Insights on the enhancement of chilling tolerance in Rice through over-expression and knock-out studies of OsRBCS3

ABSTRACT

Chilling stress is an important environmental factor that affects rice (Oryza sativa L.) growth and yield, and the booting stage is the most sensitive stage of rice to chilling stress. In this study, we focused on OsRBCS3, a rice gene related to chilling tolerance at the booting stage, which encodes the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit in photosynthesis. The aim of this study was to elucidate the role and mechanism of OsRBCS3 in rice chilling tolerance at the booting stage. The expression levels of OsRBCS3 under chilling stress were compared in two japonica rice cultivars with different chilling tolerances: Kongyu131 (KY131) and Longjing11 (LJ11). A positive correlation was found between OsRBCS3 expression and chilling tolerance. Over-expression (OE) and knock-out (KO) lines of OsRBCS3 were constructed using over-expression and CRISPR/Cas9 technology, respectively, and their chilling tolerance was evaluated at the seedling and booting stages. The results showed that OE lines exhibited higher chilling tolerance than wild-type (WT) lines at both seedling and booting stages, while KO lines showed lower chilling tolerance than WT lines. Furthermore, the antioxidant enzyme activities, malondialdehyde (MDA) content and Rubisco activity of four rice lines under chilling stress were measured, and it was found that OE lines had stronger antioxidant and photosynthetic capacities, while KO lines had the opposite effects. This study validated that OsRBCS3 plays an important role in rice chilling tolerance at the booting stage, providing new molecular tools and a theoretical basis for rice chilling tolerance breeding.

KEYWORDS:

• Rice
• OsRBCS3
• chilling tolerance
• Over-expression
• Knock-out

Introduction

Rice is one of the most important staple food crops, providing the main energy source for approximately half of the world’s population.¹ However, rice production is constrained by various abiotic stress factors, especially chilling stress, which is one of the major limiting factors for rice growth and yield.² Chilling stress affects rice growth and development, photosynthesis, the antioxidant system, hormone balance and other physiological processes, resulting in adverse consequences such as rice growth retardation, chlorosis, malformation, spikelet sterility and yield reduction.^3,4 The booting stage is the most sensitive stage of rice growth to chilling stress, which causes male sterility, anther cracking, pollen viability decline, pollination and fertilization disorders.^5,6 Heilongjiang province is the northernmost region of China. The annual mean temperature is between −5°C and 4°C from north to south. It is the province with the lowest temperature in China, and it is the northernmost cold rice-growing area in China and even in the world. Low temperatures are the number one factor limiting safe rice production in Heilongjiang, especially at the booting stage.^7,8 Therefore, improving rice chilling tolerance at the booting stage is of great significance for ensuring food security and adapting to climate change.

Rice chilling tolerance is a complex quantitative trait influenced by multiple genes and environmental factors. Using molecular biology and genomics techniques, cloning and identifying rice chilling-related genes, revealing their mechanisms and regulatory pathways, provide a theoretical basis and molecular tools for breeding rice varieties with high chilling tolerance.^9–11 Rubisco is the most important enzyme in photosynthesis and is involved in the fixation and reduction of carbon dioxide.¹² Rubisco consists of large subunits (RBCL) encoded by chloroplast genes and small subunits (RBCS) encoded by nuclear genes.¹³ In rice, five OsRBCS gene family members (OsRBCS1-OsRBCS5) have been identified. Previous studies have shown that over-expression of OsRBCS3 in rice can increase the content and activity of the Rubisco holoenzyme in leaves and thus improve the photosynthetic efficiency of rice.^14,15 On the other hand, silencing of OsRBCS2, OsRBCS3, OsRBCS4, or OsRBCS5 by the RNAi method resulted in decreased Rubisco content in rice leaves at different growth stages,¹⁴ indicating that all four genes contribute to the accumulation of Rubisco holoenzyme regardless of the growth stage and that the suppression of individual OsRBCS gene expression cannot be fully complemented by other OsRBCS genes. Among the five OsRBCS genes, OsRBCS1 has a very low expression level,¹⁶ while OsRBCS3 has been found to be down-regulated under chilling stress,¹⁷ suggesting that OsRBCS3 may play a crucial role in the rice chilling response. However, no functional validation or mechanistic analysis of the OsRBCS3 gene in rice chilling tolerance has been performed. This study aimed to fill this gap and explore the role and mechanism of the OsRBCS3 gene in rice chilling tolerance at the booting stage.

