Overexpression of Orange Gene (OsOr-R115H) Enhances Heat Tolerance and Defense-Related Gene Expression in Rice (Oryza sativa L.)

In plants, the orange (Or) gene plays roles in regulating carotenoid biosynthesis and responses to environmental stress. The present study investigated whether the expression of rice Or (OsOr) gene could enhance rice tolerance to heat stress conditions. The OsOr gene was cloned and constructed with OsOr or OsOr-R115H (leading to Arg to His substitution at position 115 on the OsOr protein), and transformed into rice plants. The chlorophyll contents and proline contents of transgenic lines were significantly higher than those of non-transgenic (NT) plants under heat stress conditions. However, we found that the levels of electrolyte leakage and malondialdehyde in transgenic lines were significantly reduced compared to NT plants under heat stress conditions. In addition, the levels of expression of four genes related to reactive oxygen species (ROS) scavenging enzymes (OsAPX2, OsCATA, OsCATB, OsSOD-Cu/Zn) and five genes (OsLEA3, OsDREB2A, OsDREB1A, OsP5CS, SNAC1) responded to abiotic stress was showed significantly higher in the transgenic lines than NT plants under heat stress conditions. Therefore, OsOr-R115H could be exploited as a promising strategy for developing new rice cultivars with improved heat stress tolerance.


Introduction
Global warming is expected to significantly reduce crop yields due to high temperatures that negatively affect plant growth and development. Thermal stress can eventually lead to cell damage and death in plants due to accumulation of excess reactive oxygen species (ROS), membrane lipid peroxidation and metabolic disturbance [1,2]. Under elevated temperature, plants have shown physiological symptoms such as scorching of leaves and stems, leaf abscission and senescence, shoot and root growth inhibition or fruit damage, which lead to a decreased productivity and quality of crops [3]. Until now, numerous heat stress defense genes have been reported to mainly encode signaling molecules such as mitogen activated protein kinase (MAPK) cascades, cellular protective enzymes such as peroxidase (POD), transcription factors such as heat shock transcription factors (HSFs), and cellular stress proteins such as heat shock proteins (HSPs) [4][5][6][7]. These genes have been reported to protect plant cells by enhancing resistance with maintaining ROS homeostasis in the cell or regulating various genes on the signal pathway under thermal stress [6,7]. It was reported recently that the function for carotenoids relates to the response of plants to environmental stress [4,5]. In addition, reactive oxygen species (ROS) generated under stress conditions oxidizes carotenoids and converts into various oxidation products including aldehydes, ketones, endoperoxides, and lactones [4]. This demonstrates that carotenoid accumulation and potential signaling in plant cells plays a positive role in heat stress response. The Orange (Or) gene encoding a cysteine-rich DnaJ protein has been reported to regulate carotenoid accumulation and abiotic stress resistance in various plant species [8][9][10][11][12][13][14][15][16]. The Or protein functions as a holdase chaperone, a post-transcriptional regulator of phytoene synthase (PSY), and a rate-limiting enzyme on the carotenoid biosynthetic pathway [12,17]. Moreover, Or protein has been reported to interact with oxygen-evolving enhancer protein 2-1 (PsbP) in the chloroplast and protect it from heat-induced denaturation [17,18]. An allele carrying a single nucleotide polymorphism (SNP), in which the 96th amino acid of the Or protein substituted arginine (Arg) to histidine (His), is associated with high-carotene accumulation in melon [9]. In addition, overexpression of AtOr protein (Q8VYD8; AtOr-R96H) leads to high level accumulation of carotenoids in Arabidopsis and tomatoes (Solanum lycopersicum) [13,19]. In the sequence comparison of the Or gene between the low-carotenoid and high-carotenoid lines in carrots (Daucus carota), SNP substituted from T to C has been identified, and it is reported that a codon TTG (leu) is changed to TCG (ser), resulting in high carotene accumulation [20]. In a recent experiment, we have found out that the site-directed mutagenesis of IbOr gene in sweet potato (Ipomoea batatas [L.] Lam), leading to Arg to His substitution at position 96 (based on the CmOr protein) in the IbOr protein (IbOr-R96H), showed significantly higher carotenoid contents and enhanced environmental stress tolerance [21,22]. In this study, to understand the physiological role of OsOr gene in rice, the carotenoid content and abiotic stress tolerance were investigated in overexpressing plants, OsOr-WT and OsOr-R115H. Our results show that in the overexpressing plants, OsOr-WT and OsOr-R115H, carotenoid content was not changed compared to NT plants, but heat stress tolerance was enhanced. In addition, these lines showed a high level of transcription of genes related to ROS scavenging enzymes under stress conditions. Interestingly, the OsOr-R115H transgenic line showed lower malondialdehyde (MDA), lower relative electrolyte leakage (REL), and higher proline content in leaves compared with NT plants.

