Over-expression of a protein disulfide isomerase gene from Methanothermobacter thermautotrophicus, enhances heat stress tolerance in rice

Accepted Manuscript Over-expression of a protein disulfide isomerase gene from Methanothermobacter thermautotrophicus, enhances heat stress tolerance in rice

Xin Wang, Jie Chen, Changai Liu, Junling Luo, Xin Yan, AihuaAi, Yaohui Cai, Hongwei Xie, Xia Ding, Xiaojue Peng PII: DOI: Reference:

S0378-1119(18)31103-X https://doi.org/10.1016/j.gene.2018.10.064 GENE 43320

To appear in:

Gene

Received date: Revised date: Accepted date:

12 June 2018 25 September 2018 22 October 2018

Please cite this article as: Xin Wang, Jie Chen, Changai Liu, Junling Luo, Xin Yan, AihuaAi, Yaohui Cai, Hongwei Xie, Xia Ding, Xiaojue Peng , Over-expression of a protein disulfide isomerase gene from Methanothermobacter thermautotrophicus, enhances heat stress tolerance in rice. Gene (2018), https://doi.org/10.1016/ j.gene.2018.10.064

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ACCEPTED MANUSCRIPT Over-expression of a protein disulfide isomerase gene from Methanothermobacter thermautotrophicus, enhances heat stress tolerance in rice

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Authors: Xin Wang1, Jie Chen1, Changai Liu1, Junling Luo3, Xin Yan1, AihuaAi1, Yaohui Cai2, Hongwei

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Key Laboratory of Molecular Biology and Gene Engineering of Jiangxi

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Address:

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*Corresponding author: Xia Ding and Xiaojue Peng

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Xie2, Xia Ding1*, Xiaojue Peng1*

Province, College of life science, Nanchang University, Nanchang, PR China.

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2. Jiangxi Super-rice Research and Development Center, Nanchang, China 3. Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of

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Agriculture of People's Republic of China, Oil Crops Research Institute, Chinese

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Academy of Agricultural Sciences, Wuhan, China. 86-791-83969537

E-mail:

[email protected]

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Tel:

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Corresponding author. Tel: +86-791-83969537; Fax: +86-791-83969537; E-mail: [email protected]

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Abstract

High temperature (HT) stress is a major environmental stress that limits agricultural

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production worldwide. Discovery and application of genes promoting high temperature

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tolerance is essential to enhance crop tolerance to heat stress. Proteins associated with chaperone and protein folding plays an important role in the high temperature stress

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response of plants. MTH1745 (MtPDI), a disulfide isomerase-like protein (PDI) with a chaperone function and disulfide isomerase activity from Methanothermobacter

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thermautotrophicus delta H, was selected for studying the heat stress tolerance using an

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ectopic expression method in rice. Through molecular identification via quantitative realtime PCR and western blot, we demonstrated that the MtPDI gene was expressed stably in transgenic rice. Heat stress tolerance and survival ratio were significantly improved in

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seedling transgenic rice. At the same time, proline content, superoxide dismutase (SOD) and

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peroxidase (POD) activities were increased in MtPDI transgenic rice with a reduced malondialdehyde (MDA) content. Moreover, increased content of thiols group was

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discovered in transgenic plants. These results indicate that heterologous expression of MtPDI from extremophiles could confer heat stress tolerance of transgenic rice through the

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accumulation of proline content, the synergistic increase of the antioxidant enzymes activity and elevated production of more thiols group, which finally ameliorated the oxidative damage.

Keywords

Heat tolerance; Protein disulfide isomerase; MtPDI; Transgenic rice 2

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ACCEPTED MANUSCRIPT Introduction With industrialization, carbon dioxide level is increasing, temperatures are rising and the resultant global warming is becoming a serious ecological problem. The rise in temperatures, especially in night time temperatures, results in serious impacts on global agricultural production including crop plant growth, development, fertility, and quality (Lobell and

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Asner, 2003).

