, Xiamen 361102, China, 3Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634, USA. †These authors contributed equally to this work. §Present address: Chengke Luo, Development Center of New Technique Application and Research, Ningxia University, Yinchuan 750021, China. Lijia Guo, Key Laboratory of Integrated Pest Management on Tropical Crops, Ministry of Agriculture, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. *Correspondence: [email protected]

Abstract Domain of unknown function 1644 (DUF1644) is a highly conserved amino acid sequence motif present only in plants. Analysis of expression data of the family of DUF1644containing genes indicated that they may regulate responses to abiotic stress in rice. Here we present our discovery of the role of OsSIDP366, a member of the DUF1644 gene family, in response to drought and salinity stresses in rice. Transgenic rice plants overexpressing OsSIDP366 showed enhanced drought and salinity tolerance and reduced water loss as compared to that in the control, whereas plants with downregulated OsSIDP366 expression levels using RNA interference (RNAi) were more sensitive to salinity and drought treatments. The sensitivity to abscisic acid (ABA) treatment was not changed in OsSIDP366-overexpressing plants, and OsSIDP366 expression was not affected in ABAdeficient mutants. Subcellular localization analysis revealed that OsSIDP366 is presented in the cytoplasmic foci that

INTRODUCTION Abiotic stress such as drought and salinity significantly reduces plant growth and limits crop yield (Roy et al. 2011). To adapt to these stresses, plants express a set of stressresponsive genes to reduce the damage and to maintain growth and reproduction. Drought and salinity are the major factors constraining the rice yield. They threaten rice survival and cause large yield losses by inducing pollen sterility and spikelet death (Flowers 2004; Dolferus et al. 2011; Swamy and Kumar 2013). As a model plant for monocot species and one of the major staple food crops in the world, rice has been subjected to intense studies with regard to the genes responsible for drought and salinity tolerance, which has led to improvement in stress tolerance and will also have significant impact on rice productivity. A number of abiotic stress-related genes have been studied in rice and Arabidopsis to address the molecular mechanism of abiotic stress responses. Arabidopsis ABI1, the protein phosphatase PP2C, has been shown to regulate the activity of SnRK2s via the ABA signaling during stress (Umezawa et al. 2009). Transgenic rice plants overexpressing the stress responsive gene SNAC1 significantly improves drought and salt tolerance (Hu et al. 2006). Overexpression May 2016 | Volume 58 | Issue 5 | 492–502

colocalized with protein markers for both processing bodies (PBs) and stress granules (SGs) in rice protoplasts. Digital gene expression (DGE) profile analysis indicated that stress-related genes such as SNAC1, OsHAK5 and PRs were upregulated in OsSIDP366-overexpressing plants. These results suggest that OsSIDP366 may function as a regulator of the PBs/SGs and positively regulate salt and drought resistance in rice. Keywords: Abiotic stress; DUF1644; processing bodies; rice; stress granules Citation: Guo C, Luo C, Guo L, Li M, Guo X, Zhang Y, Wang L, Chen L (2016) OsSIDP366, a DUF1644 gene, positively regulates responses to drought and salt stresses in rice. J Integr Plant Biol 58: 492–502 doi: 10.1111/jipb.12376 Edited by: Zhizhong Gong, China Agricultural University, China Received Mar. 17, 2015; Accepted Jul. 7, 2015 Available online on Jul. 14, 2015 at www.wileyonlinelibrary.com/ journal/jipb © 2015 Institute of Botany, Chinese Academy of Sciences

of constitutively active ABA-independent transcription factor DREB2A enhances drought tolerance in transgenic Arabidopsis plants (Sakuma et al. 2006). Recently, cytoplasmic mRNAprotein structures, including the stress granules (SGs) and processing bodies (PBs) have been reported to play roles in plant stress responses. Under the stress condition, nontranslated mRNA has been found to be temporarily sequestered into PBs or SGs, which could be degraded or returned to translation depending on the environmental condition. For instance, dehydration stress activated MPK6 phosphorylated PB protein DCP1 and promoted interaction of phospho-DCP1 with DCP5, leading to mRNA decapping in Arabidopsis (Xu and Chua 2012). SG component Tudor-SN (TSN) has been implicated in the regulation of stress responsive mRNA levels during salt stress (Frei dit Frey et al. 2010; Yan et al. 2014). The CCCH tandem zinc finger proteins AtTZF1 and OsTZF1 have recently been shown to be localized to PBs or SGs, and found to mediated stress responses by controlling mRNA turnovers of stress-related genes (Pomeranz et al. 2010; Lin et al. 2011; Jan et al. 2013). These results indicate the important roles of PBs and SGs in mRNA regulation in plant stress responses. Despite the completion of sequencing and annotation of the rice genome, many genes have not been characterized at the biochemistry and biological function levels. One of the www.jipb.net

