Molecular characterization of two small heat shock protein genes in ...

Mol Biol Rep (2013) 40:6709–6720 DOI 10.1007/s11033-013-2786-x

Molecular characterization of two small heat shock protein genes in rice: their expression patterns, localizations, networks, and heterogeneous overexpressions Deok-Jae Ham • Jun-Chul Moon • Sun-Goo Hwang Cheol Seong Jang

•

Received: 21 February 2013 / Accepted: 14 September 2013 / Published online: 28 September 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Heat stress is an example of a severe abiotic stress that plants can suffer in the field, causing a significant detrimental effect on their growth and productivity. Understanding the mechanism of plant response to heat stress is important for improving the productivity of crop plants under global warming. We used a microarray dataset that is deposited in the public database to evaluate plant responses to heat stress, and we selected the top 10 genes that are highly expressed under heat stress in rice. Two genes, OsSHSP1 (Os03g16030) and OsSHSP2 (Os01g04380), were selected for further study. These genes were highly induced in response to salt and drought but not in response to cold. In addition, OsSHSP1 and OsSHSP2 gene transcripts were induced under abscisic acid and salicylic acid but not under jasmonic acid and ethylene. Subcellular localization of proteins of 35S::OsSHSP1 were associated with the cytosol, whereas those of and 35S::OsSHSP2 were associated with the cytosol and nucleus. Heterogeneous overexpression of both genes exhibited higher germination rates than those of wild-type plants under the salt treatment, but not under heat or drought stress, supporting a hypothesis regarding

functional specialization of members of small heat-shock protein family over evolutionary time. The network of both genes harboring nine sHSPs as well as at least 13 other chaperone genes might support the idea of a role for sHSPs in the chaperone network. Our findings might provide clues to shed light on the molecular functions of OsSHSP1 and OsSHSP2 in response to abiotic stresses, especially heat stress. Keywords Chaperone network Heat stress Gene family Salt stress Small heat shock protein Abbreviations OsSHSP Oryza sativa small heat shock protein HSPs Heat-shock proteins sHSP Small heat-shock protein PEG Polyethylene glycol ABA Abscisic acid JA Jasmonic acid SA Salicylic acid

Introduction Deok-Jae Ham and Jun-Chul Moon have contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s11033-013-2786-x) contains supplementary material, which is available to authorized users. D.-J. Ham J.-C. Moon S.-G. Hwang C. S. Jang (&) Plant Genomics Lab., Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-713, Korea e-mail: [email protected] J.-C. Moon Agriculture and Life Sciences Research Institute, Kangwon National University, Chuncheon 200-713, Korea

Changes in the concentrations of carbon dioxide and other greenhouse gases have caused the earth’s average surface temperature to rise by approximately 0.8 °C over the last century, with approximately 0.6 °C of the increase occurring over just the past 3 decades. Surface air warming is proposed to rise by an additional 1.8–4.0 °C in the twenty first century [1]. Global warming is a big problem for all living organisms, including plants, animals, and humans. Heat stress is an example of a severe abiotic stress that plants can suffer in the field, causing a significant detrimental effect on their growth and productivity.

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For example, Peng et al. [2] reported that rice yields declined by 10 % for each 1 °C increase in the growingseason minimum temperature on the basis of the relationship between weather data and rice yield over 2 decades. Similarly, in China, changes in temperature have affected yields of 3 major crops—rice, wheat, and maize—according to crop and climate data from representative stations during 1981–2000 [3]. Therefore, new strategies for development of tolerant and adapted crops will be needed for sustainable rice production under global warming. Various types of heat-inducible genes, such as heatshock proteins (HSPs), heat transcription factors (HSFs), and heat-shock elements (HSEs) have been suggested to act as molecular chaperones. In particular, HSPs are known to be responsible for protein folding, translocation, assembly, degradation, and stabilization in a broad range of cellular processes under normal and/or stress conditions [4]. Five major families of HSPs, Hsp70 (DnaK), charperonins (GroEL and Hsp60), Hsp90, Hsp100 (Clp), and small HSP (sHSP) families, have been grouped according to their molecular mass [4]. In plants, the sHsps family with low molecular mass (approximately 12–40 kDa) is known to be more diverse than other HSPs/chaperone families [4]. This family is defined by a conserved C-terminal domain of 80–100 residues referred to as the a-crystallin domain (ACD) [5]. In early studies, at least 6 classes of members of plant sHSPs have been defined based on localization in different cellular compartments and amino acid sequence similarity [6]. These sHSPs are known to be localized in the cytosol or nucleus (C I, C II, and C III) [7, 8], plastid (P) [8], endoplasmic reticulum (ER) [9], and mitochondria (M) [10, 11], respectively. To date, 12 subfamilies of plant sHSPs have been defined in addition to the 6 previously defined classes, including one subfamily (Po) with a putative peroxisomal type 1 targeting signal [7, 12] and 5 additional sHsp groups targeting the mitochondria or the cytosol/nucleus [12]. Recently, Ouyang et al. [13] identified at least 39 members of the rice sHSP family and surveyed their expression patterns during vegetative and reproductive developmental stages via a genome-wide based assay. However, molecular functions of members of the rice sHSP family remain unclear. Heat stress responses are generally thought to possess multiple signaling pathways for thermotolerance. Signaling molecules such as salicylic acid (SA) and abscisic acid (ABA) have been reported to be involved in plant response to heat in ways that are different from the HSP pathway [14]. Several lines of evidence propose that each abiotic stress (e.g., cold, drought, and salt stress) elicits a unique response [15]. Heat stress has a range of effects on cell function, suggesting that many processes in thermotolerance are complex. For example, during seed germination,

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high stress might slow down or completely inhibit germination, depending on the intensity of the stress [16]. In an effort to characterize the plant heat-shock response, we utilized a microarray dataset that is deposited in the public database. We selected 2 rice sHsp genes that are highly expressed under heat shock and performed molecular analyses, including RT-PCR, subcellular localization, heterogeneous overexpression, and co-expression network.