Growth Conditions and Stress Treatments
Dongjin (Oryza sativa L.ssp. japonica) was used as a wild-type, cultivated in greenhouse facilities at Hankyong National University, Korea, as described by Jung et al. [23]. The expression levels of OsOr were investigated in roots, leaves, stems, panicles, and immature seeds at 7 days after pollination in Dongjin cultivar. The expression pattern of the OsOr in response to abiotic stresses including 48 • C heat, 200 mM NaCl, and 20% PEG6000 drought 100 µM GA 3 , 100 µM ABA, and 4 • C cold was monitored as previously reported by Park et al. [17]. Abiotic stress treatments were performed for 0 h, 3 h, 6 h, 12 h, and 24 h, and then samples were collected and stored at −80 • C. All experiments included three biological replicates.

Gene Cloning and Generation of Transgenic Rice
The full-length cDNA of OsOr (Os02g0651300) was amplified with specific primer sets for RT-PCR (Supplementary Table S1). The PCR product was cloned into pMD18-T vector (TAKARA, Seoul, Korea) and sequenced. Site-directed mutagenesis was performed to obtain point mutants by substituting H for the 115th amino acid R of the OsOr gene as previously described by Kim et al. [22]. Briefly, OsOr-R115H, which replaced the ORF (Open Reading Frame) (OsOr-WT) and major SNPs of the OsOr gene, was subcloned downstream of the binary vector pGWB5 expressed by the cauliflower mosaic virus (CaMV) 35S promoter, respectively. The constructed vector was transformed into Agrobacterium tumefaciens strain EHA105, which was introduced by infection with rice embryogenic callus, as previously described [24]. The transformed callus and plants were selected using 6 mg/L phosphinothricin and confirmed by PCR analysis, as previously reported [24]. Positive transgenic plants were first screened by PCR, and then further confirmed by TaqMan PCR to selection of single copy insertion of T-DNA and qPCR analysis to determine the expression of the transgene. In addition, the transgenic homo lines of T 3 generation were used for the analytical experiment.

Phenotype Analysis
Pollen viability was evaluated as previously described [25] from transgenic plants with OsOr gene and wild-type plants at flowering time for pollen germination analysis. Nikon SMZ800N microscope (Nikon Metrology Inc., Seoul, Korea) was used to observe pollen germination. The amylose content of whole grain was measured using the AMY-LOSE/AMYLOPECTIN kit (Megazyme Ltd., Bray, Ireland) according to the manual provided by the manufacturer. The morphology of starch grains was observed by scanning electron microscopy (SEM). Images were captured using a Quanta FEG 450 instrument (ZFE, Graz, Austria). Xanthomonas oryzae pv. oryzae (Xoo) inoculation and determination of bacterial populations were evaluated as previously described [26,27] for a wild-type plant and transgenic plants. In addition, 30 plants were randomly selected from each of the transgenic lines and NT plants, and the seed setting rate, pollen fertility, 1000-grain weight, and yield per plant were investigated.

Analysis of Carotenoid Contents
Carotenoids were extracted from rice leave tissues using 0.01% solution of butylatedhydroxytoluene in acetone and analyzed using an Agilent 1100 HPLC system (Hewlet-tePackard, Palo Alto, CA, USA) according to the method described by Lim et al. [28].

