Overexpression of Rubisco activase gene from Suaeda salsa improves the salt-tolerance and photosynthesis in Arabidopsis

Rubisco activase (RCA) is the key regulatory enzyme in photosynthetic carbon assimilation that governs the activation state of Rubisco, which is the rate-limiting enzyme in CO 2 fixation. In general, salinity seriously inhibits photosynthesis and yield in glycophytic crops, it paradoxically enhances photosynthetic efficiency in halophytes, such as Suaeda salsa ( S. salsa ). However, the potential mechanism still remains unknown. Here, we cloned and characterized the Rubisco activase gene ( SsRCA, 1425 bp) from the euhalophyte S. salsa, which encodes a 475-amino-acid protein. The SsRCA gene expression level and the RCA protein content were increased by 246% and 20%, respectively, under NaCl condition in S. salsa . To investigate the function of SsRCA, we generated SsRCA -overexpressing Arabidopsis thaliana lines. Compared with the wild type (WT), the RCA activity in transgenic lines exhibited 64% higher, and the net photosynthetic rate (Pn) were elevated by 41%, at the 100 mM NaCl stress conditions. Meanwhile, under NaCl stress, the transgenic plants showed lower Na + and MDA content, enhanced K + and proline accumulation, and reduced oxidative damage compared to WT. These results demonstrated that SsRCA overexpression enhanced the salt tolerance of plants by optimizing Rubisco activation efficiency. Our findings will provide a novel halophyte-derived genetic resource for engineering crops with improved photosynthetic resilience in saline environments. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Introduction Soil salinization is a growing global threat to agricultural productivity, and it is projected that 50% of arable land could become unsuitable for cultivation by the end of the 21st century due to salt accumulation (Litalien and Zeeb 2020; Hou et al. 2020). Salt stress impairs plant growth and development by disrupting physiological processes, particularly photosynthesis, thus ultimately leading to significant yield losses (van Zelm et al. 2020). Photosynthetic inhibition of plants under saline conditions were due to ionic toxicity, osmotic effects, stomatal closure, and even photosynthetic structure damage that restricted the efficiency of CO 2 assimilation (Chaves et al. 2009; Munns and Tester 2008). Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is the key enzyme that catalyzing the initial step of CO 2 fixation in plant photosynthetic metabolism. Despite its pivotal role, Rubisco exhibits inherently low catalytic efficiency and requires activation by Rubisco activase (RCA), which is a member of the AAA+ ATPase superfamily that maintains Rubisco in its functional conformation (Andersson and Backlund 2008; Bhat et al. 2017; Prywes et al. 2023; Pasch et al. 2025). Proteomic studies have underscored the dynamic regulation of Rubisco and RCA under salt stresses, and highlighted their importance in sustaining carbon assimilation, thus enhanced plant biomass and yield (Silveira and Carvalho 2016). Emerging evidence positions RCA as a multifunctional stress-responsive regulator. In rice, the photosynthetic efficiency and the productivity were significantly improved in plants with RCA overexpression, while the crop yields failed to enhance in Rubisco overexpression ones, thus indicating the indispensable role of RCA in optimizing Rubisco activity (Suzuki et al. 2009). Additionally, RCA was verified to be involved in stress response, for example, RCA could confer thermotolerance of some plants by stabilizing photosynthetic machinery under heat stress (Crafts-Brandner and Salvucci 2000). In transgenic rice with RCA overexpression, higher photosynthetic rates and biomass were maintained under elevated temperatures (Wang et al. 2010; Qu et al. 2021). Furthermore, the expression of RCA was elevated in heat-stressed wheat leaves, and which may contribute to preserve the carbon assimilation under heat stress conditions (Law and Crafts-Brandner 2001). In Tobacco ( Nicotiana tabacum ), the RCA activation was increased in DfRaf -overexpressed plants, and their thermotolerance was enhanced too (Song et al. 2022). RCA was also confirmed the role in mitigating drought effects by delaying leaf senescence and sustaining photosynthesis in rice (Fan et al. 2022), and improving cold tolerance in cucumber with the enhanced biomass accumulation (Bi et al. 2017). Notably, GhRCAβ2 was verified to respond to diverse abiotic stresses in cotton, including salinity, cold and heat, which suggested the RCA’s