Overexpression of Rice Metacaspase, OsMC4, Increases Endoplasmic Reticulum Stress Tolerance in Transgenic Rice Calli

: Endoplasmic reticulum (ER) is an important organelle responsible as protein synthesis regulator in plant. High salinity can also lead to the activation of ER stress, caused by the accumulation of misfolded protein. This could lead to a stress response mechanism, unfolded protein response (UPR). Failure of UPR to reverse the effect of protein misfolding will activate Programmed Cell Death (PCD). Metacaspase genes regulate programmed cell death (PCD) in plants. The present study was focused on comprehensive gene analyses of the expression patterns of type II rice metacaspase ( OsMC ) genes in response to the endoplasmic reticulum (ER) and salinity stress in rice leaf and OsMC4 in callus. A strong evidence of unfolded protein response (UPR) during tolerance to both ER and salinity stress was found in the present study. Overexpression of OsMC4 in rice callus as a fusion protein with TagRFP and controlled by the CaMV35 promoter caused major changes in the expression of the stress ER-marker genes, protein disulfide isomerase ( PDI ) and Binding immunoglobulin Protein ( BiP ) , and OsMC4 in overexpressing calli. These expression analyses of the OsMC family provide valuable information for further functional studies on the biological roles of OsMC s in PCD related to ER and salinity stress responses. Author Contributions: Conceptualization, Z.M.S. and N.F.M.Y.; methodology, Z.A.S., Z.A.R., S.Y.S., and N.F.M.Y.; validation, Z.M.S., Z.A.S., N.A.S., and N.F.M.Y.; formal analysis, N.F.M.Y.; investigation, N.F.M.Y and N.F.S.; resources, Z.M.S., Z.A.S., Z.A.R.; data curation, N.F.M.Y.; writing — original draft preparation, N.F.M.Y.; writing — review and editing, Z.M.S., P.G., M.A.M.R., N.A.A.R., and N.F.M.Y.; visualization, N.F.M.Y.; supervision, Z.M.S., Z.A.S., and N.A.S.; project administration, Z.M.S.; funding acquisition, Z.M.S., N.A.S. All authors have read and agreed to the published version of the manuscript.


Introduction
Endoplasmic reticulum (ER) is a factory for protein synthesis in the cells. Folding of nascent peptides, packaging and delivering them to the targeted placement in the body are part of the ways in which this large organelle controls the protein homeostasis network [1,2]. When the capacity in the ER is overloaded, and the demand for protein folding increases, there will be distruption in the ER homeostasis [2]. This will cause excessive accumulation of unfolded protein in the ER which leads a situation referred as ER stress [3]. There are various stress responses that could occur together and linked to the ER [4].
One of the tolerance mechanisms in plants during ER stress is unfolded protein response (UPR), which is activated upon the accumulation of misfolded proteins in the ER [ 5]. Excessive accumulation of misfolded proteins can trigger a stress response in plant first demonstrated in Norway spruce, Picea abies. The metacaspase gene of Picea abies, mcII-Pa was found to have a very crucial function in making sure that cell degeneration, cell proliferation and terminal differentiation during embryogenesis are properly balanced [20].
Genome-wide characterizations in plant species revealed that metacaspases families are made up of multiple genes in plants. For instance, Arabidopsis has nine metacaspases [21,22], while eight members were found in potato (Solanum tuberosum L.) [23]. Through various molecular and phylogenetic studies, two different types of plant metacaspases, type I and type II, differentiated by their protein structures have been identified. Type I metacaspase is characterized by an N-terminal extension prodomain from 80-120 amino acids in length and two CxxC-type zinc finger domains. It is easy to differentiate between type I and type II metacaspases since the prodomain and domain are totally absent in type II metacaspases [22]. Both types, however, possess a p20 and p10 subunits in their C-terminal regions. These regions can still be differentiated between the two since the subunits in type I is slightly shorter than the one in type II [24]. In order to understand the critical role of metacaspases in plants, and how they involve in growth and stress responses, various gene functional studies were done by gene overexpression or silencing. For example, when Metacaspase 9 (Camc9-C) in pepper (Capsicum annuum) was silenced, it was found that the cell death was 30% higher in control plants as compared to the plant with silenced Camc9-C [25]. Meanwhile, when Metacaspase 8 in Arabidopsis thaliana (AtMC8) was overexpressed, upregulation of PCD was observed in the cells after being induced with oxidative stress, while seeds and seedlings with knocked out AtMC8 showed increased tolerance towards herbicide methyl viologen. This suggested that AtMC8 was involved in PCD pathway when exposed with oxidative stress [26].
There are eight members of metacaspases in rice (OsMC) [24,27]. Type I metacaspases in rice are OsMC1-3 and type II metacaspases are OsMC4-8. In this study, the focus will be given to OsMC4 as one of type II metacaspase since type II metacaspases are similar to effector caspases in animals, which functions to execute PCD by cleaving target substrates within cells [24,28,29]. Previous studies in rice metacaspases were focused on abiotic stresses such as salinity and drought, and biotic stresses such as fungal infection. We performed gene expression analysis specifically on type II metacaspase gene in response to ER stress to study where it belongs in the UPR pathway and how rice respond to this stress. Gene expressions during plant response towards salinity stress were also analyzed since salinity was tightly related to inducing ER stress in plants [7].