Materials and methods Plant growth and treatments Rice (Oryza sativa spp. japonica cv. Donganbyeo) seedlings were allowed to grow on mesh supported in plastic containers with 1/2 Murashige and Skoog (MS) solution under the conditions of 14-h light/10-h dark at 28 °C in a growth chamber. For heat stress, 2-week-old seedlings were transferred into a chamber at 40 °C and then harvested at 0.5, 1, 3, 6, 12, and 24 h. For salt and drought stress, 2-week-old seedlings were supplied with 200 mM NaCl and 20 % polyethylene glycol (PEG), respectively. Two-week-old seedlings were kept at 4 °C without light for the cold-stress treatment, and at room temperate (26 °C) in the dark for the control. Leaf tissues were harvested at 0, 6, 12, 24, and 48 h. For the phytohormone treatments, seedlings (2-week-old) were transferred to containers with each of 0.1 mM abscisic acid, 0.1 mM jasmonic acid (JA), and 1 mM SA. For ethylene treatment, seedlings were transferred to airtight containers and then 10 lL/L-1 of ethylene gas was injected with a syringe, whereas the other seedlings were transferred to airtight containers without ethylene gas as controls. Leaf tissues were harvested at 0, 3, 6, 12, and 24 h. Semi-quantitative RT-PCR Total RNA was isolated using Trizol reagent (Invitrogen) in accordance with the manufacturer’s protocols. Firststrand cDNA synthesis was conducted using a PrimeScriptTM RT-PCR kit (Takara, Japan) according to the recommended protocols. The final products were stored at -20 °C until used. The quantity of template cDNA was adjusted by assessing the expression of the gene encoding an actin protein (Os03g50885) as an internal control. The primer sequences of the actin protein were as follows: forward primer 50 -TTCGCCGGAGATGATGCGCC-30 and reverse primer 50 -GGTGAGGAGGACGGGGTGCT-30 (Supplementary Table 1). The PCR program was conducted as follows: 5 min at 95 °C, followed by 30 cycles of 30 s of denaturation at 95 °C, 30 s of annealing at each of

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the appropriate temperatures, and 30 s for polymerization at 72 °C with a 5-min final extension step at 72 °C. All RTPCRs were carried out at least in triplicate. Cloning of full-length cDNAs Full-length sequences were retrieved from the Rice Genome Annotation Project database (http://rice.plantbiology.msu. edu/analyses_search_locus.shtml). The primer sequences for cloning of full-length cDNAs were as follows: forward primer 50 -CGTGCCGGGGCTGAAGAAGG-30 and reverse primer 50 -TGACGGTGAGCACGCCGTTC-30 for Os03g16030, and forward primer 50 -CGGCCACCTCCGACAACGAC-30 and reverse primer 50 -CCATGCCGGCCTTCACCTGG-30 for Os01g04380. The full-length clones were amplified by PCR using Pfu DNA polymerase (Takara, Japan) by using the following PCR cycle: 5 min at 95 °C followed by 40 cycles of 20 s of denaturation at 95 °C, 20 s of annealing at each of the appropriate temperatures, and 15 s for polymerization at 72 °C with a 5-min final extension step at 72 °C. The PCR products were separated on 1 % agarose gels and purified with the DOKDO PrepTM Gel Extraction Kit (ELPIS-Biotech. Inc., Korea) in accordance with the manufacturer’s protocol. The purified products were cloned into the pBIN35S-GFP vector (Lim et al. unpublished) via incubation at 16 °C for 1 h. Aliquots of the mixture were transformed into heat-competent E. coli cells and then plated on Luria–Bertani agar plates with 50 mg/L kanamycin as the antibiotic. Plasmids were extracted using a DOKDO PrepTM Plasmid Mini-Prep Kit (ELPIS-Biotech. Inc., Korea) in accordance with the manufacturer’s protocol and then sequenced by a commercial service (Macrogene, Korea). Heterogeneous overexpression in Arabidopsis Transformants (Arabidopsis thaliana) with each of the over-expressed genes were generated by the floral dipping method [17]. The transformed seedlings (T3) were grown in Petri dishes with 1/2 MS medium containing 1 % sucrose and 0.8 % agar, under a 14-h light/10-h dark cycle at 28 °C in a growth chamber. The over-expression of the transgenic lines was confirmed using One-step 5X RT-PCR Master premix (ELPIS-Biotech. Inc., Korea). For heat stress, 10-day-old seedlings were transferred to a water bath maintained at 38 °C for 90 min and then 45 °C for 60 min, and returned to the recovery conditions of a 14-h light/10-h dark cycle at 28 °C in a growth chamber (basal thermotolerance). The acquired thermotolerance tests were performed by heating the plants in a water bath initially at 38 °C for 90 min, then at 28 °C for 120 min, followed by a final heating at 45 °C for 3 h [14]. For the salt-stress treatment, seeds were grown in Petri dishes with 1/2 MS

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medium containing 1 % sucrose and 0.8 % agar and either 0, 100, 150, or 200 mM NaCl under 14-h light/10-h dark conditions at 28 °C in a growth chamber. The germination rate of each sample was scored at 1 day intervals for 10 days. For drought-stress assays, seeds of each sample were sown on 1/2 MS medium containing different concentrations (-0.25, -0.5, -0.7, or -1.2 Mpa) of PEG8000. PEG plates were prepared as per the methods described by Verslues and Bray [18] and Verslues et al. [19]. Each plate was prepared with 50 mL 1/2 MS medium containing 1.5 % agar; plates were solidified and overlaid with 75 mL of liquid PEG containing 0 (-0.25 Mpa), 250 (-0.5 Mpa), 400 (-0.7 Mpa), or 550 (-1.2 Mpa) g/L PEG8000. The plates were kept overnight, and then, the PEG solutions were removed from the plates. Germination rates were scored at 1 day intervals for 7 days. For evaluating the root elongation rate, seeds were germinated on 1/2 MS medium for 3 days and transferred into independent plates containing different concentrations. Root growth rates were monitored at 1 day intervals for 6 days and analyzed using Image J software. Subcellular localization Subcellular localizations of heat-stress inducible genes were examined by expressing the fusion proteins with green fluorescent protein (GFP) via transient expression assays in tobacco leaf (Nicotiana benthamiana). The coding regions of the full-length cDNAs amplified with appropriate primer pairs were cloned into the pBIN35S-EGFP vector and then transformed into the Agrobacterium tumefaciens strain (GV3101). In order to enhance transient expression in tobacco leaves, one construct encoding the p19 protein, which is known to be a viral-encoded suppressor of gene silencing [20], was transformed into A. tumefaciens. Transformed A. tumefaciens were grown in 25-mL YEP solutions supplemented with 50 mg/L kanamycin at 28 °C overnight. Each A. tumefaciens sample was centrifuged and washed 3–4 times with infiltration buffer (10 mM MES, 10 mM MgCl2, pH 5.6), after which 200 lM acetosyringone (Sigma-Aldrich, St. Louis, MO, USA) was added to the samples. Each construct mixed with the p19 protein was injected into the intercellular space through the stomata using a 1-mL syringe. Localization of fluorescent proteins was observed 3–5 days after infiltration, when GFP fluorescence was optimal, by using a confocal laser-scanning microscope (LSM; 510 META NLO; Carl Zeiss, Germany) at the Korea Basic Science Institute, Chuncheon Center. In silico analysis In order to study genes that were functionally related to OsSHPSP1 and OsSHSP2, 1013 CEL files of Affymetrix GeneChip genome arrays of O. sativa (GPL2025) were

