Expression and promoter analysis of the OsHSP16.9C gene in rice

Accepted Manuscript Expression and promoter analysis of the OsHSP16.9C gene in rice Yan Zhang, Baohong Zou, Shan Lu, Yuan Ding, He Liu, Jian Hua PII:

S0006-291X(16)31516-9

DOI:

10.1016/j.bbrc.2016.09.056

Reference:

YBBRC 36435

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 6 September 2016 Accepted Date: 12 September 2016

Please cite this article as: Y. Zhang, B. Zou, S. Lu, Y. Ding, H. Liu, J. Hua, Expression and promoter analysis of the OsHSP16.9C gene in rice, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.09.056. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Expression and promoter analysis of the OsHSP16.9C gene in rice

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The State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing

Agricultural University, Nanjing 210095, China.

Plant Biology Section, School of Integrated Plant Science, Cornell University, Ithaca,

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2

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New York 14853, USA. #

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Yan Zhang1#, Baohong Zou1#, Shan Lu1#, Yuan Ding1, He Liu1, and Jian Hua1,2*

These authors contributed equally to this work.

*Correspondence: Dr. Jian Hua, The State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China.

Abstract

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[email protected], [email protected].

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Small heat shock proteins (sHSPs) are molecular chaperones important for stress tolerance. In this study, heat induction of a rice sHSP gene OsHSP16.9C is analyzed

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to understand the molecular mechanisms underlying gene expression regulation in heat shock responses in rice. Promoter deletion analysis of the OsHSP16.9C using the luciferase (LUC) reporter gene in transgenic rice identifies a critical role of a promoter fragment containing an imperfect heat shock element (HSE) in heat induction. HSE was shown to be important for heat induction of AtHSP18.2, a homolog of OsHSP16.9C in Arabidopsis. In addition, the rice OsHSP16.9C promoter confers heat induction of the reporter gene expression in Arabidopsis. These data 1

ACCEPTED MANUSCRIPT suggest that the heat induction mechanisms of OsHSP16.9C and AtHSP18.2 are similar in rice and Arabidopsis. The transgenic reporter line generated offers a system to genetically dissect signaling events in heat induction in rice.

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Keywords: rice, small HSP, heat-shock response, HSE 1. Introduction

High temperature has a large negative impact on plants at various growth stages as it

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perturbs cellular homeostasis and physiology. Plants utilize a complex signaling

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network to minimize the negative impacts of heat stress [1]. Rapid induction of genes, including heat shock proteins (HSPs), are essential part of the heat-shock response [2]. HSPs are molecular chaperones highly conserved among diverse species and are induced by heat rapidly [3]. The abundant HSPs effectively repair and refold damaged

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proteins and thus play a central role in protein homeostasis and thermotolerance in plants and other organisms [4-7].

HSP expression is regulated by heat shock factors (HSFs). Phosphorylation of HSF

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by high temperature induces HSF to enter the nucleus and bind to the heat shock

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elements (HSEs) in the promoter region of the HSP genes [4]. HSE is characterized as multiple adjacent repeats of the pentanucleotide 5’-nGAAn-3’ or 5’-nTTCn-3’, with 5’-nGAAnnTTCnnGAAn-3’ as the perfect type [8]. HSEs contain at least two pentanucleotide nGAAn or nTTCn can also be functional bound by the HSF trimer [9]. HSF has also been shown to bind motifs other than HSE. For instance, AtHSFA1a can not only bind to perfect HSEs but also six novel types of motifs, including gap-type 1, gap-type 2, TTC-rich type 1, TTC-rich type 3, and two types of stress 2

ACCEPTED MANUSCRIPT responsive element [10]. Small HSPs (sHSPs) range from 15 to 30 kDa in molecular weight are more abundant than HSP proteins of other families such as HSP70, HSP60, HSP90 and

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HSP100 in plants [11,12]. The sHSP proteins in Arabidopsis thaliana can be divided into six classes based on sequence homology and their intracellular localization [13,14]. Except class III sHSPs are nuclear localized, classes I and II are cytosol