Positive regulation of stress tolerance by OsSIDP366 examples is a subset of gene families with domains of unknown functions (DUFs) (Project 2005). The names of DUFs were given when protein families with no functional annotation were added to the Pfam database, protein families with DUFs occupy a big portion of the Pfam database (about 22% in 2010) (Bateman et al. 2010; Punta et al. 2012). Different DUF proteins play various roles in plant development and stress responses. For example, TBR and TBL3, members of DUF231 genes in Arabidopsis are involved in cellulose synthesis and deposition of the secondary cell wall (Bischoff et al. 2010), while ESK1, another member of DUF231 acts as a negative regulator of cold acclimation (Xin et al. 2007). DUF724 proteins are highly expressed in pollen grains and the shoot apex, and most members of this family are localized in the nucleus and have potential biological function in regulation of the polar growth of plant cells via RNA transportation (Cao et al. 2010). DUF784 genes are involved in plant embryo sac development during fertilization (Jones-Rhoades et al. 2007). Arabidopsis RING-DUF1117 E3 ubiquitin ligases genes AtRDUF1 and AtRDUF2 are induced by ABA and drought stress, and single or double mutants of AtRDUFs exhibit reduced tolerance to ABAmediated drought stress (Kim et al. 2012). Overexpression of the wheat DUF662 domain-containing transcription factor TaSRG in Arabidopsis enhances salt tolerance (He et al. 2011). OsDSR2, a rice stress repressive DUF966 gene negatively regulates abiotic stress via ABA signaling (Luo et al. 2014). Rice DUF1618 genes have different expression patterns in different cultivars and may be involved in stress responses (Wang et al. 2014). The DUF1644 gene family (Pfam: PF07800) may respond to salinity and drought stresses according to the analysis of abiotic stress-induced genes in rice from published microarray databases (https://www.genevestigator.com/) (Hruz et al. 2008). The DUF1644 gene family is highly conserved in plants, and is presented only in the plant kingdom. The DUF1644 domain contains 9–10 highly conserved cysteine residues and is approximately 160 amino acids in length (Pfam database, http://pfam.sanger.ac.uk/). No member of the DUF1644 family has been characterized to date with regard to its biological function. In this study, we investigated the biological function of the DUF1644 family member OsSIDP366 (Stress induced DUF1644 protein, LOC_Os06g47860) in response to abiotic stress. Our results showed that overexpression and downregulation of OsSIDP366 directly affected the salinity and drought tolerance of rice. OsSIDP366 was localized in the cytoplasmic foci and colocalized with the PBs/SGs markers in rice cells. Taken together, OsSIDP366 may function as a component of PBs/SGs and acts as a positive regulator of responses to abiotic stress in rice.

RESULTS Sequence and phylogenetic analysis of OsSIDP366 Base on RGAP 7 database (http://rice.plantbiology.msu.edu/), OsSIDP366 contains a 954 bp open reading frame encoding a protein of 318 amino acids with theoretical molecular mass of 36.5 kDa. Sequence analysis using Introproscan database (http://www.ebi.ac.uk/Tools/pfa/iprscan5/) showed that OsSIDP366 has a DUF1644 domain, containing a C2H2 and a www.jipb.net

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Ring finger domain (Figure 1A). Blast search in the National Center for Biotechnology Information (NCBI) database showed that DUF1644 genes are presented specifically in the plant kingdom, with 10 members in Arabidopsis and nine in rice. Phylogenetic tree analysis of their amino acid sequences in Arabidopsis and rice indicated that DUF1644 proteins can be classified into four subgroups. Rice homologs of DUF1644 proteins are mainly distributed in subgroup I, II and III, and OsSIDP366 is presented in subgroup I (Figure 1B). Expression pattern of OsSIDP366 Since OsSIDP366 was induced by multiple abiotic stresses, the expression of OsSIDP366 under various stress treatments including drought, high salinity, cold, heat, and H2O2 was examined. As shown in Figure 2A, the mRNA level of OsSIDP366 was upregulated by high salinity treatment, with its peak at 12–24 h after treatment. For drought stress treatment, OsSIDP366 transcript was found to be significantly increased at 6 h and the high expression level lasted to 24 h. OsSIDP366 expression was also induced by treatments with heat and H2O2. In contrast, OsSIDP366 was firstly suppressed then slightly increased by cold stress. OsSIDP366 transcription under various phytohormone treatments was also examined. Its expression was induced significantly by ABA, SA and 2,4-D, and slightly induced by MeJA, IAA, KT and GA3 (Figure 2B). To further study OsSIDP366 expression under abiotic stress, we generated transgenic rice plants expressing the GUS reporter gene under the control of the OsSIDP366 promoter. The GUS activity was increased significantly in the POsSIDP366::GUS transgenic plants by treatments with NaCl and mannitol (Figure 2C). Furthermore, GUS expression was stronger in roots than in shoots, which was consistent with the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis (Figure 2C, D). The same result was observed by ABA treatment (Figure 2D). These results suggest that OsSIDP366 is involved in rice abiotic stress responses. We further investigated tissue-specific expression of OsSIDP366 by measuring its transcript level using qRT-PCR in different tissues at both seedling and flowering stages under normal growth conditions. The results showed that OsSIDP366 is highly expressed in young root, mature leaf and sheath (Figure 3A). Meanwhile, GUS activity was also detected in most tissues of POsSIDP366::GUS transgenic plants at seedling and flowering stages, such as roots, leaves, leaf sheaths, pollen, seeds at the early developing stage and coleoptile, but lower or no activity was detected in internodes, mature seeds, lemmas and paleas (Figure 3B), consistent with mRNA transcription analysis by qRT-PCR. Subcellular localization of OsSIDP366 To determine the subcellular localization of OsSIDP366, we generated the OsSIDP366:GFP fusion construct under the control of 35S promoter. The non-fusion construct expressing green fluorescent protein (GFP) alone was used as a control. As shown in Figure 4A, in transgenic rice overexpressing OsSIDP366:GFP, the green fluorescence was predominantly localized in cytoplasm and formed multiple cytoplasmic foci, while the free GFP was found throughout the cell. The size, shape and numbers of the cytoplasmic foci varied in different types of cells, which resembled cytoplasmic PBs and SGs, and were distinct from the nucleus (Figure S1). May 2016 | Volume 58 | Issue 5 | 492–502