Analysis of Heat Tolerance of Transgenic Rice
To induce heat stress, leaf discs were excised from the fourth leaves of 2-week-old transgenic and NT rice plants and treated to high temperature (48 • C) in the dark for 24 h.
To measure tissue levels of H 2 O 2 and visualize the degree of damage caused by heat stress, leaf discs were immersed in 3,3'-diaminobenzidine (DAB) solution (1 mg/mL, pH 3.8) for 5 h at 25 • C under continuous light, as described previously [18]. To measure the extent of cellular damage or membrane disruption, ion leakage was quantified using an ion conductivity meter (MTD, Schwerzenbach, Switzerland). For quantitative analysis of total area of DAB staining region, images were processed using ImageJ.

Physiological Parameters Measurements of Transgenic Rice
To investigate whether the transgenic lines can be used as oxidative stress biomarkers, the relative electrolyte leakage (REL), proline, and malondialdehyde (MDA) content were detected based on the previously described methods. Briefly, 500 mg of rice leaves were pulverized in 2 mL of the chilled reagent (0.25% (w/v) thiobarbituric acid in 10% (w/v) trichloroacetic acid). The extracts were treated at 100 • C for 30 min, cooled at room temperature, and centrifuged at 12,000× g for 15 min. The absorbance of the supernatant was measured at 450, 532, and 600 nm. The MDA content was calculated according to the formula: 6.45 × (OD 532 − OD 600 ) − 0.559 × OD 450 [29][30][31].

qRT-PCR Analysis
Total RNA was isolated from different tissues of rice plants using a RNeasy plant mini kit (Qiagen, Seoul, Korea, www.qiagen.com) respectively, and single-strand cDNA was synthesized with random oligonucleotides using a reverse transcription system (Bioneer, www.bioneer.co.kr, E-3122 (M-MLV Reverse Transcriptase) based on a previously reported method [18]. Primers used in the experiment are listed in Supplementary Table S1. qRT-PCR analysis was performed according to the manufacturer's instructions using Light-CyclerR 480 Real Time PCR system (Roche, CA, United States). "Relative expression" levels were calculated using the 2 −∆∆CT method, and OsActin (Q10DV7) was used as a reference gene in rice. All experiments included three biological replicates, each with two technical replicates.

Statistical Analysis
Data collected in this study were analyzed by one-way analysis of variance (ANOVA) using the Statistical Analysis System (SAS version 9.4). Values are mean ± SE (n = 3) and statistical significance was set to p < 0.05 according to Duncan's multiple range test [32].

Expression Profile of OsOr Gene
The full-length cDNA of OsOr was cloned into PMD18-T vector after PCR analysis using gene specific primer sets, and confirmed by sequencing. This gene consists of a 996 bp ORF encoding a putative protein of 332 amino acids, and GenBank's accession number is AK099767 (Supplementary Figure S1A,C). The OsOr protein had an estimated molecular mass of 34.3 kDa and pI of 8.36 (Supplementary Figure S1C). The genetic similarity between the sequences of OsOr and Or genes of various plant species showed the highest homology with the Or gene of morning glory (Ipomoea nil) (TA6874_35883) and was a 97% identity at the amino acid level (Supplementary Figure S1B). In addition, OsOr showed 73-80% sequence homology between several plant-derived Or genes, including the putative Or genes of tomato (L. esculentum), grape (Vitis vinifera), Arabidopsis thaliana (At5g61670), and cauliflower (B. oleracea var. botrytis) (Supplementary Figure S1C). The domain annotation of the OsOr protein contained a motif with two transmembrane domains, a plastid-targeting transit sequence, and a repeating cysteines (CxxCxGxGx) characteristic of the DnaJ protein known as a chaperone. To determine the expression pattern of OsOr in rice, we analyzed the expression profile of OsOr gene in roots, stems, leaves, panicles, and immature seeds using qRT-PCR. The results showed that OsOr gene was expressed in all tissues tested, and this gene expression was highly detected in stems and lowly detected in immature seeds ( Figure 1A). In addition, we investigated the expression pattern of the OsOr gene in response to H 2 O 2 , PEG, heat, cold, ABA, and GA 3 stresses. The results suggested that H 2 O 2 , PEG, heat, and cold stresses except GA 3 stress induced the expression of OsOr at all treatment time points ( Figure 1B). Under H 2 O 2 , PEG, and cold stress conditions, the expression level of OsOr was rapidly induced from the early stage and showed the plateau expressions for 3-24 h following the treatment. Under heat stress condition, the expression level of OsOr was rapidly induced, and gradually increased to 24 h of treatment. In addition, the cold stress condition slightly significantly induced the expression of OsOr, but the GA 3 stress conditions did not show significant difference in the OsOr expression level. Therefore, the results of expression profile for OsOr indicated that OsOr gene might be important for plant tolerance against H 2 O 2 , PEG, heat, and cold stresses.