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localized [15,16], and classes IV, V, and VI are in the plastids [15], endoplasmic

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reticulum [17,18] and mitochondria respectively [19]. Transcriptional regulation of sHSPs have been analyzed for the Arabidopsis class I sHSP gene AtHSP18.2 (AT5g54590) as it is highly inducible by heat [10,20]. Regulatory elements HSE and several gap-type 1 motifs have been identified in the promoter of AtHSP18.2 for heat

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induction[10,20] and it has been used as a reporter system for dissecting heat response signaling in Arabidopsis [21].

Up till now, no empirical experiments have been carried out to investigate the

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molecular regulation of sHSPs by heat shock (HS) in rice, and therefore it is not yet

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known if similar regulatory components are utilized in the monocot rice as in the dicot Arabidopsis. Here we characterized heat induction of the rice sHSP gene OsHSP16.9C (LOC_Os01g04360) by transgenic approach, and our data suggest a similar regulatory mechanism on rice and Arabidopsis sHSP genes. 2. Materials and methods 2.1. Plant growth conditions Oryza sativa. cv Nipponbare and Arabidopsis thaliana ecotype Columbia-0 (Col-0) 3

ACCEPTED MANUSCRIPT were used as wild type in this study. Rice plants were grown under soil culture in a growth chamber with a 12/12 hour (h) day/night cycle at 28/26ºC. Arabidopsis plants were grown in soil with 16/8 h day/night cycle at 22ºC. Proper irrigation was kept

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during the life cycle. For luciferase (LUC) analysis, leaf disks of rice (about 2 cm × 1 cm) or whole young leaves of Arabidopsis were detached from plants and placed in 1/2 Murashige and Skoog (MS) agar medium. Samples on the plate were treated at

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37ºC for 1 h under darkness. For RNA expression analysis, young rice seedlings were

2.2. Quantitative real-time PCR

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treated at 45ºC and leaf tissues were collected for RNA preparation.

Total RNA from roots, stems, young leaves, old leaves, nodes and panicles were extracted using the TRIzol reagent (TaKaRa, http://www.takara-bio.com/). The first

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strand of complementary DNA (cDNA) was synthesized using the Transcript 1st strand cDNA synthesis kit (Vazyme, Nanjing, China). Quantitative real-time PCR (20 µl reaction volume) was carried out using 1 µl of cDNA, 0.4 µl of each primer and 10

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µl of 2 × SYBR Premix Ex Taq Kit (TaKaRa, http://www.takara-bio.com/) in a

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Bio-Rad CFX96 real-time PCR detection system (Bio-Rad Laboratories, USA). The OsActin1 gene was used as an internal control. Data were analyzed following the method previously reported [22]. 2.3. Protein subcellular localization The full length coding sequences of OsHSP16.9C were amplified by PCR and cloned into the BamHI-XbaI sites of the pA7-GFP plasmid [23] to make the OsHSP16.9C:GFP fusion under the control of the CaMV35S promoter. Constructs 4

ACCEPTED MANUSCRIPT were introduced into rice protoplasts as previously described [24]. Transfected protoplasts were incubated overnight in 6-well plates in the dark. The GFP fluorescence was imaged using a confocal laser-scanning microscope (LSM 510; Carl

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Zeiss, http://www.zeiss.com/). 2.4. Phylogenetic tree analysis

The protein sequence of AtHSP18.2 from TAIR (http://www.arabidopsis.org/) was

(http://www.ncbi.nlm.nih.gov/BLAST/).

Phylogeny

reconstruction

was

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Blastp

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used to identify homologous genes in Arabidopsis thaliana and Oryza sativa by using

performed by neighbor-joining analysis. Poisson’s correction was chosen as the distance parameter, as specified in the program MEGA 5.5. The inferred phylogeny was tested by 1000 bootstrap replicates.