broad function in stress adaptation (Chao et al. 2024). In glycophytes, the photosynthetic efficiency was inhibited by salt stress, just like in tomato seedlings, the photosynthetic rate was significant inhibited by salinity with the reduced Rubisco and RCA activity, while the photosynthetic rate was promoted in plants by exogenous selenium apply (Zhang et al. 2023). And in wheat, the RCA isoforms’ content was increased under prolonged salinity condition, but which did not increase under drought stress (Bayramov 2017), thus indicating the stress-specific regulation of RCA in plants. As for halophytes such as mangroves, in which the unique strategies were employed to sustain photosynthesis under hypersaline conditions, including the upregulation of Rubisco activation and ATP synthesis (Lopes et al. 2023). Similarly, for the euhalophyte Suaeda salsa, the expression level of RCA and the activity of carbon assimilation-related enzymes were elevated within 48 hours of 200 mM NaCl exposure (Li et al. 2022). However, the promotion mechanisms of salinity that associated with the photosynthetic performance and enzyme activity still remains unresolved. To investigate the functional role of SsRCA in photosynthetic carbon assimilation and salt tolerance of S. salsa, we isolated the SsRCA gene from S. salsa, and generated the Arabidopsis overexpression lines. Our findings demonstrated that SsRCA overexpression could enhance the photosynthetic capacity and salt-tolerance in transgenic Arabidopsis by enhancing the Rubisco activation. This work advances the understanding of salt-adaptation mechanisms in halophyte and provides a genetic resource for developing salt-tolerant crops to address soil salinization challenges. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Materials and methods jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Plant material and growth condition S. salsa seeds were collected from saline soils (N37 ◦ 20′ E118 ◦ 36′) in the Yellow River Delta in Shandong Province, China, and the dried seeds were stored at <4◦C till use. The seeds were sown in plastic pots (with drainage holes) filled with washed river sand. The seedlings were cultured in a greenhouse under natural light. The temperature was 24 ± 4◦C during the day and 18 ± 4◦C at night. After a 30-day pre-cultivation, the seedlings were treated with Hoagland solution containing 0, 200 and 400 mM NaCl, respectively. Arabidopsis thaliana ecotype Col-0 (Columbia-0) seeds and SsRCA OE lines were disinfected by shaking with 70% ethanol twice (5 min each time) and 95% ethanol three times (4 min each time). After 2 d of vernalization at 4◦C, the seedlings were cultivated in the same a growth chamber (RXZ-500, Ningbo, China) with 22/18◦C (day/night) under a 16 h/8 h photoperiod. The seedlings were cultured on 1/2 MS medium for 7 days and transplanted into pots filled with nutrient soil. Pre-cultured for 10 days, the seedlings were treated with Hoagland solution containing 0 and 100 mM NaCl. Quantitative Real-Time PCR assay Total RNA of different seedlings was extracted from leaves with Fast Pure Cell/Tissue Total RNA Isolation Kit (RC101, Vazyme, China). cDNA was reverse transcribed from the RNA with SPARK script Ⅱ RT Plus Kit (Spark Jade, China). The relative mRNA expression was analyzed using Quantitative real-time PCR with the Universal SYBR Green Fast qPCR Mix. The actin gene (GenBank ID: EU429457) of was used as an internal standard for detection in S. salsa (Ma et al. 2009a). And the Arabidopsis actin gene was used to detect the expression in Arabidopsis and transgenic plants. The expression level of each sample was calculated using the 2 -△△C(T) method (Nolan et al. 2006). And at least three biological replicates were performed in this study. Bioinformatics analysis and transformation of S. salsa RCA The full-length sequence of SsRCA was cloned with primers SsRCA -S and SsRCA -A (Table S1). The cDNA of S. salsa was used as the template. The online tool EXPASY was used to translate the protein encoded by the gene. The coding protein sequences were applied in NCBI-BLAST to analyze sequence similarity, and MEGA6.0 was employed to construct a phylogenetic tree to evaluate the homology of the proteins encoded by the SsRCA gene. SsRCA gene was connected to the vector pCAMBIA1300 driven by the 35S promoter. The recombinant vector was introduced into Arabidopsis via Agrobacterium–mediated gene transfer. Measurement of physiological indexes under salt treatment The seedlings (OE 1, OE 5, OE 7 and WT) grown on 1/2 MS basic medium for 5 d were separately