Functional Characterization of Rubisco Activase Genes in Kandelia candel Under the Stress of Flooding and Salinity

1. Introduction

Flooding and salinity stress are critical abiotic factors that severely impair plant growth and agricultural productivity [14]. In recent years, driven by intensified climate change and soil salinization, significant progress has been made in understanding plant responses to either flooding or salinity stress alone, including identifying key physiological adaptations and stress-related genes [2,3,4]. However, in natural environments (e.g., in coastal farmlands), these stresses often occur simultaneously, and their combined effects pose a more complex challenge [5]. How do plants integrate molecular and physiological responses to cope with the combined flooding and salinity stress, and what are the crucial antibiotic resistance genes (ARGs) involved in these adaptive mechanisms? Addressing this question is essential for understanding plant stress resilience and developing stress-resistant crop varieties.

As we know, two typical strategies for adaptation to flooding have been developed in plants over the course of long evolution including ‘escape’ and ‘quiescence’ [6]. The plants with the escape strategy respond to flooding stress by rapid elongation of stems and leaves, enabling plant organs to breach the water surface for access to oxygen and light. In contrast, the quiescence strategy allows plants to maintain essential life processes by reducing metabolic rates and optimizing carbon allocation [7]. Notably, the successful implementation of both strategies is heavily reliant on a continuous supply of energy [8].

Flooding stress often leads to multiple secondary stresses, especially reduced light intensity and impaired gas diffusion, which significantly diminish the efficiency of photosynthesis [9]. It has been reported that the certain aquatic plants have evolved special adaptive structures, such as the continuous gas film formed on the surface of rice leaves, which maintaining effective gas exchange under flooding stress [10].

Furthermore, as a terrestrial plant, maize exhibit a compensatory increase in photosynthetic rate during the initial stages of flooding (within 6 days) [11]. Our research on the mangrove species K. candel further reveals that this species can sustain high photosynthetic efficiency under effects of flooding stress through the synergistic action of protein phosphorylation modifications and transcriptional regulatory networks, continuously accumulating sucrose to provide sufficient carbon skeletons and energy for stress-resistant metabolism [12].

In response to salinity stress, plants also need to mobilize substantial energy for the regulation of ion homeostasis and the synthesis of osmoprotectants [13]. Based on the chloroplast proteome study of K. candel, it was found that salinity stress primarily induces the upregulation of photosystem-related proteins, while key enzymes of the Calvin cycle maintain stable expression. This differential regulation effectively mitigates the inhibition of net photosynthetic rate caused by salinity stress [14]. Therefore, maintaining an adequate energy supply is a crucial pathway for plants to withstand adverse stress conditions [15].

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39), located in the chloroplast stroma, is the key rate-limiting enzyme in the photosynthetic carbon assimilation pathway, and its activity directly influences plant adaptability to environmental stress [16,17]. The catalytic function of this enzyme requires a specific activation process mediated by Rubisco activase (RCA) [18]. RCA provides energy through ATP hydrolysis, first dissociating ribulose-1,5-bisphosphate (RuBP) bound at the active site of RuBisco, and subsequently promoting the formation of a catalytically active ternary complex (ECM) between RuBisco, CO₂, and Mg²⁺ [19]. As a member of the AAA⁺ superfamily, RCA exhibits typical ATPase activity [20]. In most higher plants, the RCA gene produces two main transcripts through alternative splicing: RCA1 (long form) and RCA2 (short form), which encode α (45-48 kDa) and β (41-43 kDa) isoforms of different molecular weights, respectively [21]. Phylogenetic analysis indicates that plants such as Arabidopsis [22], rice [23], and soybean [24] express both isoforms, while species like maize [25], tobacco [26], and cucumber [27] retain only the β isoform. It was recently reported that RCA functions as an important regulatory factor in plants in responses to temperature and drought stress [28]. Maintaining photosynthetic homeostasis is a key strategy for plant tolerance to flooding and salinity stress [29]. Our previous studies have confirmed that K. Candel can sustain normal photosynthesis to meet energy demands under individual flooding and salinity stress conditions [12,14]. Therefore, we hypothesize that RCA, which regulates photosynthesis, may play a significant role in the plant's response to the combined flooding and salinity stress.