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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 using the RMA method (http://www.rproject.org/). In order to retrieve the top 1,000 probes with high correlation with OsSHSP1 and OsSHSP2 we evaluated Pearson’s correlation coefficient values using the normalized microarray dataset. Subsequently, we identified the module genes of OsSHSP1 and OsSHSP2 using the R package Weighted Correlation Network Analysis (WGCNA) following the procedure described in Zhang and Horvath [21]. Amino acids of coexpressed genes were retrieved in the TIGR database (http://rice.plantbiology. msu.edu/) and then the subcellular localization of these genes were predicted by WoLF PSORT (http://www.psort. org/). The subcellular network of genes coexpressed with OsSHSP1 and OsSHSP2 was constructed using R package ARACNE [22] and visualized using Cytoscape software [23]. For genome-wide transcriptome analyses of coexpressed genes in response to abiotic stresses, including drought, salt, and cold (GSE6901), heat (GSE14275), anoxia (GSE6908), and chilling [24], the microarray data were retrieved from the GEO database (http://www.ncbi. nlm.nih.gov/geo/). Functional enrichment analyses of coexpressed genes were conducted by agriGO [25]. The orthologs of the OsSHSP1 and OsSHSP2 genes were retrieved using BLASTp with a cut-off\1e-10 from 6 plant species: Glycine max, Sorghum bicolor, Brachypodium distachyon, Medicago truncatula, and A. thaliana, in the Phytozome database (http://www.phytozome.net). Amino acids were aligned using the ClustalW program (ftp;//ftp.ebi.ac./uk/pub/software/clustalw2/2.1/).

Results

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Expression patterns of these genes were further examined against heat stress via semi-quantitative RT-PCR (Fig. 1). Five genes (Os04g01740, Os04g28420, Os03g16030, Os01g04380, and Os02g15930) were highly induced from 30 min to 24 h after the treatment, whereas foue genes (Os04g06590, Os05g06920, Os12g25200, and Os01g03360) were slightly decreased or were not expressed. One gene (Os03g14180) was slightly induced from 1 to 24 h after the treatment. Two genes (Os3g16030 and Os01g04380) encoding sHSPs were selected for further study and were named OsSHSP1 and OsSHSP2 (O. sativa small heat shock proteins 1 and 2), respectively. A total of 48 genes from 6 plant species were retrieved and aligned. We observed the highly conserved motif known to be of the Hsp20/alpha-crystallin family domain in the C-terminal regions (Supplementary Fig. 1). Responses of OsSHSP1 and OsSHSP2 to abiotic stresses The responses of OsSHSP1 and OsSHSP2 to other abiotic stresses such as salt, cold, and drought are questionable (Fig. 2a). OsSHSP1 and OsSHSP2 were highly induced after salt stress treatment, as was OsSalT, which is known to be highly induced by this stress. Expression of the OsSHSP1 gene increased from 6 until 48 h after treatment. The OsSHSP2 gene was slightly increased at 24 h after the treatment. Similarly, the drought treatment caused induction of expression of both OsSHSP genes as well as of OsSalT. The OsSHSP1 and OsSHSP2 genes were induced from 6 h until 48 h after the drought treatment. In contrast, under coldstress treatment, OsSHSP1 and OsSHSP2 exhibited no induction or a slight decrease even though the OsDREB1A gene was induced from 12 until 48 h after treatment. Expression patterns of the OsSHSP genes under phytohormone treatments

Isolation of heat-shock inducible genes In order to retrieve rice heat-inducible genes, we chose a genome-wide transcriptome analysis for heat-shock treatment (GSE14275) from the GEO database. Based on the relative expression levels of the stress treatment versus the control groups, the top 10 genes with the highest ratios (9.37- to 5.14-fold) were selected as candidate heatinducible genes (Table 1). The locus numbers of these genes are listed as follows: Os04g01740, Os04g28420, Os03g16030, Os01g04380, Os03g14180, Os04g06590, 05g06920, Os12g25200, Os01g03360, and Os02g15930. We subsequently retrieved other genome-wide transcriptome analyses related to five abiotic stresses: drought, salt, cold, chilling, and anoxia. The expression patterns of the genes did not exhibit any striking tendency in response to these stresses (Table 1).

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We subsequently examined responses of OsSHSP1 and OsSHSP2 to phytohormone treatments, including ABA, SA, JA, and ethylene (Fig. 2b). Under ABA treatment, expressions of OsSHSP2 and OsSalT increased from 3 until 24 h after treatment, whereas OsSHSP1 showed a steady pattern of expression. In addition, OsSHSP1 and OsSHSP2 were induced at 12 h after SA treatment whereas OsPRI1b, which is known to be induced by the treatment, was induced at 3 h after treatment. In contrast, under JA stress treatment, the OsSHSP1 and OsSHSP2 genes exhibited no induction whereas OsPBZ1, which is regulated under the treatment, was induced from 12 h until 24 h after JA treatment. Finally, OsSHSP1 and OsSHSP2 exhibited no induction after ethylene treatment, whereas OsERF3, which is regulated by ethylene, was induced from 3 until 24 h after ethylene treatment.