This study used two japonica rice cultivars with contrasting chilling tolerances as materials: Kongyu131 (KY131), a japonica rice cultivar with strong chilling tolerance, and Longjing11 (LJ11), a representative chilling-sensitive cultivar in Heilongjiang Province.^6,18,19 Our previous study confirmed OsRBCS3 as a chilling-responsive gene by DGE (Digital Gene Expression Tag Profiling) analysis of KY131 and LJ11.¹⁸ This study validated its expression under chilling stress by qPCR. Over-expression and knock-out lines of the OsRBCS3 gene were also constructed using over-expression and CRISPR/Cas9 technology, respectively, and their chilling tolerances at the seedling and booting stages were evaluated. The impacts of OsRBCS3 gene over-expression or knock-out on rice antioxidant capacity and photosynthetic capacity at the booting stage were also measured. This study provides new molecular markers and strategies for rice chilling tolerance breeding.

Material and methods

Plant materials and growth conditions

Two Oryza sativa L. japonica, cv. cultivars, Longjing11 (LJ11) and Kongyu131 (KY131), were obtained from the Rice Research Institute of Heilongjiang Academy of Agricultural Sciences (Jiamusi, China). LJ11 is a chilling-sensitive cultivar that shows approximately 94% spikelet sterility under chilling stress during the booting stage.¹⁹ KY131 is a strongly chilling-tolerant cultivar.⁶

Generation of OsRBCS3 over-expression transgenic lines

The full-length coding sequence of OsRBCS3 was cloned and inserted into the pGWB12 vector (Supplementary Figure S1a) using Gateway technology and transformed into LJ11 by Agrobacterium-mediated genetic transformation to generate OsRBCS3 over-expression (OE) plants. The pGWB12 vector was kindly provided by the Northeast Institute of Geography and Agroecology of the Chinese Academy of Sciences. Positive transgenic lines were confirmed by PCR followed by sequencing. All plants were grown in the greenhouse or field at the Hulan Rice Experimental Base of Heilongjiang University.

Generation of OsRBCS3 knock-out mutants

The OsRBCS3 gene was edited by CRISPR/Cas9 gene editing technology in KY131 to generate OsRBCS3 knock-out (KO) mutants. The Cas9/gRNA vector (Supplementary Figure S1b) was purchased from ViewSolid Biotech (No. VK-005-01). Edits were verified using PCR and Sanger sequencing with primers designed by Primer Premier 6.0 software (Supplementary Table S1). Positive KO mutants were used for phenotypic analysis. All plants were grown in the same conditions as OE plants.

qPCR analysis

Flag leaves of different rice lines were used to extract total RNA with TRIzol reagent (Takara). A NanoDrop-2000C (NanoDrop) was used to measure the RNA quantity. Following the manufacturer’s instructions, a ReverTreAce ® qPCR RT Kit (Toyobo) was used to synthesize cDNA from RNA. A 7500 real-time PCR system (Applied Biosystems) performed the qPCR using SYBR ® Green PCR master mix (Toyobo). The results used the 2^−∆∆Ct method²⁰ and OsActin as an internal reference gene. Primer Premier 6.0 software was used to design the qPCR primers (Supplementary Table S1).

Evaluation of chilling tolerance

Seedling-stage chilling tolerance assay. Plants were subjected to cool air treatment.²¹ Seeds of the LJ11, OE, KY131 and KO lines were dried at 45°C for 3 days to break seed dormancy and synchronize seed maturity. Seeds were then soaked in sterile distilled water for 5 days at 4°C to equalize seed moisture content. Seeds were germinated in a constant temperature incubator at 28°C for 7 days, and uniform seedlings were selected and sown at equal distances in white porcelain plates. Each material had 30 plants in the experimental group and 30 plants in the control group. The plants were grown at 25°C with 12 h of light per day until the three-leaf stage and then transferred to an artificial climate chamber for 4 days of chilling stress treatment at 4°C. After the treatment, the condition of the seedlings was checked, and the dead seedlings were removed, leaving only the seedlings with uniform growth. The survival rate of each line was measured after the chilling treatment.