In the context of global climate change, the genetic engineering of key photosynthetic enzymes has emerged as a significant strategy for enhancing crop yields [30]. As a direct regulatory factor for RuBisco activation, RCA is considered an ideal target for genetic improvement of photosynthesis [31]. Given the substantial differences in activity among the RCA_s from various plant species, exploring efficient RCA gene resources from the plants in specialized habitats holds a considerable application potential [32]. However, the alternative splicing and functional characteristics of the RCA gene exhibit notable species-specific diversity [33,34]. The variable splice forms and functions of the RCA gene in mangrove plants that thrive in flooding and saline habitats within the intertidal zone remain poorly understood.

K. candel has evolved unique mechanisms for tolerance combined flooding and salinity stress during its long-term adaptation to intertidal environments, harboring a wealth of gene resources related to flooding and salinity stress [35,36]. Herein, we are the first to isolate the RCA gene from K. candel, identifying two variant splice variants of the RCA gene, designated KcRCA_l and KcRCA_s. Gene expression analysis and transgenic experiments showed that both KcRCA_l and KcRCA_s are the key genes involved in alleviating the adverse effects of the flooding and salinity stresses.

2. Materials and Methods

2.1. Materials and Treatments

Seedlings of Kandelia candel (L.) were collected from the Zhangjiangkou Mangrove National Nature Reserve in Zhangzhou, Fujian Province (23°55'N, 117°26'E). Healthy, mature, and similarly sized seedlings were selected and planted in nutrient pots (10 cm × 12 cm) filled with clean sand, with one seedling per pot. A total of 30 pots were arranged in a single plastic basin (42 cm × 30 cm × 14 cm) and irrigated with Hoagland nutrient solution. The seedlings were watered twice daily to replenish evaporated water, and the Hoagland nutrient solution was replaced every three days. After the seedlings developed two pair of true leaves, they were subjected to combined flooding and salinity stress treatments. Building on previous research regarding salinity stress in K. candel, we employed a treatment of 500 mmol•L^-1 NaCl in this study [14]. Seedlings with relatively uniform heights (average height difference ± 3 cm) were selected and completely submerged in a Hoagland nutrient solution contenting 500 mmol•L^-1 NaCl, with the solution level maintained 5 cm below the surface. Non-stressed seedlings serving as the control. All seedlings were grown in a greenhouse at an air temperature of 27–32 °C under natural sunlight. Leaf samples from both treantment and control groups were collected after 6, 12, 24, and 48 hours of treatment. The collected leaf samples were rapidly frozen in liquid nitrogen for 10 minutes and subsequently stored at -80 °C for RNA extraction. Each biological replicate consisted of 60 seedlings, with three biological replicates for each treatment.

2.2. Cloning of RCA Gene

Total RNA was extracted from the leaves of K. candel subjected to flooding stress using the TaKaRa Mini BEST Plant RNA Extraction Kit, following the manufacturer's instructions. The quality and integrity of the total RNA were assessed using 1% agarose gel electrophoresis and a NanoDrop ND-2000 spectrophotometer. cDNA synthesis and genomic DNA removal were performed using the TransScript® Uni One-Step gDNA Removal and cDNA Synthesis SuperMix, and the products were stored at -20°C. Based on the longest ORF sequence of RCA gene (Gene ID: CL9747Contig1, Supplementary Table S1) established in the preliminary transcriptome database of K. candel [37], cloning primers were designed using Primer Premier 5.0 software (Supplementary Table S2). PCR amplification of cDNA was conducted, followed by purification. The PCR products were ligated into the cloning vector pMD18-T and transformed into competent E. coli Trans 5α cells. Positive clones were screened and sequenced.