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Table 1 List of the candidate 10 highly inducible genes by heat stress and summary of expression profiles in various stresses. (Color table online) Locus Os04g01740

Stress

Biological

Annotation

Probe

process

Heat shock protein 82

Response to stress

Drought

Salt

Heat

Cold

Chilling

Anoxia

Os.11039.3.S1_at

−0.068033

0.0834544

9.3701755

0.0285698

−0.295464

0.1186847

Os.11039.1.S1_s_at

−0.444304

−0.154966

9.2760619

−0.483441

−0.05702

−1.503176

Os.11039.2.S1_x_at

−0.023625

0.1351138

6.7102431

0.0037391

0.0532671

−0.89564 −0.160575

Os.49651.1.S1_s_at

−0.207804

−0.150655

6.3983548

−0.121469

−0.365937

Os04g28420

Peptidyl-prolyl isomerase

Unknown

Os.25639.1.S1_at

2.1859712

2.2516071

8.1333019

2.4615295

−0.496938

2.3083797

Os03g16030

Hsp20/alpha crystallin family protein

Unknown

Os.37773.1.S1_at

0.7439881

2.8581003

6.5832049

1.0343494

−0.290815

3.9579664

Os.37773.1.S1_x_at

0.9212522

2.7839907

6.4929288

1.1014386

−0.027482

3.6724678

Os01g04380


Unknown

Os.4775.1.S1_at

−0.187113

0.6117814

6.7789321

0.4515514

3.0965916

−0.078592

Os03g14180


Unknown

Os.8926.1.S1_at

−0.049748

−0.171784

8.8147488

−0.017937

−0.024309

2.1592272

Os04g06590

Expressed protein

Unknown

OsAffx.25891.1.S1_at

3.3471356

0.9308196

6.5143965

0.1226745

−0.29316

1.7488228

Os.26930.1.S2_x_at

1.0916657

0.1535678

5.7564836

0.3395368

−0.1106

1.317163

Os05g06920

RelA-SpoT like protein RSH4

Unknown

Os.16903.1.A1_at

−1.325874

−1.010719

5.4825781

−1.348698

−2.424241

−5.476094

Os12g25200

Chloride transporter

Chloride transport

Os.27207.1.S1_at

−0.190577

0.0698422

5.1407532

1.335959

−1.921081

−1.026699

Os01g03360

BBTI5

Unknown

Os.37729.1.S1_s_at

0.9078103

0.7134254

5.5520827

−0.220092

0.1444057

−1.135623

Os02g15930

Expressed protein

Unknown

Os.51778.1.S1_at

0.6448199

0.6574683

5.1523365

0.8340736

1.8931467

0.5239581

0

1/2 C

1 T

C

3 T

C

6 T

C

12 T

C

24 h T

C

T

Heat

C

OsActin Os04g01740 Os04g28420 Os03g16030 Os01g04380 Os03g14180 Os04g06590 Os05g06920 Os12g25200 Os01g03360 Os02g15930

Fig. 1 Expression patterns of 10 rice heat-inducible genes in response to heat. Two-week-old rice seedlings were treated with heat (40 °C). The rice actin protein (Os03g50885) was used as an internal control. C and T indicate control and heat-treated plants, respectively. The arrows indicate selected genes for further work

Subcellular localizations of the OsSHSP genes In order to examine the subcellular localizations of OsSHSP1 and OsSHSP2, their full-length clones were fused with enhanced GFP (EGFP) at the C-terminal region under the control of the CaMV35S promoter. pBIN35EGFP was used as the control, and EGFP fluorescence signals were clearly detected in the cytosol and nucleus. Interestingly, the proteins of 35S::OsSHSP2 were distinctly localized in the nucleus and the cytosol. On the other hand, 35S::OsSHSP1 was localized only in the cytosol (Fig. 3). Heterogeneous overexpression in Arabidopsis Several individual plants overexpressing OsSHSP1 and OsSHSP2 were selected on the basis of expression via RT-PCR analysis (Fig. 4a). We examined the thermotolerance of the overexpressing plants. The overexpression

plants of OsSHSP1 and OsSHSP2 as well as wild-type (WT) and empty vector plants were not survival after heat treatment at 45 °C; all plants survived under 38 °C treatment (Fig. 4b, c). To test their acquired thermotolerance, 3 individual lines were subjected to 38 °C treatment for 90 min, followed by 45 °C for 180 min. No significant difference in survival rate was found between transgenic plants with OsSHSP1 and OsSHSP2 and WT plants (Fig. 4b, c); survival rates of transgenic plants of OsSHSP1 and OsSHSP2 were 45–52 and 47–49 %, respectively, similar to those of WT plants (42.5 %) and empty vector transgenic plants (35S::EGFP) (43 %). Subsequently, we examined the tolerance of plants against salt stress. Under control (0 mM NaCl) and 100 mM NaCl treatment, all transgenic and WT plants germinated, at 3 and 4 days after treatment, respectively. In contrast, under 150 and 200 mM salt treatments, both transgenic plants of OsSHSP1 and OsSHSP2 exhibited higher germination rates than WT and empty vector transgenic plants (Fig. 5). For example, high germination rates of the OsSHSP1- (80–87 %) and OsSHSP2-overexpressing plants (73–93 %) were observed after 10 days under the 200-mM salt treatment, compared to 43 % for WT and 47 % for empty vector transgenic plants. In order to evaluate drought stress tolerance of the overexpressing plants, their germination and root-elongation rates were tested under different water-deficient conditions. All seeds, including transgenic lines and WT, germinated at 3 days after plating with -0.25 and -0.5 MPa. However, seeds on PEG plates with -0.7 and -1.2 Mpa did not germinate (data not shown). For evaluating root elongation under water-deficient conditions, all seeds of transgenic lines and WT were germinated and then