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Figure 1. Protein analysis of OsSIDP366 and phylogenetic analysis of DUF1644 family in rice and Arabidopsis (A) The predicted domains of OsSIDP366. (B) Phylogenetic tree of DUF1644 members from rice and Arabidopsis. Protein sequence alignment was performed using ClustalX 2.0 and phylogenetic tree was created by MEGA 5.2 software using the neighbor-joining method. Scale bar indicates the number of expected amino acid substitutions per site per unit of branch length.

To determine whether OsSIDP366 was associated with PBs and SGs, we tested if OsSIDP366 colocalized with the PBs and SGs marker protein in rice protoplasts. We selected Arabidopsis AtDCP2, an mRNA decapping protein as a PB marker and AtPABP8, a poly(A) binding protein as an SG marker. OsSIDP366 was fused to the C-terminus of GFP, whereas AtDCP2 and AtPABP8 were fused to C-terminus of DsRed. Both constructs were driven by the ubiquitin promoter and co-expressed in rice protoplasts. As shown in Figure 4B, the green fluorescence from GFP:SIDP366 overlapped with the red fluorescence from DsRed:AtDCP2 or DsRed:AtPABP8. We next confirmed the association of OsSIDP366 with rice PBs and SGs, by testing the colocalization of OsSIDP366 with the rice homologous markers OsDCP2 for PBs and OsPABPC1, 2, 3 for SGs. Similar to their Arabidopsis counterparts, rice PBs and SGs markers colocalized with OsSIDP366 in rice protoplasts (Figure 4C). Taken together, these results indicate that OsSIDP366 may be a component of PBs and SGs. Overexpression of OsSIDP366 enhances salt and drought tolerance in rice In order to understand the function of OsSIDP366 in abiotic stress responses, overexpression and knockdown (RNAi) transgenic rice lines were generated. Both overexpression and RNAi constructs were under the control of the 35S promoter and carried the hygromycin-resistance gene for selection of transformation. Positive transgenic lines were May 2016 | Volume 58 | Issue 5 | 492–502

confirmed by PCR using hygromycin resistance gene-specific primers. OsSIDP366 expression level in transgenic plants was verified by qRT-PCR. All 28 overexpressing transgenic lines showed higher OsSIDP366 transcription levels than the wild type plants. Meanwhile, 17 of the 42 RNAi transgenic lines had significantly lower OsSIDP366 expression levels than the wild type. Two independent overexpression lines (OE2, OE17) with high expression and three RNAi lines (Ri8, Ri23, Ri31) with low expression were chosen for further analyses (Figure S2). We first evaluated the salt tolerance of transgenic plants at the seedling stage. After 4 d of germination on 1/2 MS medium, the seeds were grown on the medium containing 200 mM NaCl for 10 d. The relative shoot length of seedlings was used as a criterion to evaluate the stress tolerance, because the overexpression lines grew a little shorter than the wild type under the normal growth conditions. We found that the relative shoot length of overexpression lines was higher than that of the wild type plants, whereas the relative shoot length of RNAi lines was lower than that of the wild type plants under salt stress (Figure 5A, B, NaCl). These results suggest that overexpression of OsSIDP366 improves tolerance to salt stress in rice. Because osmotic stress occurs as a physiological stress at the late stage of drought treatment, transgenic plants were subjected to stress with 150 mM mannitol on 1/2 MS medium. Plants of the OsSIDP366 overexpression lines showed higher relative shoot length than the wild type. On the contrary, the www.jipb.net

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Figure 2. Expression pattern of OsSIDP366 in rice treated with abiotic stresses and phytohormone (A) Expression of OsSIDP366 under abiotic stresses. (B) Expression of OsSIDP366 under phytohormones treatments. ABA, MeJA, SA, IAA, KT and GA3 was applied at a concentration of 100 mM and 2,4-D at 20 mM. (C) GUS activity of POsSIDP366::GUS transgenic plants under NaCl and mannitol stresses. (D) Expression of OsSIDP366 under salt, drought and ABA treatments in different tissues of rice. Relative expression levels of OsSIDP366 were measured by qRT-PCR at indicated time points. Error bars indicate SD from three replicates.