transplanted into nutrient soil. After 10 days of adaptation to growth, they were treated with different concentrations of NaCl (0 and 100 mM NaCl) for 1 week. Leaf tissue (0.1 g fresh weight per replicate) was harvested from five seedlings to measure physiological indicators, respectively. The Na +, K +, proline, malondialdehyde (MDA) contents were measured (Leng et al. 2021). In addition, the 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining were used to detect H 2 O 2 and O 2− . Measurement of plant height After 14 days of salt treatment, the plant height was measured with a ruler above the surface of nutrient soil, respectively. At least five biological replicates were performed for each treatment. Measurement of chlorophyll content After 7 days of salt treatment, 0.3 g of mature leaves were harvested to measure the chlorophyll content. The absorbance values at 645 nm and 663 nm were determined with UV-120-02 Spectrophotometer, Shimadzu, Kyoto, Japan. Measurement of the photosynthetic parameters The expanded leaves of control and NaCl-treated S. salsa and transgenic A. thaliana were selected to measure the net photosynthetic rate (Pn), intercellular CO 2 concentration (Ci), stomatal conductance (gs), and transpiration rate (E) by a portable photosynthetic tester (CIRAS-3, PP-Systems, USA). Each measurement was performed on the same cloudless sunny morning (8:30-11:30 a.m.). In addition, the following conditions were maintained: CO 2 concentration (390 µmol mol −1 ), air humidity (60%), PARi (1800 µmol m −2 s −1 ) and leaf temperature (25◦C). And at least five biological replicates were performed for each treatment. Measurement of the Rubisco and RCA Activity 0.3 g leaves were put into a pre-cooled mortar, 0.5 ml of pre-cooled Tris buffer was added, and then grinded thoroughly. The homogenate is transferred to the centrifuge tube, then the mortar is rinsed twice with 1 ml Tris buffer, and the washing liquid is transferred to the centrifuge tube. The sample was put into a centrifuge, the temperature was adjusted to 4°C, the speed was adjusted to 4000 rpm, the centrifuge was centrifuged for 15 min, the supernatant was collected, and it was subpackaged and stored in a -20°C refrigerator. The activity of Rubisco and RCA were determined at 450 nm by a microplate reader (Spectra Max M5, Molecular Devices, San Jose, CA, USA) according to the instructions of the plant enzyme-linked immunoassay kit (Shanghai Huding Biotechnology Co., LTD., Shanghai). Three replicates were performed for each treatment. Statistical analysis The vegetative physiology and photosynthesis assessments were performed randomly with five replicate plants, and the qPCR analyses were performed with three replicates; the data are presented as means ± standard deviation (SD). The data were analyzed by the statistical software package SPSS Statistics (version 17) based on the ANOVA (one-way) method. Different letters in the figures indicate a significant difference among the mean values ( P < 0.05) by Duncan’s test. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf NaCl enhanced the photosynthetic parameters in S. salsa In S. salsa treated with 200 mM NaCl, the net photosynthetic rate was 10.7 μmol CO 2 m -2 s -1 ，which was significantly increased (by about 81%) than the control plants (plants treated with 0 mM NaCl), in the control plant the result was 5.9 μmol CO 2 m -2 s -1 (Figure S1a). For 400 mM NaCl treated plants, the net photosynthetic efficiency was also increased than control plants, which was only increased by about 29% (Figure S1a). The change trend of stomatal conductance and transpiration rate is the same as that of net photosynthetic rate, while the change trend of intercellular carbon dioxide concentration is opposite to them (Figure S1b, c, d). It indicated that the photosynthesis of S. salsa could be significantly elevated by a certain concentration of salinity, such as 200 mM NaCl. NaCl enhanced the activity of Rubisco and RCA in S. salsa At the 200 mM NaCl condition, the activity of Rubisco in S. salsa leaves was 7.3 U/g FW, which was increased by 43% than the control plants. While the activity of Rubisco was only increased by 19.6% when plants treated with a higher salinity (400 mM NaCl) (Figure 1a). Furthermore, the RCA activity was also increased by 20% under 200 mM NaCl treatment than the control plants, and there was no significant difference between control and 400 mM NaCl treated plants (Figure 1b). The change trend of the activity of Rubisco and RCA was similar to that of the net photosynthetic rate. It was speculated that the change of the activity of Rubisco and RCA was the main factor affecting the photosynthetic change of S. salsa . 