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Conventional rice breeding has increased the yield potential and yield stability in

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cultivated rice. However, the stress-resistant rice traits, such as heat tolerance, drought tolerance, salt tolerance and herbivore resistance, have not been very successfully improved

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using conventional breeding (Singh and Grover, 2008). Increasing heat stress tolerance will require a combination of genetic engineering biotechnology and improved conventional

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breeding. In response to this challenge, identifying novel genes related to heat stress and developing thermo-tolerance-improved crop plants using various genetic approaches is an alternative strategy. Many studies have shown that over expression of heat shock proteins

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(HSPs) can improve the tolerance of transgenic to heat shock, which can be attributed to its

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function of HSPs in protecting cells against damage from high temperatures by stabilizing and/or re-folding-denatured proteins as molecular chaperones (Queitsch et al. 2000;

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Katiyar-Agarwal et al. 2003; Neta-Sharir et al. 2005; Xu et al. 2011; Kim et al. 2012). Archae constitutes one of the three major domains of life, which is a highly diverse and

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abundant group of organisms that thrive in extreme environments such as hot springs, salt lakes, and volcanic craters, it possesses many unique genes and proteases for stress tolerance (Maupin-Furlow, 2013; Sarmiento et al. 2015). Hyperthermophiles, unusually heatadapted archaea, have potential to tolerate severe high temperature over 100 ℃(Stetter et al. 1993). However, cloning and transformation of heat stress genes from these extremophiles remain limited.

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ACCEPTED MANUSCRIPT PDIs (disulfide isomerase-like protein) are ubiquitously expressed in almost all tissues, which catalyze the formation of disulfide bonds and function as the chaperon (Gruber et al. 2006). Typical PDIs are composed of two approximately 57 kDa subunits in most eukaryotic species (Houston et al. 2005). Molecular structures analysis showed that PDI family proteins have five discrete domains: a, b, b’, a’ and c (Darby et al. 1996). The a and a’ domains share

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high sequence similarity to those of thioredoxin, each containing an active -Cys-Gly-His-Cysmotif (Kemmink et al. 1997). In the anaerobic archaea Methanothermobacter

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thermautotrophicus delta H, protein disulfide isomerase (old_locus_tag="MTH1745",

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locus_tag="MTH_RS08370”, Genbank accession: AAB86215.1) is characterized with 151 amino acid residues and a CPAC active site called MtPDI, exhibiting both molecular

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chaperone and disulfide isomerase activities (Ding et al. 2008). Previous reports have shown that disulfide bonds usually contribute to the stability of a folded protein conformation

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(Darby and Creighton, 1997). Furthermore, the ectopic expression of MtPDI was found to enhance the heat tolerance of Escherichia Coli (E. coli) cells significantly (Ding et al. 2008).

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However, the MtPDI protein ability to enhance the eukaryote organism ability to confer heat

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tolerance is unclear.

In this study, MtPDI gene was optimized to the rice codon usage and introduced into rice. We investigated the response of MtPDI transgenic plants and wild-type (WT) plants to

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heat stress, evaluated the malondialdehyde (MDA) and proline content, superoxide

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dismutase (SOD) and peroxidase (POD) activities, and detected free thiols group in plants. Materials and methods Plant materials and growth conditions The japonica rice variety, Zhonghua 11 were provided by Jiangxi Super-rice Research and Development Center, Nanchang, Jiangxi Province, China. T0, T1, and T2 progeny transgenic plants were grown at 26°C under 16 h of daylight in a greenhouse. Total soil was mixed and

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ACCEPTED MANUSCRIPT equal amount soil were put into the pot to plant the rice. Because the Trans2 plants displayed obvious phenotypic changes to heat stress, the T2 generation of Trans2 seeds from homozygous MtPDI plants were selected for subsequent physiological studies. For hightemperature treatment, 2-week-old Zhonghua 11 and T2 generation seeds of Trans2 MtPDI homozygous seedlings were exposed to 42°C (treatment) or 26°C (control) for 6 h, 8 h, 12 h

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and 24 h, respectively. Survival rate is the percentage of plant still alive after heat stress treatment. Ten independent transgenic plants and 10 Zhonghua 11 were tested in each

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replicate. The leaves were sampled and stored at -80°C immediately after treatment. Heat

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treatment were applied in three independent biological replicates.