RNAi lines exhibited lower relative shoot length than the wild type (Figure 5A, B, Mannitol), suggesting a positive role of OsSIDP366 in plant responses to osmotic stress. We further evaluated the drought tolerance of OsSIDP366 transgenic rice by testing the drought sensitivity of seedlings in barrels. Five-leaf stage seedlings of transgenic plants and the wild type control were subjected to drought stress by withdrawing water supply for 12 d, followed by re-watering. More OsSIDP366-overexpressing plants (75%–85%) recovered at 7 d after re-watering than the wild type (42.3%), suggesting that overexpression of OsSIDP366 confers drought tolerance in rice (Figure 6A, B). In contrast, 20%–30% of RNAi plants recovered after re-watering, which was notably lower than the wild type (40%–55%) (Figure 6C, D). In addition, we compared the water loss rates of detached leaves from transgenic and wild type plants (Figure 6E, F). The www.jipb.net

overexpression lines lost water slower than wild type, while the water loss rate of RNAi lines was higher than the wild type. These results suggest that the enhanced drought tolerance of OsSIDP366 overexpressing rice may be associated with the reduced rate of water loss. OsSIDP366-mediated abiotic stress tolerance is mainly ABA independent Since ABA signaling plays an important role in plant abiotic stress responses, and OsSIDP366 expression is induced by ABA (Figure 2B), we sought the relationship between OsSIDP366 and ABA signaling. We checked the transcript level of OsSIDP366 in ABA-deficient mutants psh3-1 and psh3-2 (Fang et al. 2008; Du et al. 2013) under drought stress, and did not detect a significant change in the OsSIDP366 transcript level between the wild type and ABA mutants before and after May 2016 | Volume 58 | Issue 5 | 492–502

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Figure 3. Tissue specific expression pattern of OsSIDP366 (A) Tissue-specific expression analysis of OsSIDP366 by qRTPCR. Relative expression levels of OsSIDP366 were measured by qRT-PCR. Error bars indicate SD from three replicates. (B) Histochemical analysis of GUS activity in POsSIDP366::GUS transgenic plants. i: mature root; ii: mature leaf; iii: sheath; iv: stem and internode; v: young leaf; vi: young root; vii: coleoptile; viii–xi: Seeds of different developmental stages; xii: lemma and palea; xiii: anther; xiv: pollens.

drought stress (Figure S3A). We also compared the vegetative growth after ABA treatment, and no significant difference was observed between the wild type and OsSIDP366 overexpression lines (Figure S3B). Furthermore, the expression of ABA biosynthesis genes, such as OsCRTISO, OsLCY, OsZDS and OsPSY showed no difference among OsSIDP366 overexpressing, RNAi, and wild type plants (data not shown), suggesting that OsSIDP366 may not regulate ABA biosynthesis. Therefore, though ABA influences OsSIDP366 expression, OsSIDP366 mediated stress responses may be largely independent of ABA signaling. Transcription profiles of OsSIDP366 overexpression lines To further understand the mechanism of OsSIDP366 in improving abiotic stress resistance in rice, digital gene express (DGE) profiles analysis was performed to identify the difference in gene expression between OsSIDP366 overexpression line OE2 and the wild type. Gene expression levels were normalized to RPKM, then the differentially expressed genes were filtered based on the criteria [log2(FoldChange)| > 1 and q-value < 0.005]. A total of 40 differentially expressed genes including 35 upregulated and five downregulated genes were detected in the OsSIDP366 overexpression line OE2 as May 2016 | Volume 58 | Issue 5 | 492–502