200 mM NaCl enhanced the SsRCA expressional level in S. salsa To investigate the relationship of gene expression and salinity, the relative expression levels of SsRCA gene in S. salsa leaves were detected at different treated time. Results showed that SsRCA gene’s expression was induced at 2 h with 200 mM NaCl treatment, and which could be maintained at a higher level (Figure 1c). However, no significant change was observed when plants grown with a higher concentration of NaCl (400 mM) at 2 h and 4 h, and which was decreased at 8 h and 48 h (Figure 1c). It indicated that a certain degree of NaCl can significantly elevate the expression level of SsRCA in the leaves, and thus suggested that the SsRCA gene plays an important role in promoting the photosynthesis of S. salsa . Enhanced growth and salt tolerance were observed in SsRCA -overexpressed transgenic Arabidopsis seedlings In order to study the function of SsRCA gene, we obtained the full length of the Rubisco activase ( SsRCA ) gene from S. salsa, which consists of 1425 base pairs and codes 475 amino acids (Figure 1d), moreover, phylogenetic trees showed that the SsRCA gene of S. salsa was most closely related to that of Chenopodium quinoa and Beta vulgaris (Figure 1e). Totally, nine transgenic A. thaliana overexpressed SsRCA gene was successfully gotten based on PCR and sequencing results, and three lines (OE 5, OE 7 and OE 1) were selected for further study (Figure S2). Compared with the WT, the transgenic lines displayed higher germination percentages and longer root at the germination period that treated with different concentration NaCl (Figure S3). Further experiments show that the transgenic lines especially OE 5, accumulated less Na + and MDA than WT (Figure 2a, c) and more K + and proline than WT (Figure 2b, d). These results indicated that SsRCA gene could reduce the damage caused by excessive accumulation of Na + by enhancing the transport of K +, and thus enhance the salt tolerance of transgenic plants. Moreover, the SsRCA gene further enhanced the salt tolerance of plants by reducing MDA production and increasing proline synthesis. In order to more directly observe the effects of salt stress on wild type and overexpressed lines, we also conducted DAB staining and NBT staining experiments which indicated that the accumulation of H 2 O 2 and O 2− decreased in SsRCA -overexpressed lines when compared with the wild-type under salt stress (Figure 2e, f, g, h). These results indicated that SsRCA overexpression lines were significantly less susceptible to ROS than WT, which further indicated that SsRCA gene could enhance plant salt tolerance by reducing the production of ROS. AtSOS1, AtNHX1, AtP5CS1 and AtGSTU5 are related to plant salt tolerance. The higher the relative expression levels of these genes, the higher the plant tolerance to salt stress. We found that there was no significant difference in the expression levels of wild type and overexpressed lines under 0 mM NaCl treatment. Compared with wild Arabidopsis Thaliana, the expression levels of AtSOS1, AtNHX1, AtP5CS1 and AtGSTU5 in SsRCA overexpression lines were all up-regulated to varying degrees, especially in overexpression line 5, which was about 2 times up-regulated (Figure 3). This suggests that the SsRCA gene can improve the salt resistance of Arabidopsis . Furthermore, SsRCA gene specifically increased the salt tolerance of the transgenic lines. We found that the transgenic lines were significantly higher than WT under 100 mM NaCl treatment (Figure 4). It was speculated that SsRCA gene could improve the photosynthetic capacity of transgenic A. thaliana, thereby increasing the plant height of overexpressed lines and making them show stronger salt tolerance. An increased photosynthesis was detected in SsRCA -overexpressed transgenic Arabidopsis In order to further confirm the relationship between SsRCA gene and photosynthesis, chlorophyll content and photosynthetic index were determined. The results of chlorophyll determination showed that without salt treatment, the chlorophyll content of all strains was basically the same. After 100 mM NaCl treatment, compared with the WT, the transgenic lines showed more chlorophyll a (Figure 5a), chlorophyll b (Figure 5b) and total chlorophyll content (Figure 5c), indicating that the improved photosynthetic efficiency in SsRCA -expressed plants treated with NaCl may partly related with the increased chlorophyll content in them.