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A

Mol Biol Rep (2013) 40:6709–6720 0 C

6 C

12 T

C

24 T

C

48 h T

C

EGFP

Bright field

Merged

T

Salt

OsSalT OsSHSP1 OsSHSP2

Cold

OsActin OsDREB1A

Drought

OsActin OsSalT OsSHSP1 OsSHSP2 3 C

6 T

C

12 T

C

24 h T

C

T

ABA

OsActin OsSalT OsSHSP1 OsSHSP2

35S::OsSHSP2EGFP

0 C

35S::OsSHSP1EGFP

OsSHSP1 OsSHSP2

B

35S::EGFP

OsActin

JA

OsActin OsPBZ1 OsSHSP1 OsSHSP2 ethylene

OsActin OsERF3 OsSHSP1 OsSHSP2

Fig. 3 Subcellular localization of the OsSHSP1-EGFP and OsSHSP2-EGFP fusion proteins in tobacco leaves. Agrobacterium strain (GV3101) harboring 35S::OsSHSP1-EGFP and 35S::SHSP2-EGFP constructs were transiently expressed. Each construct mixed with the p19 protein was injected into the intercellular space through the stomata in Nicotiana leaves. Images were captured and merged by z-series optical sections after 3 days of agro-infiltration. The 35S::EGFP construct was used as a control

SA

OsActin OsPRI1b OsSHSP1 OsSHSP2

Fig. 2 Expression level of OsSHSP1 and OsSHSP2 genes in rice plants subjected to three abiotic stresses and four hormonal treatments. a Two-week-old rice seedlings were treated with salt (200-mM NaCl), and subjected to cold (4 °C) and drought (20 % PEG). Treated plants were sampled at 0, 6, 12, 24, and 48 h. OsSalT and OsDREB1A were used as reliable stress-inducible genes for each abiotic stress treatment. b Treated plants were sampled at 0, 3, 6, 12, and 24 h. OsSalT, OsPBZ1, OsERF3 and OsPRI1b were used as reliable stress-inducible genes for each hormonal treatment, respectively. The rice actin protein (Os03g50885) was used as internal control. C and T indicate control and stress-treated plants, respectively. The experiments were performed with three biological replicates

transferred into independent plates containing different PEG concentrations (Fig. 6). Root growth was monitored at 1 day intervals for 6 days (Supplementary Fig. 2). Overexpressing plants of OsSHSP1 and OsSHSP2 exhibited lower root elongation rates than WT plants under -0.5 and -0.7 MPa. The respective root elongation rates of OsSHSP1 and OsSHSP2 were 4.10–4.77 and 4.03–4.48 under -0.5 MPa, and 1.81–2.01 and 2.21–2.76 under -0.7 MPa. In contrast, the root elongation rate of WT plants was 5.56 ± 0.18 under -0.5 MPa and 2.97 ± 0.40

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under -0.7 MPa. Under -0.25 MPa (not including PEG), no significant differences in root elongation rates were found between plants that overexpressed OsSHSP1 (5.75–6.91) and OsSHSP2 (6.54–8.56) and WT plants (7.33 ± 1.55). Under -1.2 MPa, roots of neither transgenic nor WT plants were elongated; instead, all roots withered above 90 % until 6 days (Supplementary Fig. 2). In silico analysis of highly correlated genes with OsSHSPs In order to analyze genes functionally related to OsSHSP1 and OsSHSP2 on the basis of transcriptome profiling, genes that were highly correlated to both genes were retrieved using Pearson’s correlation coefficient across 1,014 CEL files of rice microarray datasets; the top 1,000 probes with high correlation coefficients were retrieved for each gene. Among them, module genes, including 66 probes corresponding to 55 genes for OsSHSP2 and 36 probes corresponding to 25 genes for OsSHSP1, were subsequently retrieved using the WGCNA method (Supplementary Fig. 3). Interestingly, 23 genes were common to both modules, whereas 30 genes were observed in the OsSHSP1specific module and 2 in the OsSHSP2-specific module (Table 2).

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A

35S::

B WT

EGFP

35S:: OsSHSP1-EGFP-1

35S::

35S::

OsSHSP1-EGFP-2

OsSHSP1-EGFP-3

38 C, 90 min

28 C, 5 d

28 C, 10 d

45 C, 60 min

28 C, 5 d

28 C, 10 d

38 C, 90 min

28 C,

28 C, 10 d 43% ± 2.3

43% ± 6.2

WT

EGFP

C

35S::

45% ± 4.0

35S:: OsSHSP2-EGFP-1

52% ± 4.7

35S::

45 C, 180 min

5d

2h

48% ± 4.7

35S::

OsSHSP2-EGFP-2

OsSHSP2-EGFP-3

38 C, 90 min

28 C, 5 d

28 C, 10 d

45 C, 60 min

28 C, 5 d

28 C, 10 d

38 C, 90 min 28 C, 10 d 42% ± 10.4

43% ± 2.3

49% ± 13.2

48% ± 2.3

45 C, 180 min 28 C,

2h

5d

47% ± 4.7

Fig. 4 Heterogeneous over-expression of OsSHSP1 and OsSHSP2 and their thermotolerance tests. WT, 35S::EGFP (GFP-empty vector), and 3 independent lines of T3 transgenic plants (35S::OsSHSP1-EGFP and 35S::OsSHSP2-EGFP) were grown on 1/2 MS plates containing 0.8 % agar for 10 days. These plates were heated to 38 °C for 90 min, 45 °C for 90 min (basal thermotolerance), or heated to 38 °C for 90 min, cooled 28 °C for 2 h, then heated to 45 °C for 3 h (acquired

thermotolerance) in a water bath. a RT-PCR analysis of four and five independent T3 transgenic plants for OsSHSP1 and OsSHSP2 respectively, as well as WT and 35S::GFP. The AtUBC gene was used in PCR as an internal control. The phenotypes of WT, GFP-empty vector plants, and transgenic plants of OsSHSP1 (b) and OsSHSP2 (c). Images were captured 5 days after heat-shock treatment. The mean ± SD indicate the average survival rates

Expression profiles of 55 genes, including those pertaining to both modules, were evaluated in response to six abiotic stresses. As expected, the highest average log2 fold

change (FC) values of gene expression were found under heat stress (3.57), whereas the lowest average values (0.03) were found under cold stress. Under other stress treatments,

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Discussion Previously, 39 members of rice sHSP family were retrieved from the genome, and their gene structure, gene expression, and phylogenic relationships were analyzed [13].