2.3. Bioinformatics Analysis of RCA Gene

The relative molecular weight, isoelectric point, and other physicochemical properties of the proteins were analyzed using the ExPASy online tool. The TargetP 1.1 Server was utilized to analyze the transit peptide segments of the proteins. The InterProScan online tool was employed to analyze the domains and functional sites of the amino acid sequences. The NetPhos 3.1 Server was used to predict phosphorylation sites in the proteins. Homologous amino acid sequences were identified using the BLAST tool in NCBI. A phylogenetic tree was constructed using MEGA 6.06 software. The software and websites used for the bioinformatics analysis of KcRCA_l and KcRCA_s protein structures are listed in Supplementary Table S3.

2.4. Measurement of H₂O₂ and O₂^•− Contents and Expression Analysis of KcRCA_l and KcRCA_s in K. Candel under Combined Stress Condition

The contents of H₂O₂ and O₂^•− were measured at defferent time points for both treatment and control groups of K. Candel under combined flooding and salinity stress conditions. The O₂^•− content was measured using the hydroxylamine oxidation method [38], while H₂O₂ content was quantified using the Micro Hydrogen Peroxide (H₂O₂) Assay Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Following the methodology described in Section 2.2. RNA was extracted from the leaves of K. candel for both treatment and control groups at various time points, and cDNA was subsequently synthesized. Primers were designed based on the sequenced KcRCA_l and KcRCA_s sequences, with the Actin gene of K. Candel serving as the internal control. The expression levels of KcRCA_l and KcRCA_s in the leaves under different treatments were analyzed using RT-qPCR with the SYBR® Premix ExTaq™ (Tli RNaseH Plus) kit, and relative gene expression levels were calculated using the 2^−ΔΔCT method. The primer sequences are provided in Supplementary Table S4.

2.5. Arabidopsis Transformation Assay

The Gateway technology was employed to construct the expression vector for Arabidopsis. Primers with BP adapters were designed (Supplementary Table S5), and the target genes KcRCA_l and KcRCA_s were ligated to the entry vector pDONR207 via BP reaction, resulting in the plasmids pDONR207-KcRCA_l and pDONR207-KcRCA_s. The target genes were then ligated to the expression vector pEarleyGate104 via LR reaction, yielding the expression vectors pEarleyGate104-KcRCA_l and pEarleyGate104-KcRCA_s, which were transformed into Agrobacterium tumefaciens EHA105 competent cells to obtain positive strains. The floral dip method was utilized for Agrobacterium-mediated transformation of Arabidopsis, resulting in T₀ generation seeds [39]. T₀ seeds were screened for resistance to Basta (glufosinate) across multiple generations to obtain T₂ generation transgenic plants, which were confirmed as positive by PCR. The transgenic plants were cultivated in a growth chamber maintained at 22 ℃and 16 h light/8 h dark photoperiodand. The expression levels of the RCA genes were assessed by RT-qPCR in T₂ transgenic plants and wild-type(WT) plants, with the Actin gene of Arabidopsis serving as the internal control, following the methodology outlined in section 2.4. Primers were designed based on the mRNA sequences of Arabidopsis RCA-α and RCA-β isoform available in the GeneBank database (AEC09714.1, AEC09716.1). The primer sequences are provided in Supplementary Table S4.

2.6. Flooding and Salinity Tolerance Assay of Transgenic Arabidopsis

Thirty-day-old T₂ transgenic plants and wild-type(WT) plants were subjected to combined flooding and salinity stress treatment, with a salt concentration of 200 mmol·L^-1 NaCl. The treatment method was consistent with that described for K. Candel in Section 2.1. Samples were collected at 0 h, 6 h, 12 h, 24 h, and 48 h, and RT-qPCR was employed to detect the expression levels of the KcRCA_l and KcRCA_s genes in transgenic plants, following the methodology and primer described in section 2.4. The number of surviving plants was counted separately for transgenic and wild-type plants after 3 and 7 days of treatment, and the survival rates were calculated. The cultivation conditions for both the control and treatment groups were identical to those outlined in Section 2.5. Each biological replicate consisted of 45 seedlings, with three biological replicates for each treatment.

