Over Expression of the Carbonic Anhydrase Gene Confers Salinity Tolerance with Improved Yield in Rice

 
Carbonic anhydrase (CA) is the enzyme that accelerates the reversible hydration reaction of carbon dioxide converted to bicarbonate (Ren et al., 2021). They are essential in regulating carbon dioxide levels and ensuring optimal plant development and metabolic mechanisms (Vicente et al., 2022). The proper functioning of carbonic anhydrases (CAs) is crucial for integrating enzymes into various processes involved in regulating pH, CO₂ and HCO₃" concentrations and water and electrolyte balance and also essential in maintaining homeostasis in living organisms (Occhipinti and Boron, 2019). This particular enzyme assumes a crucial role in the process of C4 photosynthesis. The cytosolic carbonic anhydrase (CA) gene in Flaveria bidentis cells provides evidence for the enzyme’s crucial role in the C4 photosynthetic pathway. Salt stress upregulates the expression of the OsCA1 gene encoding carbonic anhydrase in rice seedlings and total CA activity. Over-expression of OsCA in Arabidopsis thaliana mutant plants increase salt tolerance. This suggests that OsCA may enhance plants ability to cope with salt stress, providing a potential avenue for developing salt-tolerant crops (Kandoi et al., 2022).
 
In the present study, CA overexpressing transgenic rice plants (Oryza sativa L., cv. IR64) were developed which shows enhanced photosynthesis, tolerance to salinity stress with increased yield and improved biomass.

Cloning and transformation of carbonic anhydrase (CA) gene in IR64 rice
 
The carbonic anhydrase (CA) gene was used to develop transgenic rice (Oryza sativa cv. IR64) plants using the tissue culture technique. The coding region of CA gene (0.8 kbp) from rice was PCR-amplified using forward and reverse primers (5'-ATGGGAAGTAAATCATATGAT-3' and 5'-TTTCACCTACTACCTCAGCATAA-3'). The gene (0.8 kb) was cloned in a reporter gene- free plant transformation vector pCAMBIA1300 in the place of hygromycin to generate a complete reporter and antibiotic marker-free plasmid pCAMBIA1300-CA. The above construct (pCAMBIA1300-CA) was used for the Agrobacterium tumefaciens (LBA4404) transformation method (Sahoo and Tuteja, 2012). After development of transgenic rice plants, all the analysis were compared with WT plant as control (C).
 
Molecular analysis (PCR, Southern blot and qRT-PCR) of T1 transgenic CA rice plants
 
The genomic DNA was extracted from the healthy leaves of marker-free CA transgenic plants and used to check the integration of gene by PCR and Southern blot analysis. About 25 µg of genomic DNA was used for the Southern blot experiments.  First the genomic DNA was digested with BamHI restriction enzyme and resolved on 0.8% agarose gels, followed by transfer to a negatively charged nylon membrane (Hybond-N⁺, Amersham, Inc.),  as described  by  Sambrook et al., (1989). The probe was radiolabelled by the gene amplification method, using α–[³²P] dCTP. Hybridization with the probe was conducted using the method described by Sambrook et al., (1989).
 
The expression of CA gene in different transgenic lines were studied by qRT-PCR experiment. The 21 days old seedlings of transgenic (CA) and control (C) rice (Oryza sativa cv. IR 64) plants were used for this experiment. Total RNA was isolated from 100 mg of samples with TriZOL LS reagent (Invitrogen Life Technologies USA).  The total RNA obtained was used as template for cDNA synthesis. The first strand cDNA was synthesized from 5 μg of total RNA using Superscript II Reverse Transcriptase (Invitrogen Life Technologies USA) with oligo (dT) primer according to the manufacturer’s instructions. PCR reactions were performed on Step One Real-Time PCR system (Applied Biosystems). Using Power Syber Green PCR master mix (Applied BioSystems), a 20 μl reaction mixture containing 10 pM of each gene specific primer pair (α-tublin forward 5’-GGTGGAGGTGA TGATGCTTT-3’  and reverse 5’-ACCACGGGCAAAGTT GTTAG-3’ and with CA gene specific primers (Forward 5’- TTTCCAACCCGGTGAGG -3’ and Reverse 5’- AGCAGATA GTTGTAATT -3’). The PCR cycle was as follows: 95°C for 30 s, 58°C for 30 s and 72°C for 30 s. The quantitative variation between different samples was evaluated by the ΔΔCt method and the amplification of tubulin gene was used as internal control to normalize all data (He et al., 2021). To validate the qRT-PCR results, the experiments were repeated three times. The mean values for the expression levels of the genes were calculated from three independent experiments. The relative expression level was calculated as 2^-ΔΔCT. The 2^-ΔΔCT values for the untreated transgenic were normalized to 1.
 