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Plasmid construction and rice transformation

To enhance the expression of MtPDI gene in rice, the MtPDI gene was synthesized based on

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the Codon Usage Database that provide by Kazusa DNA Research Institute (http://www.kazusa.or.jp/codon/). The synthesized MtPDI gene was obtained from a commercial service (GenscriptBiotechnology Company, Nanjing, China). The synthesized

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MtPDI gene was digested with BamHI and ligated to the binary vector plasmid pCU (Chen et

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al. 2007).The resulting constructs were designated MtPDI. The japonica rice variety Zhonghua 11 was transformed using Agrobacterium tumefaciens EHA-105, following the

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procedures published by Hiei et al (1994).

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Real-time RT-PCR analysis

Total RNA was isolated from rice seedlings with TRIzol reagent (Invitrogen). Reverse transcription was performed using MMLV Reverse Transcriptase (Invitrogen) according to the manufacturer’s directions. The Osactin gene was used as an internal control for RT-PCR analysis. qPCR was performed with a Rotor-Gene 2000 real-time thermal cycling system (Corbett Research) using the SYBR® Green Real-time PCR Master Mix (TOYOBO). Three independent biological replicates and three technical replicates for each biological replicate

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ACCEPTED MANUSCRIPT were run. Quantitative analysis was performed using the 2(-ΔΔCt) comparative method (Livak and Schmittgen, 2001). The reaction for MtPDI using specific primers (Table S1) was performed at 95°C for 10 s followed by 40 cycles of 95°C for 10 s, 58°C for 10 s and 72°C for 10 s. The reaction for Osactin using specific primers (Table S1) and was performed similarly to MtPDI.

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Western blot analysis

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Total protein was extracted from transgenic seedling and wild type seedlings, following the procedures published by Peng et al (2012). A polyclonal antibody against MtPDI was

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obtained from a commercial service (Beijing Protein Innovation Co., Ltd, China). The

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specificity of the antiserum was tested by western blot against the total protein from thermophilic archaea M. thermautotrophicus delta H, wild type and MtPDI transgenic rice

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(Fig. 1) Equal amounts of protein from independent transgenic rice and wild type rice plants were separated on a 12% SDS-PAGE gel at 4 ℃and were then transferred onto a PVDF transfer membrane at 80 V for 2 h. The membrane was incubated in 5% w/v and non-fat

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milk, with 0.05% v/v Tween-20, in a phosphate buffered saline (PBS) for 1h, washed three

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times in PBST (PBS, 0.05% Tween-20) for 10 min each, and incubated in a 1:1000 dilution of rabbit antiserum overnight at 4°C. After four 10-min washes with PBST, the membrane was

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incubated with goat anti-rabbit IgG conjugated with alkaline phosphatase (AP) in PBST solution for 2h. After four 10-min washes in PBST, the signal was visualized by

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chemiluminescent detection (Pierce, Rockford, IL) according to the manufacturer’s protocol. Determination of proline contents Leaves of the heat-stressed and non-stressed plants were used to determine the free proline. The free proline content was measured using the method described by Bates et al with some modification (1973). Leaves were harvested, weighed (0.2g) and ground with 5.0 ml 3% sulfosalicylic acid to boiling and centrifugation. Then, 200 µl of supernatant was mix

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ACCEPTED MANUSCRIPT with 400 µl of glacial acetic acid and 600 µl of 25% ninhydrin and boiled at 100 ℃for 40 min. After toluene was added, the absorbance was measured at A520. Analysis of the MDA level Lipid peroxidation was estimated by measuring the malondialdehyde (MDA) concentration

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according to Ma et al (2013). Leaf tissues (0.5g) in 1.2 ml of 0.1 (W/V) trichloroacetic acid (TCA) was centrifuged at 12000 rpm for 20 min. An aliquot of the supernatant (0.3 ml) was

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mixed with 0.3ml of 0.5% (w/v) thiobarbituric acid (TBA), incubated at 100 ℃for 20 min,

supernatant were then recorded.