Expression Patterns of ER-stress markers, PDI and BiP, in Response to ER and Salinity Stress under Various Concentrations
To analyse the gene expression analysis for metacaspases in rice and study its relation to ER and salinity stress tolerance in rice, BiP and PDI were used as stress markers in this study to confirm the ER stress-induced in rice. UPR can be induced in the lab by adding ER-stress inducers to plant tissues such as tunicamycin (TM), dithiothreitol (DTT) and cyclopiazonic acid (CPA) [30]. Salinity stress is induced by adding Sodium Chloride (NaCl) to rice tissues [31]. In this study, salt stress is used as a mean to induce ER stress and tunicamycin was used to induce ER stress in rice by inhibiting the N-glycosylation process and disrupting the post-translational modifications of glycoproteins [30].
qPCR analysis was first done for the expression of type II OsMCs in rice leaves after being subjected with ER stress for 3 hours under five different concentrations to test which concentration induces a strong and consistent ER stress in rice, hence should be used for further experiments. To characterize the intensity of the stress induced, two stress markers, BiP and PDI were included in the analysis. As shown in Figure 1A, the expression of BiP and PDI in rice leaves treated with tunicamycin was significantly induced by up to two-fold with the 5µg/mL and 15 µg/mL doses. Since salinity and drought stress were reported [7,8] to induce ER stress in rice plants, salinity stress was induced in rice leaves as a mean to induce ER-stress. ER-stress induction was also done directly via treatment with tunicamycin to study how these stresses correlate with each other. As shown in Figure 1B, the expression of BiP and PDI were most upregulated when rice leaves were induced with 150 mM NaCl for 3 hours. The upregulation of stress markers indicated that our trial tests were sufficient for further analyses. 5 µg/mL of tunicamycin was observed to be enough to induce ER stress in leaves tissues and higher concentrations used did not show any significant difference to the amount of stress observed in rice leaves. Meanwhile, 150 mM NaCl was observed to bring significant changes to the stress level in rice leaves. Hence, rice leaves were further subjected to 5 µg/mL tunicamycin and 150 mM NaCl under various time points to induce ER stress and salinity stress on rice leaves tissues, respectively. Figure 1. Expression of PDI and BiP in rice leaf after 3-hour ER stress treatment with various concentrations of (A) tunicamycin (µg/mL) (B) NaCl. The expression patterns of PDI and BiP in both treatments were calculated with UBQ5 reference genes and shown as fold change of transcript level after setting untreated as 1. Data presented are the means ± SD from three independent experiments and letters above the bars indicate significant difference at p<0.05 when analyzed using one-way ANOVA with Tukey's post-test.