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2.5. Generation of promoter deletion lines

A 2 kb sequence upstream from the translation start codon ATG of OsHSP16.9C was chosen for the promoter analysis. Genomatix matrixes (http://www.genomatix.de/)

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was used to identify potential cis-elements that are responsive to heat. Promoter

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fragments of OsHSP16.9C various lengths were PCR-amplified and cloned into the pGWB35 vector [25] to generate promoter fusions of the LUC reporter genes ProOsHSP16.9C:LUC. These constructs were stably introduced into Agrobacterium tumefaciens strain EHA105 and subsequently transformed into the Nipponbare plants by methods previously described [26]. 2.6. Luciferase expression analysis Samples were placed in petri dishes and heat treated for the specified time. 5

ACCEPTED MANUSCRIPT Luminescence signals were captured by CCD camera (Model #ST-138S, 12-16 bits, serial, Princeton Instruments, Trenton, NJ) [27]. Luminescence intensity was quantified with the WinView software provided by the manufacturer.

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3. Results 3.1. Phylogenetic relationships of OsHSP16.9C homologs in rice and Arabidopsis The AtHSP18.2 gene is involved in thermotolerance and its induction by heat stress

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has been extensively studied in Arabidopsis [10,20]. To identify a gene suitable for

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genetic dissection of heat induction in rice, we searched for homologous genes of AtHSP18.2 in Oryza sativa and Arabidopsis thaliana by the Blastp program at NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). A total of 18 genes from rice and 12 genes from Arabidopsis were identified with an E-value cutoff of 1e-5. A phylogenetic tree

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of these 30 genes were subsequently constructed based on their protein sequence homology. Seven rice genes, 3 codings for sHSPs of 16.9 kDa and 4 of 17.3 to 18 kDa, fall into the same clade with AtHSP18.2 together with other two Arabidopsis sHSP

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genes (Fig. 1). These rice sHSP proteins likely have similar subcellular localization as

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the Arbidopsis HSP18.2 protein. We fused OsHSP16.9C with GFP and expressed the fusion protein in rice protoplasts. Fluorescent signals of OsHSP16.9C-GFP were observed in both nucleus and cytoplasm (Supplementary Fig. 1), which is inconformity with the class I sHSPs are cytosol localized in Arabidopsis. Expression patterns of these seven rice genes were examined by information at the RiceXPro dataset (http://ricexpro.dna.affrc.go.jp/) (Supplementary Fig. 2). Except for OsHSP16.9A which has the highest expression in late mature embryo, all these rice 6

ACCEPTED MANUSCRIPT genes have higher expression in ovary than in other tissues (Supplementary Fig. 2). The OsHSP16.9C gene has the most extreme tissue specificity among these seven genes, with a very high expression in ovary and little expression in other tissues

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(Supplementary Fig. 2A). We subsequently confirmed the expression pattern of OsHSP16.9C by reverse transcription (RT)-PCR using RNAs isolated from different tissues including root, stem, young leaf, old leaf, node and panicle. OsHSP16.9C

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transcript had the highest expression in panicle, which is consistent with the reported

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high expression of OsHSP16.9C in ovary (Fig. 2A). Because reproductive organs are the most sensitive tissue to heat, we chose OsHSP16.9C for further heat induction analysis among this group of sHSP genes.

3.2. Effects of heat treatments on the expression level of OsHSP16.9C

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We analyzed the expression level of OsHSP16.9C with heat treatment over time as the extent of induction of HSPs is known to be dependent on the duration of treatment [28]. Because OsHSP16.9C has a high expression in ovary, we first used the

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flowering panicle for the heat induction analysis. After heat treatment at 45ºC, the

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expression of OsHSP16.9C had a sharp rise and reached the highest level at 0.5 h, followed by a decrease at 1 h and a steady level after 2 h (Fig. 2B). We then determined whether or not heat induction of OsHSP16.9C occurs in leaf as in flowering panicle. Rice seedlings at 3-leaf stage were exposed to 45ºC and samples were collected at 0, 0.5, 1, 2, 4, 12, 24 and 48 h during the treatment. Reverse transcription PCR analysis showed that the transcript of OsHSP16.9C rose sharply at 0.5 h, peaked at 1 h, and decreased to a stable level at 12 h (Fig. 2C). The dynamics of 7