showed that there were no significant differences in net photosynthetic efficiency (Pn), stomatal conductance (Gs), transpiration rate (E) and intercellular carbon dioxide concentration (Ci) between the WT and transgenic lines without salt treatment. Although the Pn of all plants were inhibited by 100 mM NaCl treatment, the Pn of transgenic plants were significantly higher than those of the WT and mutants (Figure 6). However, the degree of inhibition of overexpressed strains was significantly lower than that of wild type. The net photosynthetic rate of wild type decreased by about 45%, whereas the OE 5 decreased by only 22% (Figure 6a). In contrast, the Ci increased significantly after salt stress, but the Ci of the transgenic lines was still significantly lower than that of the wild type (Figure 6c). It was further confirmed that SsRCA gene could improve the salt tolerance by promoting the photosynthetic efficiency of plants. The expression levels of Rbcl and RCA and the activity of Rubisco and RCA were increased in SsRCA -overexpressed transgenic Arabidopsis In the study, we analyzed the expression levels of the Rbcl and the RCA . It was found that under 100 mM NaCl treatment, the relative expression of Rbcl and RCA genes in WT decreased by 50% and 49.7%, respectively. However, the relative expression of Rbcl and RCA genes in OE 5 decreased by only 13% and 15.8% (Figure 7a, b). Moreover, we further determined the activity of Rubisco and RCA. It was found that the activities of RCA and Rubisco in overexpressed lines were significantly higher than those in WT (Figure 7c, d). It is speculated that NaCl can induce the expression of Rbcl and RCA, and then regulate the activities of Rubisco and RCA, which are closely related to the carbon fixation process of photosynthesis.

Soil salinization seriously inhibits agricultural productivity and ecological stability (Shabala et al. 2015; Zhang et al. 2023a). And the photosynthesis enzyme and carbon fixation are the most sensitive site of its interference, thus inhibits the photosynthesis efficiency and reduces the biomass accumulation in crops (Zhou et al. 2024; Chavhan et al. 2025). For example, salt stress significantly reduced the content and activity of RCA and Rubisco, thereby inhibited the carbon assimilation efficiency and yield in wheat, tomato or tobacoo (Lee et al. 2014; Zhang et al. 2022; Zhang et al. 2023b). However, it’s different in salt-tolerant species. For barley, the salt tolerance was enhanced with the increased expression of HvRCA gene, and inevitably improved its photosynthetic efficiency (Aliakbari et al. 2021, 2024). Additionally, in halophytes like Rhizophora mangle and S. salsa, their elevated photosynthesis in saline environment was related to the upregulated of RCA expression and RCA activity (Lopes et al. 2023; Li et al. 2022; Cai et al. 2025). Here, we demonstrated the SsRCA gene in enhancing photosynthetic performance by increasing the content and activity of RCA. In this study, a higher net photosynthetic rate and an enhanced activity of carbon fixation-related enzymes (RCA and Rubisco) were detected in NaCl-treated S. salsa plants (Figure S1 and Figure 1a, b), and the previous results were also confirmed our present study (Li et al. 2022; Guo et al. 2020). In S. salsa leaves treated with 200 mM NaCl, the expression of SsRCA was correspondingly up-regulated than the control ones (Figure 1c). To investigate the function of SsRCA gene in plant carbon fixation and salt tolerance, the SsRCA -overexpressed Arabidopsis was successfully obtained. qRT-PCR analysis showed that the three SsRCA OE lines, SsRCA OE1 , SsRCA OE5 , and SsRCA OE7 , expressed SsRCA at high levels (Figure S2). Rubisco will be a limited factor to photosynthetic CO 2 assimilation, only if it is activated by RCA (Singh et al. 2014). Therefore, the amount and the activity of Rubisco are the main stimulations for enhancing the photosynthetic rate. In SsRCA -overexpressed lines, the Rubisco’s activity and content