Expression Patterns of ER-stress markers, PDI and BiP, and OsMCs in Response to ER Stress under Various Time Points
Expression patterns of type II OsMCs in the leaves under ER stress were analysed after being subjected to 5 µg/mL tunicamycin for 3, 6, 9, 12, 24 and 48 hours. As shown in Figure 2B, a single peak pattern was seen on the transcript level of OsMC4, OsMC5, OsMC6, OsMC7, and OsMC8 after 6 hours of the rice leaves being subjected to ER stress. The same patterns could also be observed with the transcript level pattern for PDI ( Figure 2A). All the expression analyses peaked after 6 hours of treatment and then gradually decreased as shown in Figure 2A and 2B. OsMC8 showed the most significant increase in expression after 6 hours of treatment ( Figure 2B). About 100-fold change could be observed after 6 hours, then the expression gradually decreased to 20-fold change after 9 hours and significantly decreased after 12 to 48 hours of treatment. The expression pattern of PDI increased up to 60-fold change after 6 hours of treatment showing that the plant is substantially under a lot of stress ( Figure 2A). Given the PDI expression pattern, a single peak for the expression of BiP was expected to occur at 6 hours of treatment, but instead was observed after 9 hours of ER stress ( Figure 2B).

Figure 2. Expression of (A) ER stress markers PDI, BiP and (B)
Type II OsMCs in rice leaf after treatment with 5 µg/mL of tunicamycin to induce ER stress at 6 time points. The transcript levels of PDI, BiP and OsMCs were all calculated using UBQ5 as a reference gene and shown as fold change with the untreated sample set as 1. Data presented are the means ± SD from three independent experiments and letters above the bars indicate significant difference at p<0.05 analyzed using one-way ANOVA with Tukey's post-test.

Expression Patterns of ER-stress markers, PDI and BiP, and OsMCs in Response to Salinity Stress under Various Time Points
Since salinity was reported to be able to induce ER stress [2], rice leaves were also subjected to 150 mM NaCl to induce salinity stress in rice and observe the relationship between salinity and ER stress. Rice leaves were layered with a thin coat of 150 mM NaCl for 3, 6, 9, 12, 24 and 48 hours. As shown in Figure 3A, the expression levels of BiP and PDI were observed to have a single peak after 6 hours of treatment and decreasing after 9 up to 48 hours of treatment. We can see up to 8-fold changes on PDI and up to 10-fold changes on BiP, 6 hours after treatment as compared to the corresponding control without stress treatment. As shown in Figure 3B, up to 2-fold changes were observed on the gene expression of OsMC8 after 6 hours of treatment. The expression of OsMC8 then gradually decreased up to 48 hours of treatment. Similar patterns could be observed in the expression of OsMC4 and OsMC5, where a single peak could be seen on both genes after 6 hours of treatment before the gene expression started oscillating up to 48 hours of treatment. By contrast, fluctuations and irregular patterns were observed for OsMC7, while OsMC6 is downregulated by the salt treatment. reference genes and are shown as folds of transcript level. Data presented are the means ± SD from three independent experiments and letters above the bars indicate significant difference at p<0.05 analysed using one-way ANOVA with Tukey's post-test.

Expression Patterns of ER-stress markers, PDI and BiP, and OsMC4 on Wild Type and OsMC4 Overexpressing Rice Calli in Response to ER Stress
Interesting patterns were observed on the expression pattern of OsMC4, OsMC5 and OsMC8, warranting further investigation. In this study, we report on the overexpression of OsMC4 in rice callus. The OsMC4 gene construct was successfully amplified with the correct expected size after PCR, this gene was then used for cloning using the pGWB561 vector to express OsMC4 as a fusion protein with TagRFP at the C terminus under the control of the CaMV 35 promoter.
Approximately eight weeks after transformation in cultivated rice callus via Agrobacterium tumefaciens, and after being subcultured biweekly in selection media containing 250 µg/mL cefotaxime, 250 µg/mL carbenicillin and 50 µg/mL hygromycin, positive transformants were recovered. Positive transformants were then treated with ER and salinity stress and subsequently being analysed for gene expression analysis. Transformation was confirmed using fluorescent testing with fluorescence microscope (ZEISS) since overexpressing calli comes with TagRFP fusion, giving them green fluorescence feature visible under fluorescence microscope.
Expression levels of PDI, BiP and OsMC4 were measured and calculated using UBQ5 as a reference gene. Data was obtained after expression and calculation using three biological replicates. As shown in Figure 4A and 4B, the expression levels of BiP and PDI were observed to decrease significantly from 16-fold and 90-fold highest expression for PDI and BiP in wild type, respectively, to only up to 2-fold changes in gene expression for both genes in overexpressing calli. However, a similar single peak pattern in the gene expression pattern of PDI and BiP observed in rice leaf could also be seen after 6 hours of treatment on the wild type rice calli. By contrast, the expression levels of OsMC4 saw a huge difference between wild type and overexpressing calli, as expected ( Figure 4A and 4B). The expression pattern fluctuates throughout the experiment but there was a significant difference in fold change between wild type and overexpressing calli with only 7-fold changes as the highest gene expression in wild type and 30-fold changes in overexpressing rice calli ( Figure 4A and 4B). Comparing the results in wild type and overexpressing calli, the gene expression for stress marker genes, PDI and BiP was relatively high when OsMC4 gene expression was low in wild type. Inversely, in overexpressing calli, PDI and BiP gene expression was observed to be low when OsMC4 gene expression was high in overexpressing calli. Interestingly enough, the highest expression of OsMC4 corresponded with the increase in expression of PDI and BiP from 9 to 24 hours after treatment in overexpressing calli. In wild type, the decrease in PDI and BiP gene expression also corresponded to the decrease in OsMC4 gene expression from 6 to 12 hours after treatment.