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Determination of antioxidative enzyme activities

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quickly cooled, and centrifuged at 10,000 ×g for 10 min. The A532, A600, and A450 values of the

The activities of antioxidant enzyme were determined by homogenizing 0.5 g of leaf tissue in

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4 ml of extraction buffer containing 1% polyvinylpolypyrrolidone, 50 mM cold phosphate buffer (pH7.8)，1 mM ascorbic acid, and 10% glycerol. The homogenate was centrifuged at

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12,000 rpm for 15 min, and the supernatant was assayed. The SOD activity was measured as

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described by Kumar et al (2008). The POD activity was determined as described by Chen et al (2002).

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Determination of the free thiols group Analysis of the thiols group was performed with Dithionitrobenzoic Acid (DTNB) as described

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in Ellman et al with some modification (1959). Tissues from transgenic seedlings and wild type seedlings were ground into a fine powder in liquid nitrogen, respectively. Then about 200 mg of tissue was diluted with 1M phosphate buffered saline (PBS) combined with 10mM Dithionitrobenzoic Acid (DTNB) and incubated for 20 min at room temperature. After centrifuged at 5000×g for 5min at 4 ℃, the supernatant was collected and immediately

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ACCEPTED MANUSCRIPT assayed. The absorbance was determined at 412 nm. The thiol content was estimated from a Dithiothreitol (DTT) stand curve determined in parallel for each assay. Comparative amino acid sequence analysis The amino acid sequence of rice PDI (GeneBank accession NP_001067436.1) and Mt PDI (GeneBank accession AAB86215.1) were obtained from the National Center for

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Biotechnology Information (NCBI) GeneBank. The protein sequence and domains were

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aligned using Clustal X (version 1.8).

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Statistical analysis

All experimental data were the mean of at least three independent replicates, and

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comparisons between transgenic and WT plants were performed using one-way ANOVA with Duncan’s multiple range test. All the statistical analyses were performed using SPSS

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12.0 software.

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Results

1．Comparison of the PDI units in rice and M. thermautotrophicus

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Putative block-structural comparison between two protein disulfide isomerase (PDI) in rice and the M. thermautotrophicus was carried out (Fig. 1). PDI in rice consists of a four-domain

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structure arranged in the order of a-b-b'-a', which resembles the domain architecture of this protein in other eukaryotic organisms. Regions a, (amino acids 43-146) and a', (amino acid 385-486), the two thioredoxin-like domains possess the same active site sequence of CGHC and show much higher sequence identities with each other (~30%). In addition to these two thioredoxin-like domains, rice PDI contains two additional unclear function domains, b and b', corresponding to amino acids 154-248 and 271-365 (Fig. 1A). The PDI in M.

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ACCEPTED MANUSCRIPT thermautotrophicus consists of 151 amino acids, which are characterized by only a single thioredoxin-like domain possessing a distinct CPAC active site motif (Fig.1B). The full-length and thioredoxin-like domain conversion ratio of the amino acids residues of PDI between rice and M. thermautotrophicus were compared. As shown in Fig.1C, the full length of the amino acid residues of PDI in rice and M. thermautotrophicus share the identity of 18%.

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Furthermore, thioredoxin-like domains are minimally conserved between rice and M. thermautotrophicus, the thioredoxin-like domain of M. thermautotrophicus is 19% identity

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with a domain of rice and 11 % with to the a' domain of rice in amino acid sequence.

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Fig. 1

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2. Molecular characterization of transgenic rice lines

To study the function of the MtPDI in plants, the gene was cloned downstream of a constitutive ubiquitin promoter and was introduced into rice plants via an Agrobacterium

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tumefaciens-mediated transformation. Overall, 30 independent events were obtained and

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28 transgenic events were confirmed first by PCR (data not shown). As showed in Fig S1A, all the T2 progeny transgenic seeds of Trans2 line can grow from hygromycin solution. In

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addition, every T2 progeny transgenic evens of Trans2 were confirmed by PCR (Fig S1B). RTPCR was performed to analyze the expression of MtPDI gene in the T2 progeny homozygous