2.7. Determination of Physiological Indexes in Transgenic Arabidopsis under Combined Flooding and Salinity Tolerance

The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the leaves, as well as the contents of superoxide anion (O₂^•−) , hydrogen peroxide (H₂O₂), and malondialdehyde (MDA), were measured. SOD activity was determined using the Hydroxylamine method SOD detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); POD activity was measured using the Guaiacol microplate POD detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) ; CAT activity was assessed using the Ammonium molybdate microplate CAT detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); The methods for measuring the contents of H₂O₂ and O₂^•− outlined in Section 2.4; and MDA content was determined using the thiobarbituric acid (TBA) method [40]. RCA activity was measured using a kit developed by GENMED, and Rubisco activity was assessed using a kit from Shanghai Enzyme-linked Biotechnology Co., Ltd.

2.8. Statistical Analysis of Data

Each measurement was performed with at least three biological replicates, and all data are presented as mean ± standard deviation. Statistical significance was determined using ANOVA or the Student’s t-test via GraphPad Prism 8 software, and difference at p < 0.05 and p < 0.01 was used to determine statistical significance.

3. Results

3.1. Isolation and Characterization of Two Distinct RCA Gene Isoforms in Kandelia Candel Leaves

The RCA gene was cloned from the K. candel leaves after being subjected to combined flooding and salinity stress for 6 hours, followed by bioinformatics analysis was performed. Using cDNA from the leaves of K. candel as a template, a 1500 bp gene fragment was successfully amplified via RT-PCR. The PCR product underwent an A-tailing reaction and was ligated into the cloning vector pMD18-T, followed by transformation into E. coli Trans 5α (Supplementary Figure S1). Positive clones were screened and sequenced, revealing the presence of two distinct RCA gene isoforms in K. candel: a long-chain isoform (KcRCA_l, GenBank accession: MG492021) and a short-chain isoform (KcRCA_s, GenBank accession: MG492022). The cDNA fragment length of the KcRCA_l gene was 1461 bp, while that of the KcRCA_s gene was 1425 bp, indicating a difference of 36 nucleotides between them. Furthermore, 30 positive clones from each treatment group (Control and Treatment) were randomly selected for sequencing, revealing that the occurrence probability of KcRCA_l was approximately 1:3 compared to KcRCA_s (Supplementary Table S6), suggesting a higher abundance of KcRCA_s in K. candel.

The cDNA sequences of KcRCA_l and KcRCA_s were analyzed using ORF Finder software, which revealed that KcRCA_l contains a premature stop codon, terminating translation at the 441 st amino acid. In contrast, the cDNA sequence of KcRCA_s encodes 474 amino acid residues, with 34 additional residues at the C-terminus compared to KcRCA_l. The KcRCA_s protein sequence contains 37 differing amino acid residues (Figure 1): QAAQQVNLPVPPGCTDPAATNYDPTARSDDGSC.

Analysis of the physicochemical properties, including relative molecular weight and isoelectric point (Supplementary Table S7), indicated notable differences between KcRCA_l and KcRCA_s. The KcRCA_l exhibited greater stability than KcRCA_s, with KcRCA_l classified as a stable protein and KcRCA_s as an unstable protein.

Using the TargetP 1.1 Server, the amino acid sequences of KcRCA_l and KcRCA_s were analyzed, revealing that the first 58 amino acid residues (relative molecular weight of 5620.42 Da) are predicted to function as a chloroplast transit peptide. The mature KcRCA_l protein is estimated to have a relative molecular weight of 42.49 kDa, suggesting it corresponds to the β isoforms of RCA (41-43 kDa), while the mature KcRCA_s protein is estimated to have a relative molecular weight of 46.10 kDa, indicating it corresponds to the α isoforms of RCA (45-48 kDa). Thus, it can be inferred that the RCA gene in K. candel leaves encodes both α and β isoforms.