ACCEPTED MANUSCRIPT gene pattern in response to heat was very similar in leaf and panicle, suggesting a similar heat regulation in different tissues. To determine whether the heat induction of the OsHSP16.9C transcript is due to

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transcriptional regulation or not. The genomic fragment 2 kb upstream from the translation start codon ATG of OsHSP16.9C was fused with LUC reporter gene and then transformed into the wild-type rice plant Nipponbare. Twenty-four transgenic

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lines of ProOsHSP16.9C:LUC (named P) were obtained and fifteen can observed the

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LUC expression by heat. three T1 lines were randomly selected for heat induction analysis. The blade tips were excised from young leaves and subjected to heat treatment in petri dishes. Fluorescence emitted by the plant tissues was observed in the transgenic lines (L1, L2 and L3) after heat treatment (37ºC) for 1 h while it was

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not observed in non-treated samples or non-transgenic plants (Fig. 3). Therefore the promoter of OsHSP16.9C is sufficient to confer the heat induced expression, which also indicates the heat induction of OsHSP16.9C at the transcriptional level in rice is

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largely similar to that of AtHSP18.2 in Arabidopsis.

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3.3. HSE containing fragment is essential for heat-induced transcription of OsHSP16.9C

To identify regulatory elements for heat induction of OsHSP16.9C, we searched for sequences matching known cis-element motifs in the 2 kb promoter sequence of OsHSP16.9C by Genomatix matrixes (http://www.genomatix.de/). Several stress response-related elements were predicted, including two ABRE (-953 to -918 bp, -1361 to -1345 bp), one HSE (-539 to -523 bp), and one HSFA1a-binding site (-68 to 8

ACCEPTED MANUSCRIPT -52 bp) (Fig. 4A). A pollen-specific regulatory element PSRE was also found at -1006 to -998 bp (Fig. 4A). Based on the previous studies of HSPs in Arabidopsis [9,10], we hypothesized that the imperfect HSE (gaGAAacTACgaGAAcc) at -539 to -523 bp

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and the HSFA1a-binding site (caGAGatTTCagGACag) at -68 to -52 bp were potential heat induction regulatory elements. These two elements were also found at similar positions in the promoters of other six rice homologous genes (Supplementary Fig. 3),

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increasing the likelihood that these are important elements.

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To determine whether these two elements regulate the heat induction of OsHSP16.9C gene, we further constructed LUC reporter genes under the control of upstream sequences of OsHSP16.9C of various lengths: 1000, 500, and 150 bp upstream of the ATG translation initiation site (Fig. 4A). These reporter constructs

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were named as P1 (1000 bp), P2 (500 bp) and P3 (150 bp) and stably transformed into rice (Fig. 4A). A total of 10, 4 and 9 lines were generated for the P1, P2 and P3 respectively (Fig. 4B). Two lines of each of the P1, P2 and P3 transgenic lines (at the

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T0 generation) were analyzed for heat induction of the reporter gene. After heat

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treatment at 37ºC for 1 h, fluorescent signals were induced in the P1 transgenic lines, but not the P2 or P3 lines (Fig. 4C). This indicates that element(s) in P1, likely the imperfect HSE, is important for heat induced transcription of OsHSP16.9C. 3.4. Heat induction is conferred by the rice OsHSP16.9C promoter in Arabidopsis To determine if the heat induction process for sHSPs is similar in rice and Arabidopsis, the ProOsHSP16.9C:LUC construct was transformed into Arabidopsis 9

ACCEPTED MANUSCRIPT wild type plant Col-0. Three randomly selected from a total of nine transgenic lines were heat treated at 37ºC for 1 h with the wild-type Col-0 as control. Fluorescent signals were induced in heat treated transgenic lines but not in the wild type plants