Phenotypic Analysis of Transgenic Rice
To further study the function of the OsOr gene, we constructed OsOr-WT and OsOr-R115H under the control of the CaMV 35S promoter and introduced them in rice embryogenic callus using an Agrobacterium-mediated method (Figure 2A). To select transgenic plants with a single copy gene, ten T0 transformants with OsOr-WT and OsOr-R115H were tested through PCR analysis on whether T-DNA was inserted as well as a TaqMan PCR analysis. Four independent T3 homozygous transgenic lines, #7 and #9 for OsOr-WT, and #6 and #10 for OsOr-R115H, were selected and used for subsequent analysis (Supplementary Figure S2; Figure 2B).  The expression level of OsOr gene was found to be statistically significantly higher in transgenic lines (#7, #9, #6, #10) than in NT plants ( Figure 2C). In addition, the flower structure and pollen germination of transgenic plants were shown similar to those of NT plants ( Figure 3A). In addition, the transgenic lines and NT plant showed similar responses to disease in the inoculation experiment with Xanthomonas oryzae pv. oryzae (Xoo) strain ( Figure 3B). In order to investigate the morphological characteristics of rice grain in seed cross-sections, the results of scanning electron microscope (Quanta FEG 450, FEI Electron Microscopy) analysis showed that the seeds of the transgenic lines were almost similar to those of the NT plants ( Figure 3C). As a result of examining starch content and amylose content of transgenic lines, there were no significant differences from NT plants ( Figure 3D). In addition, statistical analysis showed that no significant differences were detected in transgenic lines (#7, #9, #6, #10) compared to NT plants, including shoot length, root length, seed setting rate, pollen fertility, 1000-grain weight, and yield per plant (Figure 4). Taken together, our results showed that overexpression of OsOr-WT and OsOr-R115H was not significant phenotypic alterations compared to NT plants. In addition, the carotenoid content between the transgenic lines and NT plants was analyzed using high performance liquid chromatography (HPLC) analysis. The results were almost similar in the investigated lines (Supplementary Figure S3), implying that the OsOr gene was not directly affect to the carotenoid contents in rice [33].

Overexpression of OsOr-R115H Enhances Heat Stress Tolerance
To examine whether OsOr plays a role in heat stress tolerance, we investigated heat tolerance of the transgenic lines (OsOr-WT (#7, #9), and OsOr-R115H (#6, #10)) together with WT plants. As shown in Figure 5A, no significant morphological differences of leaf discs were observed between transgenic and NT plants before heat stress treatment. However, after incubation at 48 • C for 24 h, NT plants showed severe membrane damages, as evident from the dark-brown coloration of leaf discs, whereas OsOr-WT and OsOr-R115H leaf discs displayed less membrane damage. OsOr-R96H transgenic plants showed greater heat stress tolerance than NT and OsOr-WT plants ( Figure 5A). Our data showed that the oxidized DAB contents of NT leaves at 12 h after heat stress was 21.3 mg/g DW, significantly higher than those of transgenic lines (the oxidized DAB contents of OsOr-WT #7, #9, OsOr-R115H #6 and OsOr-R115H #10 lines were 10.9, 9.2, 8.7, and 7.7, respectively) ( Figure 5B). In addition, we determined the proline and chlorophyll contents in the leaves from transgenic lines and NT plants. Our results showed no difference in proline and chlorophyll contents between NT plants and transgenic lines grown under normal conditions. On the other hand, the proline and chlorophyll contents of the transgenic lines (OsOr-WT and OsOr-R115H) after heat stress were significantly higher than NT plants ( Figure 6A,B). Recently, it has reported that MDA and electrolyte leakage were strongly correlated with the degree of cell membrane damage under abiotic stress [34]. Thus, we measured the MDA and REL content from transgenic and wild type plants. The data displayed no obvious difference between transgenic lines and NT plants grown under normal conditions. However, the MDA and REL contents of OsOr-WT and OsOr-R115H transgenic plants were significantly lower than NT plants grown under heat stress conditions ( Figure 6C,D). As a result of analyzing carotenoids after heat stress in the transgenic plants with OsOr-WT and OsOr-R115H, the carotenoid content was slightly decreased compared to the results under normal conditions after 24 h of heat stress treatment. However, there was no difference between NT plants and overexpression plants. These results suggest that the carotenoid accumulation pathway in rice is due to differences from that of sweet potatoes.