were enhanced under NaCl condition (Figure 7c, d), and they also displayed an elevated photosynthetic rate (Figure 6a), together with an increased growth (Figure 4). And in OsRCA -overexpressed rice, an enhanced photosynthetic rate was observed in them (Masumoto et al. 2012; Qu et al. 2021). Thus, the elevated photosynthesis was related to the overproduction of RCA, and which was consistent with our present study. It was also reported that in CsRCA -overexpressed cucumber, the increased growth and promoted photosynthesis were detected (Bi et al. 2017), and it also endowed heat tolerance of CsRCA -overexpressed cucumbers with improved photosynthesis by enhancing the activities of Rubisco and RCA (Bi et al. 2016). In the present study, the activities of Rubisco and RCA, and the expressional levels of related-genes, were increased in SsRCA -overexpression Arabidopsis under salinity when compared with the wile type (Figure 7), together with the increased chlorophyll content and photosynthesis (Figure 5,6), which indicated that the SsRCA from halophyte S. salsa could enhance the photosynthetic capacity and salt tolerance of transgenic plants. Just like in mangrove and Nitraria sibirica, the enhanced salt tolerance in which was related to the increased photosynthesis-associated protein abundance (Shen et al. 2018; Lu et al. 2025). While for Phoenix dactylifera, the enhanced salt tolerance was attributed to the significantly up-regulated PdRCA gene (El Rabey et al.,2016), as well as in halophyte Rhizophora mangle (Lopes et al. 2023). In plants, a multifaceted mechanism will be employed to adapt salt environment, including ionic homeostasis maintenance, osmotic regulation, and oxidative stress resistance (Liang et al. 2024; Jiang et al. 2025). In our study, the increase in K + and proline content, and the decrease in Na + and MDA content in the SsRCA -overexpressed plants treated with NaCl suggested that their salt tolerance ability was enhanced (Figure 2). Together with the increased expression of salt tolerance related genes ( SOS1, NHX1, P5CS1 and GSTU5 ) indicated that the enhanced salt tolerance could be attributed to the enhanced RCA and Rubisco activity (Figure 3). And the results could also be confirmed by those of CsRCA -overexpressed Cucumis sativus, in which the plant height, leaf area and dry matter were all increased, while the MDA content was reduced than wild-type (WT) plants (Bi et al. 2017). In summary, the SsRCA -overexpressed Arabidopsis was successfully obtained. A higher photosynthetic rate under salt stress was observed in plants with SsRCA overexpression, and a higher RCA and Rubisco activities was also acquired. These indicating that salt stress could regulate RCA in S. salsa and transgenic plants, and could regulate the activities of RCA and Rubisco in them, thus enhanced the photosynthetic rate and increased the plant’s growth under salt stress condition. At the same time, Meanwhile, the salt-tolerant indices of the transgenic plants, such as the ionic homeostasis maintenance, antioxidant regulation, and stress-related genes’ expression, were also better than those of WT under salt stress conditions. This indicates that SsRCA overexpression can regulate the salt tolerance and photosynthetic performance of plants by modulating a series of physiological and metabolic processes. And the elevated expression of RCA might be a vital factor for higher CO 2 fixation efficiency under salt stress.

This work was supported by the Natural Science Research Foundation of Shandong Province (ZR2022MC182), and the Higher Educational Science and Technology Program of Shandong Province (J17KA136). Conflict of interest The authors declare that they have no conflict of interest. Authorship J.G. and M.Y. designed the studies. M.Y., X.W., and X.Z. performed the experiments. M.Y., X.W., and X. Wei analyzed the data. M.Y., and X.W. wrote the paper. JG revised the manuscript. All authors contributed to the article and approved the submitted version.