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the highly correlated genes showed more than twofold changes, with average log2 FC values as follows: salt = 1.27, drought = 1.17, and anoxia = 1.30. The chilling treatment was the exception with average log2 FC value = 0.94. We attempted to predict subcellular localizations of genes highly correlated with both OsSHSPs and to construct their subcellular networks (Fig. 7). Subcellular localizations of the genes were predicted mainly in the cytosol (21 genes, 38.2 %), nucleus (16 genes, 29.1 %), and chloroplast (10 genes, 18.2 %). Predicted subcellular localizations of OsSHSP1 and OsSHSP2 were found in the cytosol and were consistent with our results, except for the nucleus localization of OsSHSP2. A total of nine members of sHSP families were clustered as the highly correlated genes and their subcellular localizations were predicted to be in the cytosol (seven genes), chloroplast (two genes), and extracellular locations (one gene). Functional enrichment analysis was performed in order to predict the molecular functions of these highly correlated genes. The genes were associated with significant enrichments of certain biological processes, including response to stimulus (adjusted p value = 6.02e-09) and protein folding (p = 1.2e-0.6) (Supplementary Fig. 3).

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Fig. 5 Germination rate of OsSHSP1- and OsSHSP2overexpressing plants in Arabidopsis under salt stress. Each of the three independent seeds of 35S::OsSHSP1-EGFP and 35S::OsSHSP2-EGFP T3 transgenic lines and control plants (WT and 35S::EGFP) were plated on 1/2 MS containing various concentrations of NaCl (a 0 mM, b 100 mM, c 150 mM, d 200 mM). Germination rates for each concentration were scored at 1-day intervals for 10 days. Data represent mean ± SD of 3 independent experiments (n = 30). * and ** significant differences of overexpressing lines in comparison to WT at p \ 0.05 and p \ 0.01, respectively, by the Student’s t test

Mol Biol Rep (2013) 40:6709–6720

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According to their nomenclature, OsSHSP1 and OsSHSP2 were previously identified as OsHsp20-20 and OsHsp20-5, respectively. Although Ouyang et al. [13] reported expression patterns of both genes as well as those of other selective OsHsp20 genes via RT-PCR, most of their molecular functions remain unclear. It is generally known that the sHSP family is divided into 12 subfamilies based on localization in different cellular compartments and on sequence similarities. According to their classification, OsSHSP1 and OsSHSP2 were defined as CIII and CI, respectively [13]. Our results, indicating that OsSHSP1 (CIII) is localized to the cytosol and OsSHSP2 (CI), to the cytosol and nucleus, were consistent with previous demonstrations that classes CI–CIII are localized to the cytosol and/or nucleus [7, 8]. Our findings regarding expression patterns of OsSHSP1 and OsSHSP2 in response to abiotic stresses and phytohormones might provide some clues about their molecular functions. Both genes showed striking induction of their transcripts under salt, drought, and heat stress, but not under cold stress. Moreover, OsSHSP2 was induced by ABA treatment but OsSHSP1 was not. Generally, the phytohormone ABA acts as a key endogenous messenger in plants’ responses to abiotic stresses, and its accumulation causes ABA-dependent gene expression in plants [26]. In contrast, the ABA-independent gene expression pathway might regulate some heat-stress inducible genes, including OsSHSP1, but much work remains to clarify this [27]. The phytohormone SA is an important component of systemic acquired resistance and hypersensitive response, and it has been suggested to be involved in heat-stress

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Fig. 6 Root elongation rate of OsSHSP1- and OsSHSP2-overexpressing plants in Arabidopsis under drought stress. Each three independent seeds of 35S::OsSHSP1-EGFP and 35S::OsSHSP2EGFP T3 transgenic lines and control plants (WT and 35S::EGFP) were plated on 1/2 MS containing various concentrations of PEG (a -0.25 MPa, b -0.5 MPa, c -0.7 MPa). Root elongation rate of each concentration were scored at 1-day intervals for 6 days after being transferred to plats. Data represent mean ± SD of 3 independent experiments (n = 30). * and ** significant differences of overexpressing lines in comparison to WT at p \ 0.05 and p \ 0.01, respectively, by the Student’s t test

responses [16]. The induction of both genes under SA treatment supports the hypothesis that SA accumulation under heat stress causes modification of gene expression in response to this stress.

The induction of both genes in response to abiotic stresses such as heat, salt, and drought leads to the assumption that each gene-overexpressing plant is conferred tolerance to these stresses, especially heat stress. In contrast, the finding regarding significant tolerance to salt stresses, but no difference to heat and drought stresses, in OsSHSP1- and OsSHSP2-overexpressing plants was somewhat inconsistent with our expectation. These results might be explained by the hypothesis regarding functional diversity of members of a gene family during evolution. It is generally considered that dynamic expansion of members of gene families has been caused by whole-genome duplication and/or tandem duplication events [28]. To avoid functional redundancy between duplicate genes over evolutionary time, some hypotheses regarding the evolutionary fates of genes have been proposed, including pseudogenization, neofunctionalization, and subfunctionalization [29]. A host of studies regarding complex expression patterns of plant sHSPs demonstrate that there is no single pattern of gene expression for all plant sHSP genes, although most sSHP genes are induced by heat stress, further supporting evolutionary dynamics of their fate [30]. Our results might provide clues regarding the functional divergence of members of the gene family to evolve a specialization in the direction of specific biological processes [31]. In contrast, a model of the role of sHSPs in the chaperone network in preventing irreversible aggregation and in resolubilizing aggregated protein [30] might explain our finding concerning lower tolerance to heat and drought. The heterogeneous over-expressed gene might not cooperate well with its paralog partners or client proteins in Arabidopsis. A host of gene coexpression network analysis supports the notion that members of a gene cluster are functionally related [32]. Our network of genes that were coexpressed with OsSHSP1 and OsSHSP2 provides insight into their biological function. The finding of the existence of 9 sHSPs, as well as at least 13 other chaperone genes in the gene network, might support ideas about the role of sHSPs in the chaperone network [30]. However, further study is required to better understand their molecular harmony in response to abiotic stresses, especially heat stress. In addition, the expression patterns of the coexpressed genes in response to other abiotic stresses (Table 2) might provide insights into the molecular functions of the genes in the network against other abiotic stresses, especially drought and salt. However, further work would be needed to rule out our hypothesis. In conclusion, the findings presented here help to shed light on the molecular functions of OsSHSP1 and OsSHSP2 in response to abiotic stresses, especially heat stress. Further study on the role of the networks of both genes and