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(Supplementary Fig. 4). The results show that the heat induction mechanism of OsHSP16.9C is likely conserved between rice and Arabidopsis. 4. Discussion

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To better understand heat shock response in the monocot plant, we searched for genes

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that could be used as a reporter system for future genetic dissection of heat responses. The sHSP gene OsHSP16.9C was found to meet the criteria. It is most closely related to AtHSP18.2 which has been well studied for heat induction in Arabidopsis. OsHSP16.9C is highly induced by heat in both panicle and leaf with a similar

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dynamics (Fig. 2B and Fig. 2C). Its high expression in panicle also offers an opportunity to investigate heat sensitivity in reproduction in future. We show that heat induction of OsHSP16.9C transcript is due to transcriptional

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regulation (Fig. 3), which is similar to that of AtHSP18.2. This is further supported by

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the finding that the reporter driven by the rice promoter confers heat induction in Arabidopsis as well (Supplementary Fig. 4). Promoter deletion analysis revealed that fragment containing the imperfect HSE at -539 to -523 bp position mediates its heat induction (Fig. 4). This indicates that heat induction of sHSP genes likely uses the same cis-elements in rice and Arabidopsis. However, the HSE element in OsHSP16.9C is more distal than that in AtHSP18.2. In OsHSP16.9C related rice genes, this element resides in different distance from the transcription start site in the 10

ACCEPTED MANUSCRIPT promoter (Supplementary Fig. 3), maybe it is the reason for their differences in the transcription regulation and transcript stability by heat [28]. In Arabidopsis, Heat induction of AtHSP18.2 is dependent on both a perfect HSE

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element at -196 bp to -180 bp and nearby overlapping gap-type 1 HSFA1a binding sites [10,20]. One HSFA1a-binding site was also found in OsHSP16.9C promoter, however, the P2 fragment containing this site does not confer heat induction (Fig. 4),

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indicating that it is may not sufficient for heat induction of OsHSP16.9C. Closer

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inspection revealed that this element does not match the six sequences shown to be the HSFA1a-binding motifs in Arabidopsis, so it may not be a real HSFA1a-binding site. Alternatively, this element may need to work together with HSE for the heat induction. HSE elements and HSFA1a-binding sites are also found in promoters of six

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other rice genes related to OsHSP16.9C (Supplementary Fig. 3). The number and arrangement of these elements are different from each of the sHSP genes, which might have enabled a differential expression pattern on these genes.

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sHSPs are known for their extensive roles in plant abiotic stresses, and their roles in

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development have also been noticed [16,29-31]. The OsHSP16.9C and its close homologs have a very high expression in ovary, anther, pistil, embryo and/or endosperm even in the absence of heat (Fig. 2A and Supplementary Fig. 2). High expression in ovary appears to be a universal feature of these related genes in rice, suggesting a critical function of these proteins in development of reproductive organs. Consistent with our speculation, one rice sHSP gene OsHSP18.2 was recently shown to enhance seed vigor and longevity by reducing deleterious reactive oxygen species 11

ACCEPTED MANUSCRIPT accumulation in seeds [32]. It would be interesting to investigate how OsHSP16.9C and its related genes function in both non-stressed and stressed conditions especially in reproductive organs.

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The OsHSP16.9C protein is located in both nucleus and cytoplasm (Supplementary Fig. 1), which was different from the majority sHSPs in class I and II localized in cytosol in Arabidopsis [15]. Because the nuclear localization signal in the α-crystallin

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domain of Class III sHSPs can contribute not only to the nuclear localization of the

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class III sHSP proteins but also to an enhanced nuclear localization of class I and class II sHSP proteins [33,34]. We suspect there may be some class III sHSP proteins have effect on the localization of OsHSP16.9C protein.

In this study, we have also established a reporter system that can be used for

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genetic dissecting of stress responses. Mutants that disrupt heat induction of the reporter can be readily screened in a high throughput manner, and the causal genes for the mutations can be identified for further understanding heat responses in rice. The

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use of LUC reporter rather than the β-glucuronidase reporter previously employed for

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AtHSP18.2 offers a non-invasive assay and the possibility of conducting additional assays on the same plant. As sHSPs can be induced by stresses other than heat [15], this system can potentially be used to dissect signaling events in other environmental stresses.