Expression Patterns of ER-stress markers, PDI and BiP, and OsMC4 on Wild Type and OsMC4 Overexpressing Rice Calli in Response to Salinity Stress
To explore if salinity stress would give the same effect towards gene expression on the wild type and the overexpressing calli, the same gene expression analysis was done for salinity stress after rice callus was subjected to 150 mM NaCl for 3 up to 48 hours of treatment. As shown in Figure 5A and 5B, the expression levels of BiP and PDI were observed to decrease significantly from 30-fold and 45-fold for PDI and BiP as the highest expression in wild type, respectively, to only lower than 2-fold changes in PDI and 4-fold in BiP in overexpressing calli. In contrast, the highest expression of OsMC4 in wild type was only at 6-fold compared to 50-fold in overexpressing calli. The strong difference in the transcript level of the three gene expression could also be seen in ER stress in Figure 4A and 4B.  type and (B) overexpressing rice callus after treatment with 150 mM NaCl to induce salinity stress under 6 various time points. Expression patterns of PDI, BiP and OsMC4 in this treatment were all calculated with UBQ5 reference genes and are shown as folds of transcript level. Data presented are the means ± SD from three independent experiments and letters above the bars indicate significant difference at p<0.05 analysed using one-way ANOVA with Tukey's post-test.

Ion leakage analysis after the introduction of ER and salinity stress in rice
Ion leakage assay is a cell death assay done to further prove the condition of the cell during stress treatment. Under stressful condition, as a defence mechanism, cell will start dying and losing their cell membrane integrity, releasing electrolytes in response to the stress. Ion leakage assay is done to quantify the amount of electrolytes released during cell death [18]. As shown in Figure 6A and 6B, when applied with 5 µg/mL tunicamycin to induce ER stress and 150 mM NaCl to induce salinity stress, the ion conductivity level was overall higher in wild type compared to overexpressing calli. The highest reading for ion conductivity post-ER stress as shown in Figure 6A was up to 700 µS/cm in wild type and 600 µS/cm in overexpressing calli. As for salinity stress in Figure  6B, the highest reading after plant was exposed to salinity stress was 1200 µS/cm in wild type and 800 µS/cm in overexpressing calli. This corresponds to the difference in PDI and BiP expression under both stresses in overexpressing calli and wild type.
(A) (B) Figure 6. Quantification of rice plant cell death by ion leakage assay. Reading of ion leakage (µS/cm) recorded after (A) ER stress and (B) Salinity stress were induced in wild type and overexpressing calli for 3, 6, 9, 12, 24 and 48 hours, respectively. Data presented are the means ± SD from three independent experiments. Rice was treated with 5 µg/mL tunicamycin to induce ER stress and 150 mM NaCl to induce salinity stress.