compared to the wild type (Figure 7A, Table S1). GO analysis indicated that most enriched GO terms were associated with stress responses, such as “response to stress,” “response to biotic stimulus” and “defense response” (Figure 7B). qRT-PCR validation of differential expression of these genes (Figure 7C) showed that under normal growth conditions, most differentially expressed genes were upregulated in OsSIDP366 overexpression lines and downregulated in OsSIDP366 RNAi lines. When treated with mannitol, the transcript levels of SNAC1, HSP24.1 and RSOsPR10 in OsSIDP366 overexpression lines were higher than those in the wild type, whereas no significant change in the expression of these genes was observed in both wild type and transgenic plants after salt stress treatment. These results may partially explain the phenotype of OsSIDP366 transgenic plants under stress condition. Digital gene expression analysis also revealed that the transcript level of OsHAK5 was upregulated in OsSIDP366 overexpressing plants than that in the wild type under the normal growth conditions. OsHAK5 is a high affinity Kþ transporter gene, which has been reported to enhance salt tolerance of cultured tobacco BY2 cells and rice plants by increasing the Kþ/Naþ ratio in the shoot (Horie et al. 2011; Yang et al. 2014). Our qRT-PCR result showed that OsHAK5 transcript was increased after mannitol treatment, but not changed under NaCl condition (Figure 7C). We further examined the expression of OsHAK5 in OsSIDP366 overexpressing plants under 200 mM NaCl treatment in the presence of 1 mM KCl. As shown in Figure 7D, the transcript level of OsHAK5 in OsSIDP366 overexpression lines was about two-fold of that in the wild type under the normal condition. When treated with NaCl, the expression of OsHAK5 was upregulated in both wild type and overexpression lines, but the OsSIDP366 overexpression lines accumulated more OsHAK5 than the wild type, indicating that OsHAK5 may contribute to the salt tolerance of OsSIDP366 overexpressing rice.

DISSCUSSION DUFs protein families, though poorly characterized, constitute a big portion of the Pfam database and play important roles in plant development and stress responses. In this study, we examined the biological function of a rice DUF1644 gene, OsSIDP366, in abiotic stress response. OsSIDP366 contains 9–10 highly conserved cysteine residues and has been proposed to have zinc-binding activity as other DUF1644 members (Pfam database, http://pfam. sanger.ac.uk/). Our study showed that OsSIDP366 is localized predominantly in the cytoplasm and cytoplasmic foci (Figure 4). The cytoplasmic foci were colocalized with PBs and SGs, which are non-translated messenger ribonucleoprotein (mRNP) granules in the cytoplasm involved in mRNA degradation or stability during stress response and plant development (Nakaminami et al. 2012). Although the compositions of PBs and SGs are not fully clear, they share proteins and mRNAs in common, such as the tandem zinc finger proteins AtTZF1 and OsTZF1, which contain RNA-binding activity and positively regulate abiotic stress responses in Arabidopsis and rice (Pomeranz et al. 2010; Lin et al. 2011; Jan www.jipb.net

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Figure 4. Subcellular localization of OsSIDP366 (A) Subcellular localization of OsSIDP366 in transgenic plants overexpressing the OsSIDP366:GFP chimeric gene, driven by the 35S promoter. (B) Colocalization of OsSIDP366 with the Arabidopsis PBs marker AtDCP2 and SGs marker AtPABP8. (C) Colocalization of OsSIDP366 with the rice PBs marker OsDCP2 and SGs markers OsPABPC1, OsPABPC2 and OsPABPC3. Scale bars, 50 mm in A, 10 mm in B,C. DIC, differential interference contrast; DsRed, red fluorescent protein; GFP, green fluorescent protein. Arrows indicate PBs and SGs.

et al. 2013; Qu et al. 2014). Our observation that OsSIDP366 colocalized with both protein markers of PBs and SGs implies a potential role of OsSIDP366 as a regulator of PBs and SGs. However, whether the zinc finger of OsSIDP366 has DNA or RNA binding activity and how OsSIDP366 regulates rice abiotic stress response through PBs and SGs remain to be investigated in future research. According to the microarray data (Genevestigator) (Hruz et al. 2008), OsSIDP366 was an abiotic stress-induced DUF1644 gene. qRT-PCR and promoter-GUS analysis confirmed that OsSIDP366 responded to salt, drought and heat stresses (Figure 2A). Our data clearly showed that OsSIDP366 overexpressing plants are more tolerant, whereas the RNAi transgenic plants are more sensitive to salinity and osmotic stresses at the seedling stage (Figure 5). Similar phenotypes of transgenic plants were also observed using drought treatment (Figure 6A–D). Furthermore, OsSIDP366 overexpression lines showed reduced water loss, while the RNAi transgenic lines increased the water loss rate (Figure 6E, F). Taken together, these results suggest that OsSIDP366 positively regulates drought and salinity tolerance of rice. Abscisic acid is known to function as a pivotal messenger regulating plant abiotic stress, and exogenous ABA treatment was also found to induce OsSIDP366 expression (Figure 2B). However, OsSIDP366 expression was not changed in either the wild type or ABA deficient mutants under drought stress (Figure S3A). We observed no difference in plant growth between wild type and OsSIDP366 overexpression plants after ABA treatment (Figure S3B). Moreover, there was no www.jipb.net