Transcription Analysis of Stress-Related Genes
To illustrate the regulation mechanism underlying the enhanced heat stress tolerance, we measured the expression of four genes encoding ROS scavenging enzymes (OsCATA, Os-CATB, OsAPX2, OsSOD-Cu/Zn) and five genes (OsLEA3, OsDREB2A, OsDREB1A, OsP5CS, SNAC1) related to abiotic stress tolerance under normal growth and heat stress conditions using qRT-PCR (Figures 7 and 8). There was no significant difference in the expression of all genes investigated under normal growth conditions. By contrast, the expression of all genes was remarkably higher in OsOr-WT and OsOr-R115H plants than in NT plants after heat stress. These results show that OsOr overexpression induces proteins involved in ROS scavenging pathways and defense mechanisms, thereby reducing oxidative damage and improving heat resistance.

Discussion
Plant orange (Or) proteins contain an N-terminal unknown region, transmembrane domains, and a C-terminal DnaJ-like domain, and meaningful progress has been made in understanding the biological function of the orange genes [8]. Some orange (Or) genes such as BoOr (ABH07405), IbOr (KX792094), CmOr (A0A0D3MU35), AtOr (Q8VYD8), SIOr (NP_001315338), and DcOr (KZN00418) showed resistance to various environmental stresses as well as carotenoid accumulation [8][9][10][11][12][13][14][15][16]. However, the clear biological functions of these genes are still unclear and require further studies. A single nucleotide substitution in the gene encoding CmOr is correlated with the orange color fruit. This substitution results in an Arg to His change at the 108th amino acid of CmOr [9]. In previous report, overexpression of AtOr His (R90H), SbOr His (R104H), and IbOr His (R96H) with mutations at the site corresponding to the mutation in CmOr (golden SNP altering Arg to His) resulted in high total carotenoid levels and β-carotene accumulation [13,22,35]. In a sequence alignment, we found that OsOr possessed Arg at the 115th amino acid, corresponding to the 108th position of CmOr, 90th position of AtOR and 96th position of IbOr. Thus, to examine whether the substitution of the conserved Arg to His in OsOr promoted carotenoid accumulation, site-directed mutagenesis of OsOr-WT was performed to generate OsOr-R115H. In this study, we report the isolation, characterization, and physiological function of the orange (Or) gene from rice. Our results show that in the overexpressing plants, OsOr-WT and OsOr-R115H, carotenoid content was not changed compared to NT plants, but heat stress tolerance was enhanced. In addition, we did not find significant differences between OsOr-WT and OsOr-R115H. The reason why OsOr-R115H did not have a significant effect on carotenoids, unlike sweet potatoes, is thought to be due to a different mechanism of carotenoid accumulation, which is consistent with that previously reported by Yu el al. [33]. Evidence has shown that some Or genes are induced or inhibited expression by abiotic stress, and overexpression of these genes can increase the tolerance of transgenic plants to heat [8]. Similarly, our results showed rapid changes of OsOr gene expression in heat and PEG stress (Figure 1), suggesting OsOr might play a critical role in heat and PEG stress. In addition, overexpression of the golden SNP-carrying mutant IbOr allele was resulted to have enhanced abiotic stress tolerance as well as carotenoids accumulation [8]. In addition, Yu et al. (2021) reported that salt and cold stress resistance was reduced in Nipponbare rice plants overexpressing the OsOr gene [33]. Therefore, we constructed OsOr gene (OsOr-WT and OsOr-R115H) for overexpressing transgenic plants and tested their heat stress tolerance. Our data suggested that the overexpression of OsOr-WT and OsOr-R115H enhanced heat stress tolerance, and transgenic plants showed less membrane damage and aging compared to NT plants in Dongjin rice (Figures 5 and 6). In general, when plants encountered abiotic stresses such as heat and salt, REL, proline, and MDA can react quickly to survive in extreme environmental