Conflicts of interests The authors declare that they have no conflict of interest. Acknowledgements 12

ACCEPTED MANUSCRIPT The authors would like to thanks Dr. Chaofeng Huang in Nanjing Agricultural University for pGWB35 vector. This research was supported by Jiangsu Collaborative Innovation Center for Modern Crop Production and Priority Academic Program

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Development of Jiangsu Higher Education Institutions. References

[1] J. Larkindale, E. Vierling, Core genome responses involved in acclimation to high temperature,

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Plant physiol. 146 (2008) 748-761.

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[2] R.I. Morimoto, P.E. Kroeger, J.J. Cotto, The transcriptional regulation of heat shock genes: a plethora of heat shock factors and regulatory conditions, EXS. 77 (1996) 139-163. [3] M.E. Feder, G.E. Hofmann, Heat shock proteins, molecular chaperones, and the stress response: Evolutionary and ecological physiology, Annu Rev Physiol. 61 (1999) 243-282.

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[4] K.C. Kregel, Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance, J Appl Physiol. 92 (2002) 2177-2186. [5] D.S. Latchman, Heat shock proteins and cardiac protection, Cardiovasc Res. 51 (2001) 637-646.

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[6] L.H.E.H. Snoeckx, R.N. Cornelussen, F.A. Van Nieuwenhoven, R.S. Reneman, G.J. Van Der Vusse,

AC C

Heat shock proteins and cardiovascular pathophysiology, Physiol Rev. 81 (2001) 1461-1497. [7] R.I. Morimoto, Dynamic remodeling of transcription complexes by molecular chaperones, Cell. 110 (2002) 281-284.

[8] M. Fernandes, T. O’Brien, J.T. Lis, Structure and regulation of heat shock gene promoters, Cold Spring Harbor Monograph Archive. 26 (1994) 375-393. [9] F. Schoffl, R. Prandl, A. Reindl, Regulation of the heat shock response, Plant Physiol. 117 (1998) 1135-1141. 13

ACCEPTED MANUSCRIPT [10] L.H. Guo, S. Chen, K.H. Liu, Y.F. Liu, L.H. Ni, K.Q. Zhang, L.M. Zhang, Isolation of heat shock factor HsfA1a-binding sites in vivo revealed variations of heat shock elements in Arabidopsis thaliana, Plant Cell Physiol. 49 (2008) 1306-1315.

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[11] W.X. Wang, B. Vinocur, O. Shoseyov, A. Altman, Role of plant heat shock proteins and molecular chaperones in the abiotic stress response, Trends Plant Sci. 9 (2004) 244-252.

[12] W.W. de Jong, G.J. Caspers, J.A.M. Leunissen, Genealogy of the alpha-crystallin small heat-shock

SC

protein superfamily, Int J Biol Macromol. 22 (1998) 151-162.

M AN U

[13] T. Mahmood, W. Safdar, B.H. Abbasi, S.M.S. Naqvi, An overview on the small heat shock proteins, Afr J Biotechnol. 9 (2010) 927-939.

[14] E.R. Waters, G.J. Lee, E. Vierling, Evolution, structure and function of the small heat shock proteins in plants, J Exp Bot. 47 (1996) 325-338.

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[15] E. Vierling, The roles of heat shock proteins in plants, Annual Review of Plant Physiology and Plant Mol Biol. 42 (1991) 579-620.

[16] K.D. Scharf, M. Siddique, E. Vierling, The expanding family of Arabidopsis thaliana small heat

EP

stress proteins and a new family of proteins containing alpha-crystallin domains (Acd

AC C

proteins), Cell Stress Chaperon. 6 (2001) 225-237. [17] K.W. Helm, J. Schmeits, E. Vierling, An endomembrane-localized small heat shock protein from Arabidopsis Thaliana, Plant Physiol. 107 (1995) 287-288.