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Table 2 Expressed patterns of highly correlated genes with OsSHSP1 and OsSHSP2 against six abiotic stresses. (Color table online) Features

Probes

Overapped Coexpressions

Features OsSHSP1specific Coexpressions

OsSHSP2specific Coexpressions

Os.36328.1.S1_at OsAffx.10905.3.S1_at Os.4775.1.S1_at Os.519.1.S1_at OsAffx.23978.1.S1_at Os.51778.1.S1_at Os.55371.1.S1_at Os.49648.1.S1_at Os.49648.1.S1_s_at Os.8926.1.S1_at Os.37773.1.S1_at Os.37773.1.S1_x_at Os.2292.1.S1_a_at Os.2292.2.S1_at Os.2292.3.S1_x_at Os.11039.1.S1_s_at Os.11039.2.S1_x_at Os.11039.3.S1_at Os.49651.1.S1_s_at OsAffx.13615.1.S1_at OsAffx.13615.1.S1_x_at Os.25639.1.S1_at Os.7537.1.S1_at Os.16317.1.S1_at Os.8971.1.S1_at Os.5817.1.S1_at OsAffx.27969.1.S1_x_at Os.41109.1.S1_at Os.41109.1.S1_x_at Os.11112.1.S1_s_at Os.27716.2.S1_a_at Os.55330.1.S1_at Os.55330.1.S1_x_at OsAffx.19095.1.S1_at

Probes

Genes LOC_Os01g04340 LOC_Os01g04360 LOC_Os01g04380 LOC_Os01g08860 LOC_Os01g68960 LOC_Os02g15930 LOC_Os02g48140 LOC_Os02g54140 LOC_Os02g54140 LOC_Os03g14180 LOC_Os03g16030 LOC_Os03g16030 LOC_Os03g53340 LOC_Os03g53340 LOC_Os03g53340 LOC_Os04g01740 LOC_Os04g01740 LOC_Os04g01740 LOC_Os04g01740 LOC_Os04g01740 LOC_Os04g01740 LOC_Os04g28420 LOC_Os04g36750 LOC_Os04g45480 LOC_Os05g44340 LOC_Os06g09560 LOC_Os06g36930 LOC_Os07g43740 LOC_Os07g43740 LOC_Os08g40910 LOC_Os08g43334 LOC_Os11g13980 LOC_Os11g13980 LOC_Os11g31060

Description hsp20/alpha crystallin family hsp20/alpha crystallin family hsp20/alpha crystallin family hsp20/alpha crystallin family Conserved hypothetical protein Expressed protein hsp20/alpha crystallin family hsp20/alpha crystallin family hsp20/alpha crystallin family hsp20/alpha crystallin family hsp20/alpha crystallin family hsp20/alpha crystallin family HSF-type DNA-binding domain HSF-type DNA-binding domain HSF-type DNA-binding domain Heat shock Heat shock Heat shock Heat shock Heat shock Heat shock Peptidyl-prolyl isomerase hsp20/alpha crystallin family Heat shock protein STI Heat shock protein 101 Heat shock protein DnaJ HSF-type DNA-binding domain containing protein Zinc finger, C3HC4 type domain Zinc finger, C3HC4 type domain Expressed protein HSF-type DNA-binding domain hsp20/alpha crystallin family hsp20/alpha crystallin family IQ calmodulin-binding and BAG domain

Gen nes

Descriptio on

Drought 0.58 −0.18 −0.19 0.44 0.41 0.64 0.09 2.58 4.00 −0.05 0.74 0.92 0.90 1.35 2.88 −0.44 −0.02 −0.07 −0.21 0.34 −0.16 2.19 0.11 0.53 2.47 3.80 0.19 2.23 1.98 2.08 1.78 0.09 0.16 0.55

Drought

Salt

Salt −0.15 −0.20 0.61 1.61 0.15 0.66 −0.22 2.46 3.80 −0.17 2.86 2.78 0.92 1.68 2.79 −0.15 0.14 0.08 −0.15 0.08 −0.07 2.25 −0.12 0.78 2.29 3.18 0.03 3.23 2.84 2.35 1.32 0.04 0.06 1.75

Abiotic stresses Heat Cold Chilling 3.76 −0.04 −0.15 4.90 −0.07 0.80 6.78 0.45 3.10 4.55 0.32 2.66 3.10 1.55 0.09 5.15 0.83 1.89 2.73 0.35 0.52 5.37 −0.05 1.75 5.53 −0.27 1.78 8.81 −0.02 −0.02 6.58 1.03 −0.29 6.49 1.10 −0.03 1.11 −0.08 −0.16 1.49 0.15 −0.08 2.48 0.24 0.78 9.28 −0.48 −0.06 6.71 0 0.05 9.37 0.03 −0.30 6.4 −0.12 −0.37 6.14 0.18 0.07 5.98 −0.23 −0.58 8.13 2.46 −0.50 8.29 0.03 0.29 1.91 −0.05 1.16 5.11 0.36 2.24 0.55 4.19 0.01 0.48 0.03 −0.21 0.56 −0.95 1.71 0.58 −0.69 1.32 1.60 3.47 −0.13 0.84 −0.21 2.92 4.16 −0.05 −0.01 3.94 0.19 0.32 4.44 0.13 0.30

Abiotic stressess Chilling Heat Cold

Anoxia −0.22 −0.03 −0.08 1.55 0.60 0.52 −0.11 0.80 2.30 2.16 3.96 3.67 0.55 0.38 0.62 −1.50 −0.90 0.12 −0.16 −0.12 0.22 2.31 1.06 2.07 2.79 4.39 0.01 2.53 1.99 1.43 1.38 0.81 0.64 2.44