Discussion
The role of metacaspases in rice has been extensively discussed in a myriad of abiotic stress research [31]. However, the information of its role in ER stress in rice was limited as compared to other plants such as tomato [5], and Arabidopsis [32]. Role of metacaspases in salinity stress in rice is also quite new. Tunicamycin blocks glycosylation in the ER [30], while environmental stress like salinity stress could also lead to ER stress in plants [33].
The results observed in this study showed that type II metacaspases, especially OsMC4, might play a role in reducing ER and salinity stress in rice. Nonetheless, various responses were observed from each metacaspase. Some are more upregulated than the other after the stress was induced, and some are downregulated throughout the experiment. This showed that from all type II metacaspases, not all of them are directly involved in regulating this type of stress. Different types of metacaspases might involve in different types of defence mechanisms. For example, OsMC1 in rice was reported to be upregulated when rice samples were infected with blast fungus and high concentration of salicylic acid but were downregulated when pathogenic Rhizoctonia solani was introduced [31]. In this study, OsMC8 was reported to be significantly upregulated after rice leaf was being subjected to ER stress ( Figure 2B), but the gene expression was observed to be much lower after the rice leaves were introduced with salinity stress ( Figure 3B). Therefore, the upregulation of OsMC8 might imply its involvement in plant responses towards ER stress than salinity stress.
Two genes, PDI and BiP were used in this study as ER-stress marker genes. When plants are exposed to ER stress, BiP will be disassociated from ER stress sensors to initiate UPR pathway in response towards ER stress [6,7], while PDI catalyzes disulfide bonds formation or breakage during protein folding to aid in protein folding in plants during exposure to stress [10]. In this study, the upregulation of UPR-targeted genes after 6 hours of treatment for most genes and 9 hours for PDI which resulted in a single peak on gene expression patterns were an indicator that UPR pathway was activated in rice. Rice activates its defence mechanism towards ER stress by initiating the UPR pathway [34]. This was supported by the high stress level shown by the stress marker genes BiP and PDI after 6 hours of ER and salinity treatment on rice leaves but gradually decreased right after ( Figure 2B and 3B). ER stress and salinity stress can induce the upregulation of BiP transcription [35]. Certain rice metacaspases genes showed similar patterns after 6 hours of treatment. This is the case for OsMC4, OsMC5, OsMC6, OsMC7 and OsMC8 in rice leaves for ER stress ( Figure 2B) but only OsMC4, OsMC5 and OsMC8 for salinity stress ( Figure 3B). However, in the subsequent analysis using rice calli as tissue samples, for wild type and overexpressing calli, the peaks on 6 hours were mostly observed on the wild type, rather than the overexpressing calli (Figure 4 and 5). This shows how rice leaves and callus might have similar responses towards ER and salinity stress although they are both different types of tissues.
The peak for the ER-stress markers, BiP and PDI at the time points during gene expression may have been an indication that the rice leaves and callus were initially under a lot of stress, and that the stress increases in the first hours of the experiment. Subsequently, the expression gradually decreased for most of the metacaspases genes, suggesting that protein misfolding might have successfully be resolved by UPR-targeted genes ( Figure 2B and 3B). However, the expression of OsMC7 in salinity stress as shown Figure 3B, starts increasing again after the single peak pattern, and continued towards the end of the experiment. This might imply that this gene was dealing with a different stress in which the plants started releasing more OsMC7 into the system. The sudden drop in the expression of most genes after the stress treatments on rice leaves as shown in Figure 2 and 3 (A and B) might also indicate that plants were either adapting or switching to PCD to stop the rescue mechanism, whereby cells started killing itself. This could be seen in ER stress in rice leaves, whereby the expression in BiP and PDI gradually increased after the single peak, showing the stress level was increasing, and more UPR-targeted genes were released, although the OsMCs for that treatment started to decrease after the peak (Figure 2A and 2B).
A much stronger UPR and a boost in the expression of BiP could be influenced by imperfection or deformities found in the UPR signalling pathway [30]. However, in this study, the overexpression of OsMC4 has caused a much reduced expression of BiP and PDI genes, after treatment of overexpressing calli with ER. The callus with overexpressed OsMC4 showed much higher expression of OsMC4 gene in overexpressing calli. In wild type, the highest expression of OsMC4 was only 7-fold but in overexpressing calli, the highest OsMC4 expression was 30-fold ( Figure 4A and 4B). Similarly, during salinity stress, the highest OsMC4 expression was 6-fold in wild type and 50-fold in wild type ( Figure 5A and 5B). The overexpression of OsMC4 gene in rice plants might have helped increase their tolerance towards ER stress and salinity stress.
Overexpression of metacaspases genes in other plants has been demonstrated to help relieve stress under extreme abiotic stress. For example, the overexpression of AtMC8 in Arabidopsis has shown to be helpful in PCD induced by ultraviolet light and H202 [26], while silencing of metacaspases gene could hinder plants from reacting properly towards stress. In this study, overexpression of OsMC4 showed difference in the relative expression of BiP and PDI in both gene expression analysis and ion leakage assay. The ion conductivity reading in wild type is higher than in overexpressing calli. This also corresponds with the difference in PDI and BiP expressions under both stresses in overexpressing calli and wild type. Lower ion conductivity reading was observed for both BiP and PDI in overexpressing calli compared to wild type. Thus, in this study, overexpression of OsMC4 has helped increase rice tolerance towards both ER and salinity stress.