significant difference in transcript levels of ABA biosynthesis genes among wild type, OsSIDP366 overexpressing and RNAi transgenic plants. These results indicate that although ABA induces OsSIDP366 expression, the role of OsSIDP366 in abiotic stress response may be largely independent of the ABA signaling. Digital gene expression profiles analysis revealed that overexpression of OsSIDP366 resulted in the upregulation of many biotic and abiotic stress related genes, including pathogenesis-related protein genes (PRs), DnaJ genes, transcription factor SNAC1, Kþ transporter OsHAK5 and secondary metabolism genes (Table S1). As previously reported, SNAC1 acts as an important NAC transcription factor positively regulating drought stress responses by promoting stomatal closure to reduce water loss (Hu et al. 2006). Kþ transporter OsHAK5 could enhance salt resistance in rice and BY2 cells by modulating the Kþ/Naþ ratio in the shoot. Our study showed higher transcript levels of SNAC1 and OsHAK5 in OsSIDP366 overexpression plants than those in the wild type under both normal and osmotic stress conditions (Figure 7C). Moreover, in the presence of Kþ, the abundance of OsHAK5 transcript was increased in overexpression plants during salt stress (Figure 7D). OsHAK5 overexpressing exhibited enhanced rice salt tolerance and increased the Kþ/Naþ ratio in the shoot (Yang et al. 2014). These results partially explain how OsSIDP366 mediates stress tolerance by affecting differentially expressed genes. Under salt stress, the expression level of SNAC1, HSP24.1 and PR1b showed no significant change among the wild type and the transgenic plants, only the May 2016 | Volume 58 | Issue 5 | 492–502

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MATERIALS AND METHODS

Figure 5. Salt and osmotic stress tolerance in rice plants overexpressing OsSIDP366 and RNAi (A) Performance of wild type control TP309, OsSIDP366overexpressing OE2 and OE17, and RNAi Ri8, Ri23 and Ri31 plants subjected to stress with 150 mM mannitol and 200 mM NaCl on 1/2 MS medium. (B) Relative shoot length of wild type TP309, OsSIDP366 overexpressing and RNAi plants subjected to mannitol and salt stresses. Bar, 2 cm in A. Error bars in B indicate SD (n ¼ 6). Statistical significance is indicated by  P < 0.05;  P < 0.01, t-test.

expressions of RSOsPR10 and PBZ1 were suppressed in RNAi lines (Figure 7C); which suggest that these genes may not be the main regulators contributing to the salt tolerance of OsSIDP366 overexpression plants. The upregulation of PRs genes and DnaJ genes in OsSIDP366 overexpressing rice implies the possible role of OsSIDP366 in responses to biotic and other stresses. In conclusion, our data demonstrate that the DUF1644 domain-containing gene OsSIDP366 may perform as a component of PBs and SGs in abiotic stress regulation in rice. OsSIDP366 enhances plant drought and salt stress tolerance by regulating the expression of stress relative genes such as SNAC1 and OsHAK5. Additional research on DUF1644 domain and the function of OsSIDP366 in PBs and SGs will help us reveal the mechanism of how OsSIDP366 contributes to drought and salt resistance in rice. May 2016 | Volume 58 | Issue 5 | 492–502

Plant and growth and stress treatments Rice (Oryza sativa L. japonica) cultivar Taipei309 (TP309) was used as the wild type control in this work. Rice seeds were immersed in distilled water at 37 °C for 2 d, and then grown hydroponically in a growth chamber under a 14 h light/10 h dark photoperiod at 26 °C. Four-leaf stage seedlings were subjected to abiotic stress and hormone treatments. For drought stress, seedlings were exposed in the air without water supply. Salt and oxidative stresses were applied by submerging seedlings in 200 mM NaCl and 1% H2O2, respectively. Seedlings were transferred to a 4 °C and a 42 °C growth chamber for cold and heat stresses, respectively. Whole plants were sampled at 0, 1, 3, 6, 12 and 24 h after treatment. Hormone treatments were conducted by spraying leaves with 100 mM abscisic acid (ABA), 100 mM methyl jasmonate (MeJA), and 100 mM salicylic acid (SA), respectively, then sampled at 0, 0.5, 1, 3, 6, 12, and 24 h after spraying. Leaves of rice seedlings were sprayed with 100 mM indole-3-acetic acid (IAA), 20 mM 2,4-dichlorophen oxyacetic acid (2,4-D), 100 mM kinetin (KT) or 100 mM gibberellin acid (GA3), then sampled at 0, 0.5, 1, 3, 6, and 12 h after spraying. To test drought resistance of transgenic rice at the seedling stage, transgenic and wild type seeds were germinated on half strength Murashige and Skoog (1/2 MS) medium with or without 50 mg/L hygromycin, respectively. Plantlets of hygromycin-resistant seedlings and wild type control (both at shoot height of 4–5 mm) were transferred to barrels filled with a mixture of soil and sand (1:1). When the plants grew to the five-leaf stage, water supply was withdrawn. After severe drought stress (all leaves wilted, about 12 d after water withdrawing), water was added for recovery, and survival performance was photographed and recorded. Water loss rate of detached leaves was measured using the method reported previously (Xiang et al. 2008; Mao et al. 2010). Leaves of transgenic rice at the five-leaf stage were cut and weighed immediately, then exposed to air at room temperature and weighted every hour. To evaluate salt and osmotic stress tolerance, germinated seeds were transplanted in 1/2 MS medium supplemented with 200 mM NaCl and 150 mM mannitol, respectively. Relative shoot length was measured 7 d after transplanting. For ABA sensitivity test, germinated seeds were transplanted in 1/2 MS medium supplemented with 3 mM ABA. Relative shoot length was measured 7 d after transplanting. Phylogenetic analysis Protein sequences of DUF1644 family in rice and Arabidopsis were downloaded from NCBI database (http://www.ncbi.nlm. nih.gov/). Protein sequence alignment was performed using ClustalX 2.0 with default parameters (Thompson et al. 1997). A phylogenetic tree was constructed by MEGA5.2 using neighbor-joining method (Tamura et al. 2011). Bootstrap analysis was performed with 1,000 replicates to test the phylogenetic tree. Plasmid construction and rice transformation To generate the OsSIDP366 overexpression construct, the fulllength coding sequence (CDS) of OsSIDP366 was amplified www.jipb.net