conditions [34,36]. Therefore, the REL, an important parameter reflecting the degree of cell membrane damage, was significantly lower in the leaves of the transgenic plants than in the leaves of NT plants ( Figure 6D). These results suggested that the OsOr gene might protect cell membrane integrity of plants in response to heat stress. Mellacheruvu et al. [37] have reported that abiotic stress on plants causes lipid peroxidation, resulting in MDA accumulation. Our data displayed that the MDA content was higher in NT plants than in transgenic plants with OsOr-WT and OsOr-R115H ( Figure 6C), which shows that heat stress has more damage to NT plants compared to transgenic plants. Furthermore, proline has been reported to act as a stabilizer to protect plants from abiotic stress damage [38]. In our data, the proline of the transgenic plant with OsOr-WT and OsOr-R115H accumulated more compared with NT plants under heat stress conditions. These results suggested that proline might be responsible for enhancing heat stress tolerance shown by the transgenic plants overexpression OsOr. These results showed that enhanced heat stress tolerance of the transgenic plants with OsOr-WT and OsOr-R115H was at least partially related to reduced MDA content, reduced REL, and increased proline content. In addition, biochemical factors such as MDA, REL, etc. mentioned above have also contributed to significantly inducing expression of abiotic stress-response genes [39,40]. In our study, the expression level of four genes related to ROS-scavenging enzymes (OsCATA, OsCATB, OsAPX2 and OsSOD-Cu/Zn) and six genes in response to abiotic stress (OsLEA3, OsDREB2A, OsDREB1A, OsP5CS, SNAC1) were more highly expressed in the transgenic lines (OsOr-WT #7, OsOr-WT #9, OsOr-R115H #6 and OsOr-R115H #10) than in NT plants under heat stress conditions (Figures 7 and 8). Sirko et al. [41] reported that the function of OsCATA gene plays a role in protecting plants from damage caused by reactive oxygen species (ROS). It has been demonstrated that there is a positive correlation between the expression of OsP5CS gene and the accumulation of proline, and overexpression of OsP5CS increases resistance to abiotic stress in transgenic plants [42]. In addition, OsLEA3, OsDREB2A, OsDREB1A, and SNAC1 genes were reported to be sensitive to abiotic stress [43][44][45][46][47]. It was reported that the plant hormone ABA is produced by five 9-cis-epoxycarotenoid dioxygenases (NCEDs), and strigolactones are synthesized by the enzymes CCD7 and CCD8 [48]. However, because of examining the expression of OsOr gene, it was slightly upregulated by ABA stress, and there was no difference in carotenoid content in transgenic plants. Taken together, the results showed that the transgenic lines further enhanced heat stress tolerance, perhaps by strengthening the expression levels of genes related to ROS-scavenging enzymes and responding to abiotic stress under heat stress conditions. The current study also demonstrated that the OsOr gene could serve as a candidate for breeding heat stress tolerant varieties in rice. Future research should further explore OsOr gene regulatory network and functionality using CRISPR/Cas9 system mediated base-editing technique, etc., in greater depth to unravel the mechanism for controlling heat stress tolerance in rice.  Figure S2: TaqMan PCR analysis for copy number assays using TaqMan probe for single copy selection in T0 transgenic Rice. +; single copy, -; multi copy, T 3 -homozygous and T 3 -heterozygous; single copy control, NC; negative control, NT; Non-transgenic control. Figure S3: Contents and composition of carotenoids in the leaves of Or gene transgenic plants relative to NT plants in normal condition(A) and 24h heat stress condition(B). Carotenoid levels determined by HPLC analysis of leaf tissues harvested from three independent transgenic plants for each construct. Data represent mean ± SD of three independent replicates (p < 0.05; One-way ANOVA, followed by Duncan's post hoc test). Table S1: Primers used for qRT-PCR analysis in this study.