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transgenic rice. As shown in Fig. 2A, the transcript of MtPDI was detected in the transgenic plants, but not in the wild type plant. These transgenic plants were further detected using real-time RT-PCR, plant trans2 and trans3 exhibited a high level of transgene overexpression. As shown in Fig. 2B, the transcript of MtPDI increased 5 and 6-fold respectively compared with other transgenic plants. OsPDIL1-1 and OsPDIL4-1 as two of the rice PDI gene, qRT-PCR analysis showed that the expression level of OsPDIL1-1 and OsPDIL4-1 were not affected in the transgenic lines (Fig. S2). Moreover, the expression of the transgene at

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ACCEPTED MANUSCRIPT the protein level was assayed by western blot. Western blot profiles of the total proteins extracted from the transgenic plant rice were distinguished by the presence of an 18~KD protein, and which was not detected in the total protein of the wild type plant (Fig. 2C). Fig. 2

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3. Evaluation of MtPDI transgenic rice plants in heat stress The MtPDI transgenic plants exhibited heat stress tolerance. Wild type and transgenic

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plants seedlings at the four-leaf stage were transferred to a growth chamber for 42 ℃heat

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stress treatment. The treatment inhibited growth in both the wild type and transgenic plants, however, the over-expression plants exhibited leaf rolling and wilted later than the

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wild type during the course of the heat stress treatment (Fig. 3). The wild type and transgenic plants exhibited a similar survival rate after 6 hours (h) and 12 h heat stress

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treatment (Fig. 3B, 3C, Fig. 3F), whereas after 18h and 24h heat stress treatment, the survival rate of wild type plants significantly decreased compared with the transgenic

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plants. Approximately 35% of the overexpression plants survived after 18h of heat stress

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treatment, whereas the wild type only exhibited a 3.3% survival rate after the same stress treatment (Fig. 3D, Fig. 3F). After 24h of heat stress treatment, the transgenic plant still exhibited a 20% survival rate, whereas almost all the wild type seedlings died after the

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Fig. 3

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same heat stress treatment (Fig. 3E, Fig. 3F).

4. Determination of MDA and Proline content To test the degree of membrane lipid peroxidation caused by heat stress, the MDA content of the MtPDI transgenic plant and wild type were measured under heat stress and normal conditions. As shown in Fig. 4A, no obvious difference was observed in the MDA content between the transgenic plant and wild type under normal conditions, however, when

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ACCEPTED MANUSCRIPT subjected to heat stress, the MDA content increased in both the MtPDI transgenic plants and wild type plants. However, the rate of MDA content increased in the MtPDI transgenic plant was significantly lower compared with that of wild type. The MDA content in wild type was increased 4.05 times under heat stress after 6h whereas the MDA content in the MtPDI transgenic plant only increased 0.4 times in the same time. Furthermore, the MDA content

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in the PDI transgenic plants was significantly lower compared with that of wild type after heat stress treated at 6h and 12h, respectively.

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Proline changes were investigated in the transgenic plants under heat stress, 2-week-old

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seedlings were transferred to a 42 ℃growth chamber, and sampled at 0 h, 6 h, 12 h, 18 h and 24 h, respectively. As shown in Fig. 4B, the accumulation of proline under normal

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conditions was similar for between WT and transgenic plants. However, under heat stress conditions, the accumulation of proline in MtPDI transgenic plants was significantly higher

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than that in wild type.

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Fig. 4

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5. Enzymatic Activity of an Antioxidative System in Transgenic Plants Under Heat Stress We measured the antioxidant enzymatic activities of SOD and POD under heat stress. As

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shown in Fig. 5A, SOD activity levels were not significantly different between the WT and transgenic rice. When subjected to heat stress, the SOD activity in transgenic plant

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significant increased compares to the wild type plants. The results indicated that POD activities increased in WT and transgenic plants after heat stress. In transgenic plants, the POD activity was significantly higher than that in wild type plants after heat stress treated at 18 h and 24 h (Fig. 5B).