The booting stage chilling tolerance assay. All plants were subjected to standard management practices that included cultivation, irrigation, fertilization, and disease control at the Hulan Rice Experimental Base of Heilongjiang University. LJ11, OE, KY131 and KO plants were cultured to the booting stage and then moved to an artificial climate chamber for the chilling tolerance assay. Thirty plants per line were used for both the experimental and the control groups. The plants were treated at 15°C with 12 h light per day for 7 days and then moved to a greenhouse for normal recovery until rice maturity. The average seed setting rate of the main panicle was investigated to evaluate chilling tolerance.

Physiological traits under chilling stress

Flag leaves on day 0 and day 7 of the chilling stress treatment were collected for physiological determination as described in section 6.5. The samples (50 mg) of frozen flag leaves were ground with a precooled mortar in 50 mM potassium phosphate buffer (pH 7.8) containing 1% polyvinylpyrrolidone. The homogenate was spun at 15,000 × g for 20 min at 4°C, and the supernatant was used for enzyme assays. SOD activity was measured by the photochemical method of NBT as described²² with slight modifications. The reaction mixture contained 0.05 mL of crude enzyme extract, 1.5 mL of phosphate buffer solution, 0.3 mL of methionine, 0.3 mL of NBT, 0.3 mL of EDTA-2Na, 0.3 mL of riboflavin solution and 0.25 mL of distilled water. The absorbance of the mixture at 560 nm was recorded, and SOD activity was defined as the amount of enzyme needed to inhibit 50% of NBT reduction.

POD activity was determined by a previously reported method.²³ The reaction mixture consisted of 0.9 mL of guaiacol solution, 1 mL of H₂O₂, 1 mL of PBS and 1 mL of enzyme extract. The absorbance change of the brown guaiacol product at 460 nm was used to calculate POD activity. CAT activity was assessed by a previously reported method²⁴ with slight changes. The reaction mixture included 3 mL of PBS, 0.6 mL of H₂O₂ and 0.1 mL of enzyme extract. CAT activity was estimated as the decrease in absorbance at 240 nm for 1 min. MDA content was measured by the method reported²⁵ with slight alterations. In brief, rice flag leaves weighing 0.3 g were collected and homogenized in 5 mL of trichloroacetic acid containing 0.25% thiobarbituric acid. The mixture was heated for 30 min at 95°C and then cooled in ice water and spun at 10,000 × g for 20 min. The absorbance of the supernatant at 440 nm, 532 nm and 600 nm was recorded to determine the MDA content.

Rubisco activity in leaves

Rubisco enzyme activity was measured by the enzyme extract assay²⁶ using a microplate reader (Biotek Instruments) at a wavelength of 340 nm. Fresh leaf tissue (1 g) was homogenized in 0.25 M Tris – HCl buffer (pH 7.8) containing 2% DTT, 0.0025 M EDTA and 0.05 M MgCl₂. The homogenate was centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was mixed with 100 M Tris – HCl buffer (pH 8.0) containing 40 mM NaHCO₃, 10 mM MgCl₂, 5 mM DTT, 0.2 mM EDTA, 4 mM ATP, 0.2 mM NADH, 0.2 mM ribulose 1,5-bisphosphate, and 1 U each of 3-phosphoglycerate kinase and glyceraldehyde 3-phosphodehydrogenase.

Results

OsRBCS3 gene expression differed significantly under chilling stress in different japonica rice cultivars

To investigate the role of the OsRBCS3 gene in rice chilling tolerance, the expression levels of the OsRBCS3 gene under chilling stress were compared in two japonica rice cultivars, LJ11 and KY131, which have different levels of chilling tolerance. The relative expression levels of the OsRBCS3 gene in flag leaves at the booting stage were quantified by qPCR at five time points: 0 h, 2 h, 5 h, 12 h and 24 h. As shown in Figure 1, the expression level of the OsRBCS3 gene was similar between LJ11 and KY131 at 0 h, but it diverged significantly as the chilling stress time increased. In LJ11, a chilling-sensitive cultivar, the OsRBCS3 gene showed a weak response to chilling stress, and its expression level remained relatively stable. It increased by 2.07-fold, 2.33-fold and 1.67-fold at 2 h, 12 h and 24 h, respectively, compared to that at 0 h. In KY131, a chilling-tolerant cultivar, the OsRBCS3 gene exhibited a rapid and strong response to low temperature and showed a trend of increasing first and then decreasing. It increased by 2.97-fold at 2 h, reached a peak value of 3.44-fold at 12 h, and then decreased to 2.13-fold at 24 h relative to that at 0 h. These results suggest that the expression level of the OsRBCS3 gene is positively correlated with rice chilling tolerance and differs significantly under chilling stress in different japonica rice cultivars.