Plants Materials and Growth
MR219 Rice (Oryza sativa) of indica rice hybrid by Malaysian Agricultural Research and Development Institute (MARDI) were used for gene expression analyses of Metacaspase genes on Endoplasmic Reticulum (ER) stress and salinity stress. The rice plants were grown under natural lighting and weather and were watered every 2-3 days for approximately 2-3 weeks.

Treatments for Endoplasmic Reticulum Stress
Two-weeks old MR219 seedlings were submerged or layered with a thin coat of various concentrations of tunicamycin to test which concentration gives out the most stable and comparable gene expression in response to ER stress. Rice seedlings were subjected to five different concentrations of tunicamycin (5 µg/mL, 10 µg/mL, 15 µg/mL, 20 µg/mL and 25 µg/mL) for 3 hours. Each tunicamycin was prepared with 0.02% Tween20 and diluted in DEPC-treated water (Invitrogen). After gene expression analyses, rice seedlings were subjected to ER stress using 5 µg/mL as the most suitable concentration for 6 different time points (3 hours, 6 hours, 9 hours, 12 hours, 24 hours and 48 hours). The same treatment was then done to induce ER stress to wild type and overexpressing calli. Callus was submerged or layered with a thin coat of 5 µg/mL of tunicamycin solutions prepared with 0.02% Tween 20.

Treatment for Salinity Stress
The treatment was started with a treatment trial of 2-3 weeks old seedlings using 4 different concentrations of Sodium Chloride (NaCl); 50 mM, 150 mM, 250 mM and 500 mM diluted with DEPC water and 0.02% Tween20. After gene expression analyses, rice seedlings were treated with 150 mM NaCl as the most suitable concentration for 6 different time points (3 hours, 6 hours, 9 hours, 12 hours, 24 hours and 48 hours). The same treatment was then done to induce salinity stress to wild type and overexpressing. Callus was submerged or layered with a thin coat of 150 mM NaCl solutions prepared with 0.02% Tween 20.

RNA extraction and cDNA conversion
All treated samples were collected and extracted using TRIsure ™ (Bioline, UK). RNA purification was done using RNeasy plant mini kit (QIAGEN). All RNA samples were stored at ̶ 80 oC. The purified RNA was retro-transcripted into cDNA using the RT2 first strand kit (QIAGEN). cDNA samples were then ready for qPCR analysis.

qRT-PCR Analysis for Gene Expression
qPCR analysis was done using SensiFAST SYBR Hi-Rox Kit (Bioline, UK) on a StepOne Plus ™ machine from Applied Biosystems with a total volume of 10 µL. A total of 9 primers set were used for qPCR analysis post-treatment with ER and salinity stress. Two housekeeping genes; UBQ5 (accession number AK061988) and eEF1α (accession number AK061464) [36], two ER-stress marker; BiP (accession number AF006825) and PDI (accession number AB039278) [37], and 5 type II Metacaspases;, OsMC4, OsMC5, OsMC6, OsMC7 and OsMC8 [31], genes were used for this analysis. Two housekeeping genes were used at first but focus was given to UBQ5 for final analysis.