InterProScan analysis of the domains and functional sites of KcRCA_l and KcRCA_s revealed that the amino acid sequence from positions 138 to 332 contains a P-loop nucleoside triphosphate hydrolase functional domain, while the sequence from positions 163 to 304 corresponds to an ATPase, AAA-type, core functional domain, indicating ATP-binding biological function and belonging to the AAA⁺ superfamily of proteins (Supplementary Figure S2). The C-terminus of KcRCA_l lacks conserved sequences; however, the C-terminus of KcRCA_s contains a unique C-extension structure specific to the α isoforms: DPAATNYDPTARSDDGSC, which includes two cysteine residues (Cys) at positions 451 and 470 in the amino acid sequence, further confirming that KcRCA_s is an α isoform and KcRCA_l is a β isoform.

Online analysis using the NetPhos 3.1 Server predicted four phosphorylation sites in both KcRCA_l and KcRCA_s proteins, including one serine (Ser) phosphorylation site, one threonine (Thr) phosphorylation site, and two tyrosine (Tyr) phosphorylation sites. This finding is consistent with the phosphoproteomic data obtained from K. candel leaves [12].

NCBI BLAST sequence alignment analysis indicated that the sequences share over 80% similarity with 24 homologous amino acid sequences encoded by RCA genes. A phylogenetic tree constructed using MEGA 6.06 software, which included RCA genes from 24 plant species, revealed that KcRCA_l (MG492021) and KcRCA_s (MG492022) are most closely related to those of Cicer arietinum and Durio zibethinus, clustering with Populus euphratica, while showing more distant relationships with the RCA-encoding amino acid sequences of 20 other species, indicating they belong to two different subfamilies (Figure 2).

3.2. Analysis of H₂O₂ and O₂^•− Contents and KcRCA Gene Expression Levels in Kandelia candel Leaves Under Combined Stress of Flooding and Salinity

To assess the effects of combined flooding and salinity stress on the growth of K. candel, the contents of hydrogen peroxide (H₂O₂) and superoxide anions (O₂^•−) in the leaves were measured at 6, 12, 24, and 48 hours post-treatment, and morphological changes of the plants were observed and photographed after 48 hours. The results indicated that 6 hours after the stress treatment, the contents of H₂O₂ and O₂^•− in the leaves of K. candel were significantly higher than those in the control group, and remaining elevated thereafter (Figure 3, A, B). Notably, the seedlings of K. candel exhibited significant stress responses within 6 hours of combined flooding and salinity stress. After 48 hours, the leaves of the treatment group displayed slight wilting, although no obvious color changes were observed. Upon excavation, it was noted that the roots of the treatment group had turned black, with some fine roots exhibiting signs of rot (Figure 3, C, D). This indicates that the combined stress on K. candel for 48 hours resulted in significant phenotypic changes.

To verify the expression of the two splice variants of the RCA gene under stress conditions, this study analyzed the expression levels of KcRCA_l and KcRCA_s in the leaves of K. candel at various stress time points in both the treatment and control groups using RT-qPCR. Under flooding and salinity stress treatment, the expression levels of KcRCA in the leaves of K. candel demonstrated that combined stress significantly promoted the overall expression levels of both KcRCA_l and KcRCA_s (Figure 3, E, F). Notably, short-term flooding ( 6 hours) resulted in a significant enhancement of KcRCA_l and KcRCA_s expression levels in the leaves, which is advantageous for the plant's adaptation to flooded and saline environments.

3.3. Establishment and Molecular Confirmation of Arabidopsis Transgenic Plants

In this study, the RCA gene was transferred into Arabidopsis for functional verification, as no established genetic transformation system currently exists for K. candel. Transgenic Arabidopsis was successfully obtained through herbicide resistance and PCR identification. The results indicated that transgenic plants expressing KcRCA_l exhibited a specific band of approximately 1500 bp (Supplementary Figure S3), while transgenic plants expressing KcRCA_s also displayed a specific band of about 1500 bp (Supplementary Figure S4). No corresponding bands were amplified in wild-type Arabidopsis. Additionally, a specific band of approximately 1500 bp was cloned using the expression vector plasmid as a template. The PCR products from positive plants were sent for sequencing, and the results were consistent with the PCR detection, confirming that the exogenous genes KcRCA_l and KcRCA_s have been successfully integrated into the transgenic Arabidopsis. The results of vector construction (Supplementary Figure S5, 6) and Agrobacterium PCR detection (Supplementary Figure S7) are presented in the supplementary materials.