Figure 1. Expression of OsRBCS3 in flag leaves at the booting stage of rice cultivars under chilling stress.

To rule out the possibility that the sequence variation of the OsRBCS3 gene affects its expression level, the coding sequence (CDS) of the OsRBCS3 gene in LJ11 and KY131 was sequenced and aligned. The results revealed that the CDS sequence of the OsRBCS3 gene was identical between the two cultivars (Supplementary Figure S2), indicating that there was no sequence difference. This suggests that the difference in the expression level of the OsRBCS3 gene under chilling stress in different japonica rice cultivars is mainly attributed to transcriptional regulation rather than sequence variation.

The relative expression levels of the OsRBCS3 in the LJ11 and KY131 lines were measured by qPCR and normalized to LJ11 at 0 hour (set as 1). Error bars represent the SD (n = 5). LJ11 is a chilling-sensitive cultivar, and KY131 is a chilling-tolerant cultivar. HAC, hours after chilling treatment. ** indicate highly significant differences in comparison with KY131 at P  < 0.01.

Over-expression or knock-out of the OsRBCS3 gene changed rice chilling tolerance at the booting stage

To further investigate the role of the OsRBCS3 gene in rice chilling tolerance, we constructed overexpression (OE) and knockout (KO) lines of this gene using the Agrobacterium-mediated genetic transformation method (Supplementary Figure S3). We used LJ11 and KY131 as backgrounds for OE and KO lines, respectively, and obtained the T1 generation plants after positive identification and field cultivation. The expression level of OsRBCS3 was measured in flag leaves at the booting stage by qPCR. The results showed that the OE lines had 6.51-fold higher expression of OsRBCS3 than the wild-type (WT) line LJ11 (Figure 2a). The OsRBCS3 gene was successfully knocked out in KO lines (Figure 2b,c), as confirmed by sequencing results that KO lines showed a single base insertion at the target site, resulting in a frameshift mutation and premature termination of the coding region (Figure 2d). No off-target effects were found in KO lines for the other four family genes (OsRBCS1, OsRBCS2, OsRBCS4 and OsRBCS5) (Supplementary Figure S4). These results indicate that OE and KO lines of the OsRBCS3 gene were successfully generated.

Figure 2. Relative gene expression of OsRBCS3 and CRISPR/Cas9 editing in rice cultivars.

Next, the effects of the OsRBCS3 gene on rice chilling tolerance at the seedling and booting stages were investigated. Rice seedlings of wild-type and transgenic lines were subjected to chilling stress at 4°C for 4 days, followed by recovery at a normal temperature. The survival rate of each line was then measured and compared. The results showed that the OE lines had a significantly higher survival rate than the LJ11 lines (p < 0.01), indicating that their seedling-stage chilling tolerance was significantly improved (Figure 3a,c). In contrast, the KO lines had a significantly lower survival rate than the KY131 lines (p < 0.01), indicating that their seedling-stage chilling tolerance was significantly reduced (Figure 3a,c). At the booting stage, we exposed rice plants to chilling stress at 15°C for 7 days, then transferred them to a greenhouse until maturity and measured their seed setting rate. The results showed that OE lines had a significantly higher seed setting rate than LJ11 lines (p < 0.01), increasing by 131.05%, indicating that their booting-stage chilling tolerance was improved (Figure 3b,d). In contrast, the KO lines had a significantly lower seed setting rate than the KY131 lines (p < 0.01), decreasing by 51.32%, indicating that their booting-stage chilling tolerance was reduced (Figure 3b,d). These results demonstrate that over-expression of OsRBCS3 gene can enhance rice chilling tolerance at the booting stage, while knock-out of the OsRBCS3 gene can impair rice chilling tolerance at the booting stage.