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Triticum aestivum (Ta), Zea mays (Zm), Hordeum vulgare (Hv), Oryza sativa (Os), Arabidopsis thaliana (At), Vitis vinifera (Vv), Eucalyptus grandis (Eg), Glycine max (Gm), Populus euphratica (Pe), Spinacia oleracea (So), Suaeda salsa (Ss), Chenopodium quinoa (Cq), and Beta vulgaris subsp. Vulgaris (Bv). Values are presented as the means ± SD of 5 (Rubisco and RCA activity analysis) or 3 ( SsRCA expression level analysis) replicates. Different letters indicate a significant difference at P ≤ 0.05 by Duncan’s test. Figure 2 Physiological indices and detection of H 2 O 2 and O 2− in wild-type and SsRCA overexpressed plants under salt stress. (a) Na + content of the wild-type and overexpressed plants. (b) K + content of the wild-type and overexpressed plants. (c) MDA content of the wild-type and overexpressed plants. (d) Proline content of the wild-type and overexpressed plants. (e) DAB staining and (f) Quantitative analysis of DAB-positive areas (%) of the wild-type and overexpressed plants. (g) NBT staining and (h) Quantitative analysis of NBT-positive areas (%) in wild-type and overexpressed plants. Values are presented as the means ± SD of 5 replicates. Different letters indicate a significant difference at P ≤ 0.05 by Duncan’s test. Figure 3 The relative expression of salt resistance-related genes in wild-type and SsRCA overexpressed plants. (a) AtSOS1 gene relative expression analysis. (b) AtNHX1 gene relative expression analysis. (c) AtP5CS1 gene relative expression analysis. (d) AtGSTU5 gene relative expression analysis. Values are presented as the means ± SD of 3 replicates. Different letters indicate a significant difference at P ≤ 0.05 by Duncan’s test. Figure 4 Growth of wild-type and SsRCA overexpressed plants treated with 0 and 100 mM NaCl. (a) The growth phenotype of wild-type and SsRCA overexpressed plants. (b) Plant height statistics of wild-type and SsRCA overexpressed plants. Values are presented as the means ± SD of 5 replicates. Different letters indicate a significant difference at P ≤ 0.05 by Duncan’s test. Figure 5 Chlorophyll contents of wild-type and SsRCA overexpressed plants treated with 0 and 100 mM NaCl. (a) The content of chlorophyll a. (b) The content of chlorophyll b. (c) The content of chlorophyll a and b. Figure 6 Photosynthetic indices of wild-type and SsRCA overexpressed plants treated with 0 and 100 mM NaCl. (a) The net photosynthetic rate. (b) The stomatal conductance. (c) The transpiration rate. (d) The intercellular carbon dioxide concentration. Values are presented as the means ± SD of 5 replicates. Different letters indicate a significant difference at P ≤ 0.05 by Duncan’s test. Figure 7 Analysis of the expression levels of genes related to carbon fixation and the activities of Rubisco and RCA in wild-type and SsRCA overexpressed plants treated with 0 and 100 mM NaCl. (a) The relative expressive levels of Rbcl gene. (b) The relative expressive levels of RCA gene. (c) The enzyme activity of RCA. (d) The enzyme activity of Rubisco. Values are presented as the means ± SD of 3 ( Rbcl and RCA expression level analysis) or 5 (Rubisco and RCA activity analysis) replicates. Different letters indicate a significant difference at P ≤ 0.05 by Duncan’s test. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Supplementary files Supplementary file 1：Figure S1 Photosynthesis indexes of S. salsa under different concentrations of NaCl. (a) The net photosynthetic rate. (b) The stomatal conductance. (c) The transpiration rate. (d) The intercellular carbon dioxide concentration. Values are presented as the means ± SD of 5 replicates. Different letters indicate a significant difference at P ≤ 0.05 by Duncan’s test. Supplementary file 2：Figure S2 Identification of positive transgenic Arabidopsis thaliana seedlings and SsRCA gene expression analysis. (a) Hygromycin screening positive seedlings. (b) Electrophoretogram for DNA identification of SsRCA overexpression lines. (c) Expression levels of SsRCA in overexpressed strains examined by qRT-PCR. Values are presented as the means ± SD of 3 replicates. Different letters indicate a significant difference at P ≤ 0.05 by Duncan’s test. Supplementary file 3：Figure S3 Germination and growth of wild-type and SsRCA overexpressed plants treated with different concentrations of NaCl (0, 50, 100, and 150 mM) for 5 d. (a) The growth phenotypes detected at 5 days treated with NaCl. (b) The germination percentage detected at 24 hours treated with NaCl. (c) The root length detected at 5 days treated with NaCl. Values are presented as the means ± SD of 5 replicates. Different letters indicate a significant difference at P ≤ 0.05 by Duncan’s test. Supplementary file 4: Table S1 PCR primers for the SsRCA gene cloning and gene expressional level analysis. Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 285views 207downloads Citations Download citation Meixiang Yang, Xinlei Wang, Xiaoqian Zhang, et al. Overexpression of Rubisco activase gene from Suaeda salsa improves the salt-tolerance and photosynthesis in Arabidopsis. Authorea. 30 May 2025. DOI: https://doi.org/10.22541/au.174857959.94050910/v1 DOI: https://doi.org/10.22541/au.174857959.94050910/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.