Through generation resistance screening, T₂ generation transgenic Arabidopsis seedlings (Supplementary Figure S8，Supplementary Figure S9) were obtained and utilized for resistance verification of the transgenic lines. We analyzed the phenotypic characteristics and morphology of the transgenic plants. No significant differences in seedling were observed between the wild-type and transgenic plants (Figure 4, A-C). The RT-qPCR data indicated that the expression levels of the KcRCA_l and KcRCA_s genes in the transgenic plants were significantly higher than those in the wild-type plants (Figure 4, D, E). These results confirm the successful generation of transgenic Arabidopsis plants that overexpress the KcRCA_l and KcRCA_s genes.

3.4. Transgenic Arabidopsis Plants Exhibited Enhanced Tolerance to Flooding and Salinity Stress

The performance of wild-type and transgenic Arabidopsis seedlings were submerged in 200 mmol·L⁻¹ NaCl solution for flooding and salinity trentment. On Day 3 of stress, wild-type Arabidopsis leaves exhibited significant purpling and wilting, whereas the transgenic lines thrived(Figure 5, A). By monitoring the survival rates of the plants subjected to flooding and salinity stress treatments over several days, it was observed that, for the same duration of treatment, the survival rate of the transgenic plants was significantly higher than that of the wild-type plants. Furthermore, no significant difference was found between the two transgenic lines.(Figure 5,B, C). These phenotypic results indicate that the transgenic plants possess a higher tolerance to combined flooding and salinity stress compared to the wild-type plants.

In this study, RT-qPCR technology was employed to analyze the expression levels of the KcRCA_l and KcRCA_s genes in transgenic plants under combined flooding and salinity stress at various time points. The results indicated that the expression level of KcRCA_l and KcRCA_s genes were upregulate in the transgenic plants after 6 hours of treatment, remaining significantly higher than those in the control group. Although expression level decreased after 48 hours of treatment, they remained elevated compared to the control group.(Figure 6). The upregulation of RCA gene expression may be associated with the enhanced tolerance to combined flooding and salinity stress observed in the transgenic plants.

3.5. Physiological Analysis of Transgenic Arabidopsis Under the Combined Flooding and Salinity Stress

Physiological assessments of the transgenic plants' resistance for combined flooding and salinity tress and key photosynthetic enzyme activities revealed that, compared to wild-type plants, the activities of superoxide dismutase (SOD) and peroxidase (POD) in transgenic plants were significantly increased (Figure 7A,B), while catalase (CAT) activity did not exhibit significant changes (Figure 7 C). The content of hydrogen peroxide (H₂O₂) did not change significantly (Figure 7 D), whereas superoxide anion (O₂^•¯) content significantly increased (Figure 7 E) Meanwhile, malondialdehyde (MDA) content significantly decreased (Figure 7 F). These results indicate that the antioxidant capacity of plants expressing KcRCA_l or KcRCA_s has been enhanced to some extent. Furthermore, compared to wild-type plants, RCA activity in transgenic plants significantly increased, with Rubisco activity enhanced by 1.09-fold and 2.75-fold in KcRCA_l and KcRCA_s transgenic plants, respectively (Figure 7 G,H). This suggests that KcRCA_l or KcRCA_s contributes to the improvement of photosynthetic efficiency in transgenic plants.