Statistical analysis
The data were analysed by one-way ANOVA and Tukey's tests (p < 0.05) using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). qRT-PCR data were analysed using the 2−ΔΔCt method.

Molecular Cloning and Overexpression of Metacaspase Gene
To over express OsMC4, gene-specific primer was used for the first PCR to amplify the gene using DNA as the starting material. The construct was then cloned into a pGEM vector and then sent for sequencing. Sequencing results showed that a correct gene construct was obtained, hence, Gateway Entry clone was made by adding attB1 and attB2 primer sequences to the extracted DNA by PCR. PCR amplification products were then ligated into special plasmids called Gateway donor vectors, Gateway TM pDONR TM 221 Vector (Invitrogen) by adding BP Clonase enzyme mix. Our destination vector was pGWB561 (Nakagawa lab, Japan) with TaqRFP fusion. A cloning construct for OsMC4 was successfully obtained and it was then cloned into the destination vector and transformed into callus via Agrobacterium tumefaciens strain EHA105.

Plant Transformation
Callus cultivation was done in preparation for callus transformation. Firstly, seeds were peeled to expose the embryos and were plated on T1 media plates (Murashige & Skoog medium with B5 vitamins, L-Arginine, L-Glutamine & L-Asparagine). A several seed sterilization techniques were carried out to reduce the contamination rate. Firstly, seeds were left under running water for one hour before seeds were soaked and shaken in a solution containing fungicide and Tween-20 for 30 min. This step was repeated twice. After an hour, seeds were soaked and shaken in another solution containing Virkon and Tween-20 for 30 min. After that, seeds were transferred to a solution of pure Ethanol and treated for a minute. Lastly, seeds were introduced to another solution containing 70% Clorox and Tween-20 for another 30 min. Seeds were lastly rinsed 3 times with distilled water and left to dry up to 3 hours before being plated in the T1 media. Callus started to grow after 14-20 days. After the transformation of OsMC4 into rice callus, callus was transferred into co-cultivation media for 3 days. Then, callus was transferred into selection media containing 250 µg/mL cefotaxime, 250 µg/mL carbenicillin and 50 µg/mL hygromycin for selection. The overexpressing calli was subcultured into fresh selection media every two weeks and positive transformants were selected after 2-3 months. Positive transformants were visibly lighter in shades since negative transformants die in selective media with antibiotics. Positive transformants were confirmed with fluorescent testing under fluorescence microscope (ZEISS).

Ion Leakage
Ion leakage analysis was done as a cell death assay. Ion conductivity was measured after samples were treated with 5 µg/mL tunicamycin for ER stress or 150 mM NaCl for salinity stress for 3, 6, 9, 12, 24, 48 hours using compact conductivity meter LAQUAtwin-EC-11 (Horiba Ltd). 100 mg rice callus were submerged in 1 mL DEPC-treated water (Invitrogen) water in 24-well plate at room temperature for one hour prior to ion conductivity measurement after stress treatment.water for one hour. Three replicates were done for each selected time point. The conductivity meter was calibrated with standard KCl solution.

Conclusions
In order to increase our understanding of the functions of OsMCs, the present study was focused on the possible involvement of type II OsMCs especially OsMC4 in ER stress and how salinity stress was related to ER stress through comprehensive analysis of the expression changes in rice leaves and rice callus treated with both stresses. Our data first displayed how rice metacaspases might be involved in the UPR pathway during plant response to stressful condition. Three metacaspase genes, OsMC4, OsMC5 and OsMC8 showed highest gene expression 6 hours after stress treatment in rice leaf before sudden drop in gene expression which might have been related to activation of plant rescue mechanism during protein misfolding. Meanwhile, OsMC4, might play a very important role in tolerance towards ER and salinity stress after its overexpression in this study helped to increase rice tolerance towards both stresses. Overexpression of this gene significantly reduced the level of BiP and PDI expression in rice, indicating that stress level is relatively lower in the successfully overexpressing calli. Results from the analysis of the OsMCs members provide clear evidence that metacaspase, particularly the OsMC4 reduces ER and salinity stress in rice. Further functional analysis of OsMCs genes in ER and salinity stress tolerance should be done in the future to create higher stress tolerance in transgenic rice.