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Figure 6. Drought tolerance in rice plants overexpressing OsSIDP366 and RNAi (A) Performance of wild type TP309 and OsSIDP366-overexpressing lines OE2 and OE17 before and after drought stress at the seedling stage. (B) Survival rate of TP309 and OsSIDP366 overexpressing plants after drought stress (n ¼ 3). (C) Performance of TP309 and OsSIDP366 RNAi lines Ri8, Ri23 and Ri31 before and after drought at the seedling stage. (D) Survival rate of TP309 and OsSIDP366 RNAi plants after drought stress (n ¼ 3). (E) Water loss rate of detached leaves of TP309 and OsSIDP366 overexpressing lines (n ¼ 3). (F) Water loss rate of detached leaves of TP309 and OsSIDP366 RNAi lines (n ¼ 3). Bar, 10 cm in A and B. Error bars indicate SD. Statistical significance is indicated by  P < 0.05;  P < 0.01, t-test.

from rice cultivar Nipponbare by RT-PCR. The PCR product was cloned into pENTR/D-TOPO entry vector (Invitrogen), and then introduced into destination vector pH7WG2 under the control of the cauliflower mosaic virus 35S promoter by LR reaction following the manufacturer’s instruction (Invitrogen). The RNAi construct was generated by cloning the 300 bp www.jipb.net

OsSIDP366 gene-specific PCR fragment into the pENTR/DTOPO entry vector, then into the destination vector pH7GWIWG2 under the control of the 35S promoter. For expression pattern analysis, the OsSIDP366 promoter region (2.2 kb fragment upstream of the ATG start codon) was amplified from genomic DNA of Nipponbare by PCR. The PCR May 2016 | Volume 58 | Issue 5 | 492–502

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Figure 7. Transcription profile analysis of differentially expressed genes under stresses (A) Comparison of expression patterns of differentially expressed genes identified between the wild type TP309 and OsSIDP366overexpressing transgenic plant OE2. Red dots represent upregulated differentially expressed genes with significant difference. Green dots represent downregulated differentially expressed genes with significant difference. Blue dots represent genes with no significant difference. log2 (FoldChange)| > 1 and q-value < 0.005 were used as threshold to identify significant differentially expressed genes. (B) Significantly enriched GO categories with differentially expressed genes. Results were summarized in three categories: biological process, cellular component and molecular function. Asterisk ( ) indicates the enriched GO terms. (C) Validation of the differentially expressed genes from DGE analysis by qRT-PCR. The expression levels were normalized to that of the endogenous control Actin. Error bars indicate SD from three replicates. (D) Expression profiles of OsHAK5 in wild type and OsSIDP366-overexpressing lines under normal and high salinity conditions in the presence of Kþ.

product was cloned into pDONR207 by BP reaction, and the sequencing-confirmed fragment was recombined into pMDC164 vector, placing in front of the GUS reporter gene. To investigate the subcellular localization of OsSIDP366, the CDS without the termination codon was cloned into pENTR/DTOPO, then the fragment was recombined into pMDC83 vector, and fused to the N-terminus of the mgfp6 (GFP) reporter gene downstream of the 2  35S promoter. All the constructs were introduced into TP309 rice by Agrobacterium tumefaciens-mediated transformation (Hiei et al. 1994; Lin and Zhang 2005). RNA isolation and quantitative real-time PCR Total RNA was isolated from rice samples using Eastep Universal RNA Extraction Kit (Promega). For real-time PCR analysis, first-strand cDNAs were synthesized from Dnase I May 2016 | Volume 58 | Issue 5 | 492–502

treated total RNA using M-MLV 1st Strand Kit (Invitrogen). qRT-PCR was performed on ABI Stepone real-time PCR system (Applied Biosystems) using SYBR Premix Ex Taq II (TaKaRa) according to the manufacturer’s protocol. Rice Actin (LOC_Os03g50885) was used as an endogenous control. Relative expression levels were determined as described previously (Livak and Schmittgen 2001). Primers are listed in Table S2. Subcellular localization and GUS activity assay Stable transgenic rice overexpressing OsSIDP366:GFP under the 35S promoter was generated as described above. Subcellular localization of OsSIDP366:GFP in root cells of transgenic rice was observed using a confocal laser scanning microscope (LSM780; Carl Zeiss). For colocalization study, the CDS of OsSIDP366 was fused with green fluorescent protein www.jipb.net