Fig. 5

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ACCEPTED MANUSCRIPT 6. Determination of the free thiols group in MtPDI transgenic rice Thiols play an important role in protein synthesis and the activation of enzymes especially under biotic stress conditions. The PDI protein exhibits thioredox in activities, thus, we determined the thiols content in MtPDI transgenic rice. As shown in Fig. 6, the free thiols

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content increased by 20% in MtPDI transgenic rice compared with that in wild type rice.

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Fig. 6

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Discussion

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The enhancement of heat stress tolerance in crops is an important challenge for food security to facilitate adaptation to global warming. Conventional breeding of cultivated rice

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has generated increasing yield potential and yield stability. However, the stress-tolerance characteristics of rice, such as heat tolerance, drought tolerance, and herbivore resistance, are very limited. There is an urgent need to establish sustainable technologies for increasing

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the stress-tolerance potential of rice. Recently, some plant-stress tolerance-related genes

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have been identified, for example, OsRab7 and OsCA in rice, and LEA3 in soybeans were reported to confer stress tolerance of E. coli cells when expressed in host cells (Liu et al.

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2010; Peng et al. 2011; Peng et al. 2014). It has been indicated that some protective mechanisms might be common in prokaryote and eukaryote under stress conditions (Garay-

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Arroyo et al. 2000).

It is well known that extreme microorganisms, including a number of archaea, could cope with various extreme environmental conditions, which represent an underutilized and innovative source of novel stress resistant genes (Maupin-Furlow, 2013). Whether the genes from archaea can increase the stress tolerance of rice is seldom reported. It was reported that the protein disulfide isomerase (MtPDI) from thermophilic archaea M.

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ACCEPTED MANUSCRIPT thermautotrophicus contributed to heat stress resistance in E. coli (Ding et al. 2008). However, it is still unknown whether MtPDI could contribute to protecting plants from heat stress, therefore we introduced this gene into rice. Our results indicated that overexpression of MtPDI enhanced heat stress tolerance in transgenic rice. Previous reports showed that increased dosage of PDI protein helped the secretion and folding of heterologous protein

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(Bao and Fukuhara, 2001). However, whether the MtPDI protein enhanced heat stress tolerance in transgenic rice via the similar mechanism remains a subject for further

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investigation.

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Putative block-structure indicated that the PDIs in both rice and M. thermautotrophicus have thioredoxin-like domains. However, thioredoxin-like domains are minimally conserved

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between rice and M. thermautotrophicus, the thioredoxin-like domain of M. thermautotrophicus is 19% identity to the domain of rice and 11 % similar to the a ' domain

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in rice. In most eukaryotic species, PDIs catalyzes the formation of disulfide bonds and function as chaperon (Gruber et al. 2006). Adverse environments, such as heat, drought,

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cold and salinity stresses, easily disturb the complex cell physiological processes and leading

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to unfolding or misfolding of proteins. Some PDIs are induced to improve protein folding and transport, which subsequently regulate the protein networks under stresses (Zhu et al. 2014). MtPDI exhibited both molecular chaperone and disulfide isomerase activities (Ding et

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al. 2008), and ectopic over-expression MtPDI protein increased heat stress tolerance in

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transgenic rice. Therefore, we suppose that MtPDI expressing transformants exhibit more effective disulfide bond formation and chaperone activity in a heat-stress environment compared with non-expressing control transformants, which improved rice’s anti-heat-stress ability. It is well known that heat stress generally causes membrane lipid peroxidation and oxidative damage to plant cells, and antioxidant enzymes play an important role in alleviating oxidative damages (Marnett, 1999). We analyzed the heat-resistant physiological 14

ACCEPTED MANUSCRIPT index between the MtPDI trangenic plants and wild type plants. The MDA content was found to be much lower than that of wild type plants under heat stress condition, which suggests that the expression of MtPDI in rice reduced the membrane caused lipid peroxidation caused by heat stress. At the same time, the proline content and antioxidant enzymes activity were observed as being higher than that of wild type plants under heat-stress, which

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may serve as the frontline of defense against oxidative stress. Furthermore, thiols play an important role in protein synthesis and the activation of enzymes especially under biotic

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stress conditions (Foyer and Noctor, 2005). In our study, increased thiols group was found in

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transgenic plants, suggesting that the overexpression of MtPDI may enhance the production of free thiols content. Thus, we suggested that the ectopic expression of MtPDI in rice could

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protect the plant from oxidative damage under heat stress by inducing and increasing antioxidant enzyme activities, proline content, and thiols content.