Figure 3. Effects of chilling stress on the survival rate and seed setting rate of four rice cultivars (LJ11, OE, KY131, and KO).

(a) The relative mRNA expression levels of OsRBCS3 in LJ11 and OE were measured by qPCR and normalized to the expression level in LJ11 (set as 1). Values are mean ± SD (n = 5; ***p < 0.001, Student’s t-test). (b) Sequencing result of the CRISPR/Cas9 target site in wild-type rice (KY131). (c) Sequencing result of the CRISPR/Cas9 target site in the KO line, showing a single base insertion at the arrow position. (d) Amino acid sequence alignment of OsRBCS3 between wild-type and knockout rice lines. The OsRBCS3 CDS sequences in KY131 and KO were Sanger sequenced, translated into amino acid sequences based on the codon table, and aligned. LJ11 is a chilling-sensitive cultivar, OE is an OsRBCS3-overexpressing line derived from LJ11, KY131 is a chilling-tolerant cultivar, and KO is an OsRBCS3-knockout line derived from KY131 using CRISPR/Cas9 technology.

(a, c) Survival rate of rice seedlings exposed to 4°C for 4 days. (b, d) Seed setting rate of rice plants exposed to 15°C for 7 days and then transferred to a greenhouse until maturity. Values are mean ± SD (n = 30). Letters within each sample refer to one-way ANOVA tests (p < 0.01, Duncan test). LJ11 is a chilling-sensitive cultivar, OE is an OsRBCS3-overexpressing line derived from LJ11, KY131 is a chilling-tolerant cultivar, and KO is an OsRBCS3-knockout line derived from KY131. DAC, days after chilling treatment. 0 d, control (no chilling stress); 4 d, 4 days of chilling stress; 7 d, 7 days of chilling stress. (Scale bars: a-b, 5 cm.)

Effects of the OsRBCS3 gene on physiological indicators of rice under chilling stress at the booting stage

To explore the physiological role of the OsRBCS3 gene under chilling stress at the booting stage of rice, we measured the antioxidant enzyme activities (CAT, POD, SOD), MDA content and Rubisco activity of four rice cultivars (LJ11, OE, KY131 and KO) after 0 and 7 days of chilling stress (15°C). The results showed that the expression level of the OsRBCS3 gene affected the antioxidant and photosynthetic capacities of rice under chilling stress. Specifically, after chilling stress, CAT and POD activities decreased significantly in LJ11 and KO, while they increased significantly in KY131 and OE (Figure 4a, b). This indicates that overexpressing the OsRBCS3 gene can improve rice antioxidant capacity at the booting stage, while knocking out it can reduce it. SOD activity increased significantly in LJ11 and OE after chilling stress, while there was no change in KY131 and KO (Figure 4c). This suggests that the effect of the OsRBCS3 gene on SOD activity may be related to the chilling tolerance of rice cultivars. The MDA content increased significantly in LJ11 and KO after chilling stress, while there was no change in KY131 and OE (Figure 4d). Rubisco activity decreased significantly in all rice cultivars after chilling stress (p < 0.001, Student’s t-test), of which KO decreased the most (Figure 4e), by 84.72%. This indicates that knock-out of the OsRBCS3 gene may inhibit rice photosynthetic capacity at the booting stage, while over-expression can relatively maintain it. These results indicate that the OsRBCS3 gene has important physiological roles under chilling stress at the booting stage of rice.

Figure 4. Chilling stress induced changes in the activity of CAT, POD, SOD and Rubisco enzymes and the MDA content in the flag leaves of four rice cultivars (LJ11, OE, KY131, and KO).

(a-e) Enzyme activity and MDA content in the flag leaves of rice plants after 7 days of exposure to 15°C. Values are mean ± SD (n = 5). Letters within each sample refer to one-way ANOVA tests (p < 0.01, Duncan test). LJ11 is a chilling-sensitive cultivar, OE is an OsRBCS3-overexpressing line derived from LJ11, KY131 is a chilling-tolerant cultivar, and KO is an OsRBCS3-knockout line derived from KY131. DAC, days after chilling treatment. 0 d, control (no chilling stress); 7 d, 7 days of chilling stress.