[18] C. Lenne, M.A. Block, J. Garin, R. Douce, Sequence and expression of the mRNA encoding HSP22, the mitochondrial small heat shock protein in pea leaves, Biochem J. 311 ( Pt 3) (1995) 805-813. [19] P.R. LaFayette, R.T. Nagao, K. O'Grady, E. Vierling, J.L. Key, Molecular characterization of cDNAs 14

ACCEPTED MANUSCRIPT encoding low molecular weight heat shock proteins of soybean, Plant Mol Biol. 30 (1996) 159-169. [20] T. Takahashi, Y. Komeda, Characterization of two genes encoding small heat shock proteins in

RI PT

Arabidopsis thaliana, Mol Gen Genet. 219 (1989) 365-372. [21] T. Takahashi, S. Naito, Y. Komeda, The Arabidopsis Hsp18.2 promoter/Gus gene fusion in

heat shock response, Plant J. 2 (1992) 751-761.

SC

transgenic Arabidopsis plants - a powerful tool for the isolation of regulatory mutants of the

M AN U

[22] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method, Methods. 25 (2001) 402-408. [23] C. Voelker, D. Schmidt, B. Mueller-Roeber, K. Czempinski, Members of the Arabidopsis AtTPK/KCO family form homomeric vacuolar channels in planta, Plant J. 48 (2006) 296-306.

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[24] S.B. Chen, L.Z. Tao, L.R. Zeng, M.E. Vega-Sanchez, K. Umemura, G.L. Wang, A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice, Mol Plant Pathol. 7 (2006) 417-427.

EP

[25] T. Nakagawa, T. Kurose, T. Hino, K. Tanaka, M. Kawamukai, Y. Niwa, K. Toyooka, K. Matsuoka, T.

AC C

Jinbo, T. Kimura, Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation, J Biosci Bioeng. 104 (2007) 34-41.

[26] S. Toki, N. Hara, K. Ono, H. Onodera, A. Tagiri, S. Oka, H. Tanaka, Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice, Plant J. 47 (2006) 969-976. [27] V. Chinnusamy, B. Stevenson, B.H. Lee, J.K. Zhu, Screening for gene regulation mutants by bioluminescence imaging, Science S Stke. 2002 (2002) pl10-pl10. 15

ACCEPTED MANUSCRIPT [28] J.C. Guan, T.L. Jinn, C.H. Yeh, S.P. Feng, Y.M. Chen, C.Y. Lin, Characterization of the genomic structures and selective expression profiles of nine class I small heat shock protein genes clustered on two chromosomes in rice (Oryza sativa L.), Plant Mol Biol. 56 (2004) 795-809.

RI PT

[29] N. Wehmeyer, E. Vierling, The expression of small heat shock proteins in seeds responds to discrete developmental signals and suggests a general protective role in desiccation tolerance, Plant Physiol. 122 (2000) 1099-1108.

SC

[30] S.J. Guo, H.Y. Zhou, X.S. Zhang, X.G. Li, Q.W. Meng, Overexpression of CaHSP26 in transgenic

M AN U

tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance, J Plant Physiol. 164 (2007) 126-136.

[31] D.E. Perez, J.S. Hoyer, A.I. Johnson, Z.R. Moody, J. Lopez, N.J. Kaplinsky, BOBBER1 is a noncanonical Arabidopsis small heat shock protein required for both development and

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thermotolerance, Plant Physiol. 151 (2009) 241-252.

[32] H. Kaur, B.P. Petla, N.U. Kamble, A. Singh, V. Rao, P. Salvi, S. Ghosh, M. Majee, Differentially expressed seed aging responsive heat shock protein OsHSP18.2 implicates in seed vigor,

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longevity and improves germination and seedling establishment under abiotic stress, Front

AC C

plant sci. 6 (2015).