4. Discussion

4.1. Successfully Cloned Two Variable Splice Variants of RCA Gene from a Mangrove Plant

Combined stress presents a significant challenge to successful crop production, and the gene resources that enable plants to respond to such stresses are often found in species that thrive in specialized or extreme environments[41]. For instance, K. candel grows in coastal wetlands and has long been subjected to high levels of flooding and salinity stress. Consequently, it has developed a comprehensive suite of adaptive mechanisms to cope with the combined challenges of flooding and salinity, harboring a wealth of stress-resistant genes [42]. Photosynthesis, as the central process of plant energy metabolism, plays a crucial role in crop adaptation to environmental stress [43]. Previous studies have demonstrated that K. candel can sustain high photosynthetic efficiency by upregulating the expression of photosynthesis-related proteins while stabilizing the expression of key enzymes in the Calvin cycle, including Rubisco and RCA, in response to moderate salinity stress (100-200 mM NaCl) and flooding stress [10,31]. In this context, Rubisco serves as the key rate-limiting enzyme that catalyzes the carboxylation reaction of CO₂ with ribulose-1,5-bisphosphate (RuBP), and its activation is critically dependent on ATP hydrolysis mediated by Rubisco activase (RCA) [44,45]. This study successfully cloned two variable splice variants of RCA, KcRCA_l and KcRCA_s, from the leaves of K. candel for the first time. Bioinformatics analysis revealed that KcRCA_l encodes 440 amino acids (42.49 kDa) and KcRCA_s encodes 474 amino acids (46.10 kDa), both containing typical ATPase functional domains (P-loop NTP hydrolase domain and AAA⁺ ATPase core domain) [46]. Phylogenetic analysis indicated that the RCA of K. candel is most similar to that of Cicer arietinum and Durio zibethinus (>85%), clustering separately from Populus euphratica, while showing lower similarity (<70%) with the RCA genes of 20 other terrestrial plants. This confirms the conservation of the RCA gene and reveals the unique evolutionary characteristics of mangrove plants' RCA in intertidal habitats. As the first RCA gene cloned from a mangrove plant, this study provides important molecular targets for elucidating the mechanisms of salt and flood tolerance in plants.

4.2. Multifunctional Regulation of RCA in the Flooding and Salinity Tolerance Physiology of Kandelia Candel

Recent studies have shown that RCA not only maintains photosynthetic carbon assimilation efficiency by activating Rubisco but also plays a central regulatory role in plant responses to various environmental stresses [47,48]. For instance, in response to temperature stress, heat-tolerant cucumber (Cucumis sativus) significantly upregulated RCA gene expression and enhanced RCA enzyme activity, effectively maintaining Rubisco's catalytic efficiency and allowing net photosynthetic rates to remain above 80% of the control group under high-temperature conditions [27]. Under low-light stress, although the photosynthetic rate of cucumber significantly decreased, the transcription level of CsRCA exhibited a compensatory increase, significantly alleviating the reduction in Rubisco activity [49]. Additionally, in maize (Zea mays) under ozone stress, the mRNA levels of RCA in senescent leaves significantly increased in response to the stress [25]. While RCA genes have been shown to play roles in plant resistance to high temperatures, ozone, and low light, their functions in response to flooding and salinity stress have not been previously reported. Generally, environmental stress inhibits Rubisco activity, leading to a significant decrease in photosynthetic rates; however, RCA can enhance and maintain Rubisco's catalytic activity to regulate photosynthesis under stress [50,51]. This study found that the expression levels of KcRCA in the leaves of K. candel significantly increased under flooding and high salinity stress, suggesting that K. candel may compensate for the adverse effects of stress on Rubisco activity by increasing RCA expression to maintain normal photosynthesis under environmental stress. These results indicate that KcRCA in the leaves of K. candel plays a crucial role in resisting flooding and salinity stress, a finding that has been confirmed for the first time in salt-tolerant and flood-tolerant mangrove plants.