Discussion

The OsRBCS3 gene plays an important role in rice chilling tolerance at the booting stage

Rice is very sensitive to chilling stress, especially at the booting stage, which can cause a significant reduction in the seed setting rate and yield.^8,27 Therefore, revealing the molecular mechanism of rice chilling tolerance and breeding rice varieties with high chilling tolerance is of great significance for ensuring food security and adapting to climate change. Rice chilling tolerance is a complex quantitative trait influenced by multiple genes and environmental factors. To date, some chilling-related genes have been identified and functionally analyzed,^28,29 but many unknown genes and regulatory networks remain to be discovered.

In this study, we identified and characterized a rice gene related to chilling tolerance, OsRBCS3, which encodes the Rubisco small subunit (rbcS), a key enzyme in photosynthesis.³⁰ By analyzing the rice digital expression profile¹⁸ and validating it by qPCR, we found that the expression level of the OsRBCS3 gene under chilling stress was significantly different in two japonica rice cultivars with different chilling tolerances, KY131 and LJ11 (Figure 1), and positively correlated with chilling tolerance. To further investigate the function of the OsRBCS3 gene in rice chilling tolerance, we constructed over-expression (OE) and knock-out (KO) lines of the OsRBCS3 gene using over-expression and CRISPR/Cas9 technology, respectively, and evaluated their chilling tolerance at the seedling and booting stages. The results (Figure 3) showed that over-expression of the OsRBCS3 gene could improve rice chilling tolerance at the booting stage, while knock-out of the OsRBCS3 gene could reduce rice chilling tolerance at the booting stage. These results indicated that the OsRBCS3 gene played an important role in rice chilling tolerance.

The OsRBCS3 gene may improve rice chilling tolerance at the booting stage by enhancing antioxidant capacity and photosynthetic capacity

To explore the physiological mechanism of the OsRBCS3 gene under chilling stress at the booting stage, we measured the antioxidant enzyme activities (CAT, POD, SOD), MDA content and Rubisco activity of four rice lines (LJ11, OE, KY131 and KO) after 0 and 7 days of chilling stress (15°C) (Figures 4 and 5). We found that, compared with the wild type, over-expression of the OsRBCS3 gene could improve the antioxidant capacity and photosynthetic capacity of rice at the booting stage, while knock-out of the OsRBCS3 gene could reduce the antioxidant capacity and photosynthetic capacity of rice at the booting stage.

Antioxidant capacity is one of the important indicators for plants to resist chilling stress.³¹ Chilling stress can increase the production of reactive oxygen species (ROS) in plants, which can cause oxidative damage to the plant cell membrane, nucleic acids and proteins.³² Plants have a series of antioxidant enzymes (such as CAT, POD, SOD, etc.) that can scavenge ROS and maintain the redox balance of plants.³³ MDA is a product of lipid peroxidation, which can reflect the stability and damage level of the plant cell membrane.³⁴ Our results (Figure 4) showed that, compared with the wild type, of the OsRBCS3 gene could increase the CAT and POD activities and decrease the MDA content of rice at the booting stage, indicating that it could enhance the antioxidant capacity of rice and reduce the cell membrane damage caused by chilling stress. In contrast, knockout of the OsRBCS3 gene could decrease the CAT and POD activities and increase the MDA content of rice at the booting stage, indicating that it could reduce the antioxidant capacity of rice and increase the cell membrane damage caused by chilling stress.

Photosynthetic capacity is both the basis of plant growth and development and one of the important factors for plants to cope with chilling stress. Chilling stress can affect plant photosynthesis, leading to a decrease in the photosynthetic rate and accumulation of photosynthetic products.^35–37 Rubisco is the most important enzyme in photosynthesis and is involved in the fixation and reduction of carbon dioxide.³⁸ Rubisco consists of large subunits and small subunits, and the small subunits are encoded by OsRBCS genes.³⁹ Our results (Figure 4e) showed that, compared with the wild type, over-expression of OsRBCS3 gene could increase the Rubisco activity of rice at the booting stage, indicating that it could relatively maintain the photosynthetic capacity of rice and increase the accumulation of photosynthetic products. On the contrary, knock-out of the OsRBCS3 gene could decrease the Rubisco activity of rice at the booting stage, indicating that it could inhibit the photosynthetic capacity of rice and reduce the accumulation of photosynthetic products.