Positive regulation of stress tolerance by OsSIDP366 (GFP) and the CDS of AtDCP2 and AtPABP8 were fused with red fluorescent protein (DsRed) both under the control of the maize (Zea mays) ubiquitin promoter in pXUN vector. Rice homologs of Arabidopsis AtDCP2 and AtPABP8 were identified by BLAST search in NCBI database. One homolog of AtDCP2 [OsDCP2 (LOC_Os02g56210)] and three homologs of AtPABP8 [OsPABPC1 (LOC_Os09g02700), OsPABPC2 (LOC_Os08g22354), OsPABPC3 (LOC_Os04g42600)] were found. The CDSs of these genes were amplified from rice cultivar Nipponbare by RT-PCR then fused with DsRed as described above. These constructs were introduced into rice protoplasts by PEG-mediated transformation and observed as described above. Rice protoplast isolation and PEG-mediated transformation were based on the protocol reported previously with minor modifications (Zhang et al. 2011). Histochemical activity of GUS of POsSIDP366::GUS transgenic rice was detected according to the protocol described previously (Jefferson et al. 1987). Tissues and young shoots of rice were incubated in staining buffer (50 mM sodium phosphate at pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 1 mg/mL X-Gluc, 100 mg/mL chloramphenicol, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide) at 37 °C overnight and then washed with 75% ethanol to remove chlorophyll. GUS images were taken with a stereomicroscope. DGE sequencing and bioinformatics analyses Two-week-old wild type and OsSIDP366 OE2 transgenic rice seedlings were grown under normal growth conditions. Total RNA was isolated by Eastep Universal RNA Extraction Kit (Promega) according to the manufacturer’s instructions. RNA sequencing was performed on Illumina Hiseq 2000 platform by Novogene (Beijing, China). The clean reads were mapped to the Rice Annotation Project Database (RAP-DB) using TopHat v2.0.9 (Kim et al. 2013), and the abundance of mapped reads was normalized to RPKM (Reads Per Kilo bases per Million reads) (Mortazavi et al. 2008). P-values were adjusted using the Benjamini and Hochberg method. q-value of 0.005 and log2 (Fold change) of 1 were set as the threshold of significantly differential expression. Differentially expressed genes (DEGs) were used for GO enrichment analysis by GOseq (Young et al. 2010) to map all DEGs to terms in the GO database (P-value < 0.05) to identify significantly enriched GO terms. The primers for qRT-PCR were listed in Table S2.

ACKNOWLEDGEMENTS We thank Dr. Chunyan Ren at Mount Sinai School of Medicine, New York, USA and Prof. Zonglie Hong at University of Idaho, Moscow, Idaho, USA for revising this manuscript. This work was supported by Prophase Project of National Key Basic Research Program of China (2012CB126312) and Science and Technology Foundation of Guizhou Province of China ([2012] 2277).

AUTHOR CONTRIBUTIONS Guo C. and Luo C. performed most of the research and Guo C. drafted the manuscript. Guo L. and Li M. performed some gene expression analyses. Guo X. and Zhang Y. carried out www.jipb.net

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some stress analyses. Wang L. revised the manuscript. Chen L. designed the experiment, supervised the study, and revised the manuscript.

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SUPPORTING INFORMATION

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Additional supporting information may be found in the online version of this article at the publisher’s web-site. Figure S1. Non-nuclear localization of OsSIDP366 Roots of transgenic plants expressing OsSIDP366:GFP were stained with DAPI to visualize the nuclei of cells. Red arrows indicate cytoplasmic foci of OsSIDP366, and white arrows indicate nuclei. Figure S2. Transcript level of OsSIDP366 in overexpressing and RNAi lines using qRT-PCR (A, B) Transcript levels of OsSIDP366 in overexpression lines. (C, D) Transcript levels of OsSIDP366 in RNAi lines. Red triangles indicate transgenic lines selected for further study. Error bars indicate SD from three replicates. Figure S3. ABA independence of OsSIDP366-mediated abiotic stress responses (A) Expression of OsSIDP366 in ABA deficient mutants phs3-1 and psh3-2 under normal growth and drought conditions. (B) Performance of TP309 and OsSIDP366 overexpressing plants subjected to 3 mM ABA on 1/2 MS. The expression levels are normalized to that of Actin. Error bars indicate SD from three replicates. Table S1. Differentially expressed genes revealed by DGE analysis Table S2. Sequences of primers used in this work www.jipb.net