Anox xia

O Os.9238.2.S1_at

LOC_Os01g55260

AAA-tyype ATPase family

−0.26

0.25

2.73

−0.03

1.75

0.16

O Os.46024.1.S1_at O Os.6400.1.S1_a_at O Os.5574.1.S1_s_at O Os.24611.1.S1_at O Os.12257.1.S1_at O Os.6308.1.S1_at O Os.11419.1.S1_at O Os.8481.2.S1_x_at O Os.8481.4.S1_x_at O Os.12244.1.S1_at O Os.16245.1.S1_at O Os.16659.1.S2_at O Os.53622.1.S1_at O Os.50642.1.S1_at O Os.16058.1.S1_at O Os.5698.1.S1_s_at O Os.49636.1.S1_at O Os.38581.1.S1_s_at O Os.35463.1.S1_at O Os.11376.1.S1_at O OsAffx.15339.1.S1_aat O OsAffx.15339.1.S1_ss_at O Os.9950.1.S1_at O Os.11046.1.S1_at O Os.11106.1.S1_at O Os.38021.1.S1_at O Os.53368.1.S1_at O Os.37238.1.S1_at O Os.53568.1.S1_at O Os.10942.1.S1_a_at O OsAffx.18681.1.S1_ss_at

LOC_Os01g62290 LOC_Os01g65320 LOC_Os022g08490 LOC_Os022g40900 LOC_Os022g52150 LOC_Os033g02260 LOC_Os033g11910 LOC_Os033g13450 LOC_Os033g13450 LOC_Os033g15960 LOC_Os033g18200 LOC_Os033g49430 LOC_Os044g44100 LOC_Os044g48030 LOC_Os055g03910 LOC_Os055g33710 LOC_Os055g35970 LOC_Os055g38530 LOC_Os055g48810 LOC_Os066g06490 LOC_Os066g09820 LOC_Os066g09820 LOC_Os066g14240 LOC_Os066g39240 LOC_Os066g46900 LOC_Os077g43950 LOC_Os077g49470 LOC_Os088g09690 LOC_Os088g39560 LOC_Os100g28340 LOC_Os11g05170

DnaK ffamily Expresssed protein Chaperrone protein clpB 1 RNA reecognition motif containing Heat shhock 22 kDa protein DnaK ffamily DnaK ffamily Expresssed protein Expresssed protein hsp20/aalpha crystallin family y Heat shhock protein DnaJ RNA-splicing factor Pre-mR Expresssed protein Heat strress transcription facttor B-1 RNA polymerase II-associatted protein 3 WD do omain, G-beta repeat domain d Expresssed protein DnaK ffamily dnaJ doomain U-box ddomain containing heeat shock Cupin ssuperfamily Cupin ssuperfamily hsp20/aalpha crystallin family y Endothhelial differentiation-reelated factor 1 Phosphhosulfolactate synthase-related RNA reecognition motif containing Proteinn kinase APK1B, chloroplast precursor Nuclearr transcription factor Y subunit OTU-liike cysteine protease family f Heat strress transcription facttor Expresssed protein

1.07 −0.07 0.92 4.10 2.26 −0.29 −0.02 0.72 0.65 2.07 1.81 1.89 0.64 3.19 0.07 2.78 1.46 2.65 3.32 1.42 0.74 −0.04 1.12 2.03 1.75 3.91 0.50 0.82 0.53 3.37 1.31

1.08 0.16 0.86 3.15 2.79 0.43 0.23 1.38 1.11 2.34 1.06 2.06 1.71 2.85 0.32 2.13 1.41 2.38 2.61 1.28 1.61 0.97 1.66 2.76 1.52 2.03 0.49 0.59 0.53 2.38 0.83

3.07 −0.74 2.37 2.82 5.13 2.26 3.96 1.24 1.19 4.47 2.04 4.4 1.87 0.51 1.91 3.86 1.38 3.28 3.09 2.29 2.22 1.33 0.53 5.25 4.18 2.25 0.49 1.22 3.38 1.06 4.8

−0.01 −0.33 0.22 0.05 0.60 −0.31 0.24 −0.03 −0.29 0.84 −1.19 −0.27 −0.01 −1.07 0.28 0.22 −0.69 0.86 −0.41 −0.23 0.93 0.39 −0.63 −0.11 0.5 0.24 −0.46 −0.76 −0.56 −0.91 0.06

2 −0.12 2.31 0.90 1.15 2.03 2.18 0.87 0.68 1.28 0.89 −0.13 2.82 1.30 0.52 2.15 0.61 1.73 1.73 1.64 1.63 0.95 1.47 0.8 1.70 1.31 0.30 0.66 1.11 1.13 −0.46

0.52 1.06 1.82 2.31 3.68 0.49 −0.53 1.76 1.67 3.41 0.78 1.13 3.05 2.15 0.82 1.93 2.36 1.73 2.43 1.59 0.61 0.39 1.24 5.72 0.17 0.06 0.66 1.63 0.68 1.5 2.38

O OsAffx.13479.1.S1_aat

LOC_Os033g53400

Transm membrane BAX inhibiitor motif

0.37

−0.09

2.54

0.03

0.17

0.53

O Os.51758.2.S1_at

LOC_Os077g47840

Expresssed protein

0.13 1.17

0.13 1.27

1.52 3.57

0.19 0.03

0.07 0.94

0.24 1.30 0

Averaage expression value

s abiotic stresses represents the log2(FC), where red rep Coolor in response to six presents up-regulatiion, green representts down-regulation,, and black represen nts no change in expression.

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Fig. 7 Subcellular networks of genes highly correlated with OsSHSP1 and OsSHSP2. The colors of the lines indicate functional interactions of OsSHSP1 (blue) and OsSHSP2 (pink). (Color figure online)

their client proteins in preventing irreversible aggregation would provide much important information regarding the molecular mechanism of sHSPs in response to heat stress as well as other abiotic stresses. Acknowledgments This research was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

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