4.3. Molecular Characteristics and Functional Roles of Alternatively Spliced Isoforms of RCA in Kandelia Candel

The variable splicing of plant RCA genes exhibits significant species diversity. Phylogenetic analysis indicates that different plant groups have evolved unique regulatory mechanisms for RCA expression: in most higher plants (such as Arabidopsis, rice, spinach, and barley) [21,23,52,53], variable splicing of a single gene produces two isoforms of 41 kDa and 45 kDa; some species (such as tobacco and cucumber) retain only the expression of the smaller isoform [26,27]; while maize has evolved a unique 3' UTR variable splicing mechanism that generates different isoforms through post-translational modifications [54]. Additionally, plants like cotton and soybean have formed two independent RCA genes through gene duplication events [47,55]. This study reveals for the first time the variable splicing mechanism of RCA in the mangrove plant K. candel, identifying two transcripts, KcRCA_l and KcRCA_s, produced through alternative splicing. KcRCA_l forms a smaller β isoform (42.49 kDa) via a premature stop codon (UAA), while KcRCA_s encodes a complete α isoform (46.10 kDa). Notably, the α isoform contains two highly conserved cysteine residues (Cys247 and Cys392), suggesting its potential involvement in redox signaling. In response to environmental stress, K. candel can precisely maintain an α:β ratio of approximately 3:1(Supplymentary Table S6), a regulatory pattern that aligns with the reported functional division mechanism of RCA in rice [23], where the α isoform primarily activates Rubisco while the β isoform is responsible for maintaining its active state. These findings not only expand our understanding of the functional diversity of plant RCA but also provide new theoretical foundations for elucidating the unique adaptation strategies of intertidal plants. However, the specific molecular functions and regulatory networks of the two isoforms require further investigation.

4.4. Biotechnological Potential of RCA Gene Derived from Kandelia Candel for Enhancing Flooding and Salinity Tolerance in Crops

In recent years, RCA gene engineering has emerged as a significant strategy for enhancing crop photosynthetic efficiency and stress resistance. Multiple studies have demonstrated that exogenous overexpression of RCA genes can significantly improve the photosynthetic performance of transgenic plants [56]. For instance, overexpression of endogenous RCA genes in Arabidopsis and rice has been shown to enhance photosynthetic efficiency and improve adaptability to temperature stress and low-light environments [57,58,59]. This study is the first to clone the RCA gene from the highly salt- and flood-tolerant mangrove plant K. candel and validate its function through a heterologous expression system in Arabidopsis, successfully achieving RCA gene overexpression in Arabidopsis (Figure 4, D, E). The phenotypic analysis demonstrated that the transgenic plants exhibited significantly higher resistance to flooding and salinity stress compared to the wild type (Figure 5, A). Under the same stress duration, the survival rate of the transgenic lines was significantly higher than that of the wild type (Figure 5, B, C). These results indicate that overexpression of the KcRCA gene can enhance the tolerance of Arabidopsis to flooding and salinity stress. Meanwhile, this study also indicated that transgenic plants exhibited significantly enhanced antioxidant capacity, improved RCA enzyme activity, and better Rubisco activation status, thereby maintaining higher photosynthetic efficiency under stress conditions. These findings provide the first evidence that the RCA gene from mangrove plants possesses superior stress response characteristics, offering new high-quality gene resources for crop stress resistance breeding and pioneering a novel approach to crop improvement using genes from plants in unique habitats. Future research could validate the function of KcRCA in major crops, elucidate its molecular regulatory network, and develop molecular marker-assisted breeding technologies based on RCA. This study provides an important theoretical and technical foundation for utilizing specialized plant gene resources for crop improvement.

5. Conclusions

In the present study, we successfully cloned the RCA gene from the mangrove plant K. candel and identified two variable splice variants, KcRCA_l and KcRCA_s, which encode a β isoform of 440 amino acids and an α isoform of 474 amino acids, respectively. Bioinformatics analysis revealed that both isoforms contain multiple phosphorylation sites and lysine acetylation sites, suggesting potential regulation by post-translational modifications. Phylogenetic analysis indicated that KcRCA is most closely related to the RCA genes of chickpeas and durians (similarity > 85%). Expression analysis demonstrated that combined flooding and salinity stress significantly induced the upregulation of KcRCA_l and KcRCA_s expression. Through a heterologous expression system in Arabidopsis, it was confirmed that plants expressing the KcRCA gene exhibited significantly enhanced antioxidant capacity, with RCA and Rubisco activities markedly increased, thereby maintaining photosynthetic efficiency under flooding and salinity stress. Our study firstly clarified the molecular mechanism at the molecular level by which mangrove plants adapt to the unique intertidal habitat through the regulation of RCA gene expression. It not only expanded the molecular theoretical basis for plant responses to cope with combined flooding and salinity stress, but also provided gene resources of significant application value for the genetic improvement of crops against stress.