[33] R. Wollgiehn, D. Neumann, U. Zurnieden, A. Musch, K.D. Scharf, L. Nover, Intracellular distribution of small heat stress proteins in cultured cells of Lycopersicon Peruvianum, J Plant Physiol. 144 (1994) 491-499.

[34] M. Siddique, M. Port, J. Tripp, C. Weber, D. Zielinski, R. Calligaris, S. Winkelhaus, K.D. Scharf, Tomato heat stress protein Hsp16.1-CIII represents a member of a new class of nucleocytoplasmic small heat stress proteins in plants, Cell Stress Chaperon. 8 (2003) 16

ACCEPTED MANUSCRIPT 381-394.

Figure legends

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Fig. 1 Phylogenetic tree of AtHSP18.2 and its close homologs in Arabidopsis thaliana (At) and Oryzae sativa (Os). The amino sequences of these proteins have been aligned and used to construct an evolutionary tree by the software MEGA 5.5.

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Fig. 2 The expression patterns of OsHSP16.9C. (A) RNA expression in different

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tissues. (B) RNA expression in flowering panicle after 45ºC heat induction. (C) RNA expression in younger leaf at 3-leaf stage after 45ºC heat induction. All samples are from Nipponbare. Shown are relative expression level analyzed by quantitative real-time PCR. Expression is normalized by the expression level at 0 h (no heat

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treatment). OsActin1 was used as the control gene. Different letters above bars represent significant difference (p≤0.05) among samples.

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Fig. 3 Heat induces expression of ProOsHSP16.9C:LUC in rice. Shown are leaf disks from 3-leaf old seedling of wild type Nipponbare and three ProOsHSP16.9C:LUC

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lines (L1, L2, and L3) treated at 37ºC for 1 h or without (CK). The upper panel shows the leaf discs and the lower panel shows the fluorescence signal from LUC activity.

Fig. 4 Bioinformatics analysis of cis-elements in the promoter of OsHSP16.9C. (A) Diagram of the promoter and its deletion series (P, P1, P2, P3) of OsHSP16.9C as well as cis-elements predicted by Genomatix matrixes. Symbols of cis-elements are placed on the bottom of the line when the consensus sequence is found on the reverse 17

ACCEPTED MANUSCRIPT strand. TSS is the transcription start site. (B) Summary of transgenic lines generated for the P, P1, P2, and P3 constructs, and the LUC expression by heat induced. (C) LUC activity analysis of ProOsHSP16.9C:LUC lines. Leaf discs of two representative

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transgenic lines (L1 and L2) from each of the P1, P2, and P3 and the wild type (WT) obtained from 3-leaf stage seedling were placed on 1/2 MS agar medium and subject

were not subject to heat treatment with CK.

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to luminescence imaging after heat treatment at 37ºC for 1 h. Disks in the CK column

protoplasts. Bars = 10 µm

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Supplementary Fig. 1 Localization of OsHSP16.9C protein expressed in rice

Supplementary Fig. 2 Spatio-temporal profile of the seven OsHSPs from rice. The

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expression level of 7 OsHSP genes at different growth development stages and tissues as presented in RiceXPro database.

Supplementary Fig. 3 Predictions of HSEs and HSFA1a-binding sites in the

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promoters of seven OsHSP genes. Sequences 2 kb upstream of the first transcription

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start site for each gene was analyzed by Genomatix matrixes. HSE and HSFA1a binding sites are depicted.

Supplementary Fig. 4 Heat induces the expression of ProOsHSP16.9C:LUC in Arabidopsis. Shown are leaves from the wild type (WT) and three transgenic lines of ProOsHSP16.9C:LUC (L1, L2 and L3) with 1 h of 37ºC heat treatment or without the treatment (CK). The top panel shows the leaves and the bottom panel shows the luminance images of the leaves. 18

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ACCEPTED MANUSCRIPT Highlights:


A reporter system can be used to genetically dissect signaling events in heat induction in rice.




A critical role of a promoter fragment containing an imperfect HSE has been

identified in heat stress.

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Serious of sHSPs in rice show a critical function of these proteins in development of reproductive organs.

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