In summary, our results supported our hypothesis that the OsRBCS3 gene might improve plant chilling tolerance by enhancing antioxidant capacity and photosynthetic capacity under chilling stress at the booting stage. This is consistent with some previous studies.^40–43 However, some studies also reported that OsRBCS3 gene expression decreased under chilling stress,¹⁷ which might be due to differences in varieties, tissues, time points, etc. To further elucidate the chilling tolerance mechanism of OsRBCS3 gene during seedling and heading stages, we plan to conduct experiments on more representative rice varieties in the future. Therefore, the role of the OsRBCS3 gene in rice chilling tolerance needs further in-depth study.

The OsRBCS3 gene provides new molecular markers and strategies for rice chilling tolerance breeding

The results of this study show that the OsRBCS3 gene is an important gene closely related to rice chilling tolerance, and its over-expression or knock-out can significantly affect rice chilling tolerance at the booting stage (Figures 2 and 3b, d). By over-expressing the OsRBCS3 gene, we improved the chilling tolerance of LJ11, a representative rice cultivar that is sensitive to chilling in Heilongjiang province. This provides a new molecular marker and strategy for using this gene for rice chilling tolerance breeding. We also plan to over-express the OsRBCS3 gene in other representative rice cultivars that are sensitive to chilling, and create new cultivars. We can use methods such as molecular marker-assisted selection (MAS) to increase the expression level of the OsRBCS3 gene in existing excellent varieties or hybrid combinations, thereby improving their chilling tolerance, while retaining other desirable traits. This can expand the adaptation range and planting area of rice and increase grain yield and security.⁶ In addition, we can also use this gene for functional and mechanistic studies, reveal the regulatory network and signaling pathways of rice chilling tolerance, and provide clues for understanding the response and adaptation of rice to chilling stress.^29,44 In summary, we believe that the OsRBCS3 gene has high application value and potential in rice chilling tolerance breeding, and can provide new strategies and means for breeding new varieties or hybrid combinations that adapt to different climatic conditions and environmental requirements.

Conclusion

This study identified a rice gene related to chilling tolerance, OsRBCS3, which encodes a Rubisco small subunit, involved in photosynthesis. The OsRBCS3 gene expression level under chilling stress was significantly different among different japonica rice varieties, and positively correlated with rice chilling tolerance. Over-expression or CRISPR/Cas9 knock-out of the OsRBCS3 gene could significantly change rice chilling tolerance at the booting stage, with OE lines showing higher chilling tolerance. Further physiological and biochemical analysis revealed that over-expression or knock-out of the OsRBCS3 gene could also affect the antioxidant capacity and photosynthetic capacity of rice at the booting stage, thereby increasing or decreasing rice chilling tolerance. We believe that the OsRBCS3 gene plays an important role in rice chilling tolerance, possibly by improving antioxidant capacity and enhancing photosynthetic capacity and thus improving rice chilling tolerance at the booting stage. The validated OsRBCS3 gene obtained in this study can provide important gene resources and reference bases for breeding transgenic early japonica rice varieties with excellent agronomic traits, and stress resistance.

Authors’ contributions

YH and RL designed the experiments. SS and YH performed the experiments and wrote the manuscript. CT, YH and SS analyzed the data. YH, CT, SS and RL revised the manuscript. All the authors have read and approved the manuscript.

Ethical approval

The authors have not performed any experiments involving human participants or animals for this study.

Availability of data and materials

All data supporting the findings of this study are available within the paper and its Supplementary Information.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592324.2024.2318514.

Additional information

Funding

This work was supported by the Research fees of Heilongjiang provincial research institutes (CZKYF2022-1-C012, CZKYF2023-1-C021, CZKYF2023-1-A004); Heilongjiang Province Agricultural Science and Technology Innovation Leap Forward Project Youth Science and Technology Innovation Fund Project (CX22YQ24); Heilongjiang Province Agricultural Science and Technology Innovation Leapfrog Project (CX23ZD01).