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Taken together, our results established a basis for further research on rice heat tolerance and provided a potential method for improving the thermo-tolerance in rice

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through the incorporation of the novel stress resistant genes of archaea.

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Acknowledgements This work was supported by National Natural Science Foundation of China (31160270, 31401038, 31760377, 31560041), Project of Jiangxi Provincial Department

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of Education (No. GJJ13102), and State Major Special Science and Technology of Transgene (No.2016ZX08001001-002-008).

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Authors contributions Xiaojue peng designed the study and wrote the manuscript. Xin Wang performed the experiment. Jie Chen, Changai Liu and Aiai Hua helped detection enzymes activity. Hongwei Xie helped planting the rice. Junling Luo and Yaohui Cai helped observing the phenotype of plants. Xia Ding helped analyzing the data, XinYan helped writing this manuscript. All authors read and approved the final manuscript. Conflict of interest The authors declare that they have no conflicts of interest.

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ACCEPTED MANUSCRIPT Compliance with ethical standards This article does not contain any studies with human subjects or animals performed by any of the authors.

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Figure legends Fig.1 A putative block-structural comparison of PDI between rice and M. thermautotrophicus. A, The domain structure and the deduced amino acid sequence of PDI

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in rice. B, The domain structure and the deduced amino acid sequence of PDI in M. thermautotrophicus. C, The multiple sequence alignment of rice PDI and M.

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thermautotrophicus.

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Fig. 2 Expression of the MtPDI gene in transgenic rice plants. A, The MtPDI expression in transgenic plants was determined by reverse transcription analysis. B, Real-time

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quantitative PCR analysis in transgenic plants Values are mean ±SD (n=3). C, Western blot analysis of transgenic rice plant. Trans2 and Trans3, two independent T2 progeny transgenic

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Fig. 3 Heat stress tolerance in wild-type and MtPDI transgenic plants. A-E Morphology of

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wild type and transgenic seedling under normal conditions (A) and after 6 hours (B), 12 hours (C), 18 hours (D) and 24 hours (E) under heat stress. F Analysis of the survival rate of

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wild type and transgenic plant under heat stress. Bars are means of three biological replicates. Each biological replicate was the average data collected from 10 plants each of the WT and Trans2 lines. *, indicates that difference between transgenic and wild type plants is significant (p<0.05).

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ACCEPTED MANUSCRIPT Fig. 4 The MDA (A) and proline (B) content in transgenic and wild type plant under normal and heat stress conditions. Bars are means of three biological replicates. The significant level of the difference between WT and transgenic plants is indicated by *P < 0.05; ** P < 0.01.

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Fig. 5 Active analysis of the antioxidant enzyme of transgenic and wild type plant under normal and heat stress conditions. A, The level of SOD analysis between wild type and

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transgenic plants. B, The level of POD analysis between wild type and transgenic plants. Bars

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and transgenic plants is indicated by *P < 0.05; ** P < 0.01.

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Fig. 6 Free thiols content in the transgenic plant and wild type rice. Bars are means of three biological replicates. *, indicates that the difference between transgenic and wild type plants

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ACCEPTED MANUSCRIPT Abbreviations list

disulfide isomerase-like protein (PDI)

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superoxide dismutase (SOD) and peroxidase (POD) heat shock proteins (HSPs)

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wild-type (WT)

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malondialdehyde (MDA) phosphate buffered saline (PBS)

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Dithionitrobenzoic Acid (DTNB)

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trichloroacetic acid (TCA)

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ACCEPTED MANUSCRIPT Highlights

1. Ectopic expression of MtPDI in transgenic rice. 2. Heat stress tolerance and survival ratio were significantly improved in seedling

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transgenic rice.

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3. Ectopic expression of MtPDI in rice could protect the plant from oxidative damage under heat stress by inducing and increasing antioxidant enzyme activities, proline

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content, and thiols content.

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