Salinity tolerance index (TI)
 
The TI of the 200 mM NaCl-treated CA transgenic (L7 and L9) and control plants were calculated using the following formula:
   
Leaf disk senescence assay and chlorophyll content
 
Healthy and fully expanded rice leaf squares of 1 cm x 1 cm dimension were taken from similar age transgenic lines (L7 and L9) of transgenic and control plants. The discs were floated in 100 and 200 mM solution of NaCl for 72 h. The experiment was performed at room temperature with three biological replicates as previously described (Tuteja et al., 2013).
 
Biochemical analysis of antioxidant activities of marker-free CA transgenic lines
 
The seeds of CA transgenic (L7 and L9) and control (C) plants were placed in hydroponics for germination. After plants had grown for 21 days, they were shifted to another hydroponics filled with 200 mM NaCl for 24 h. All our experiments were conducted in the greenhouses of the Centurion University of Technology and Management, Bhubaneswar, Odisha, India,  where 16 h light photoperiod and 8 h dark at 25°C was  maintained. After 24 h salt stress, plant tissues were used for biochemical analysis of e.g., catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol peroxidase (GPX), proline, hydrogen peroxide and lipid peroxidation; in addition, we also measured the relative water content (RWC) as well as the electrolytic leakage. All the parameters were measured using the methods described earlier by Garg et al., (2012).
 
Measurement of photosynthetic activities and agronomic characteristics of CA transgenic plants
 
The rate of photosynthesis, photosynthetic yield, intercellular CO₂ concentration, CO₂ release, stomatal conductance and the rate of transpiration were recorded using an infrared gas analyser (IRGA; LI-COR, http://www.licor.com) on sunny days between 11:00 AM and 12:00 noon. For these measurements, plants were grown in metal pots filled with soil and all the pots were kept inside another big horizontal tank filled with 200 mM NaCl solution. All the parameters were measured using the expanded leaves of mature (60 d old) plants. After 12 days of salt stress, different agronomic  parameters (plant height, number of tillers/plant, number of panicle/plant, number of filled grain/ panicle, number of chaffy grains/panicle, straw dry weight, 100 grain weight, root length, root dry weight, leaf area and plant dry weight) were measured using methods described earlier (Tuteja et al., 2013).
 
Statistical analysis
 
Data was obtained from at least three independent experiments. The mean values are presented and standard errors included. One-way analysis of variance (ANOVA) was performed on the data using SPSS (12.0 Inc., USA) to determine the least significant difference (LSD). The means were interpreted by Duncan’s multiple range tests (DMRT).

Molecular analysis of marker-free PDH45 transgenic lines
 
The marker-free CA transgenic IR64 rice plants were developed using the pCAMBIA1300-CA gene construct (Fig 1a). Phenotypically the transgenic rice plants were not significantly different from control plants (Fig 1b). The desired CA gene (0.8 kb) fragment was detected by PCR (Fig 1c). The Southern blot results confirmed the integration of CA gene in transgenic rice plants in all the two transgenic lines (L7 and L9) (Fig 1d). The real-time PCR (qRT-PCR) provided <“5 fold induction in the transcript level of CA in transgenic lines (L7 and L9) (Fig 1e).  The salinity tolerance index of CA transgenic rice lines was found to be higher (75.3 and 72.8% in L7 and L9 respectively) in comparison with control plants (30.2%) (Fig 1f). The damage caused in the leaf pieces by salinity stress was observed in all the plants after 72 h. The reduction of chlorophyll content in leaf tissues was lesser in transgenic lines as compared to C plants (Fig 1g). The lesser chlorophyll content in the leaf tissues of C plants as compared to transgenic lines provides strong evidence towards tolerance against salinity stress (Fig 1h). We have generated transgenic rice lines overexpressing carbonic anhydrase gene using Agrobacterium-mediated transformation with higher afûnity for CO₂. Overexpression of CA gene under the control of 35S promoter resulted in higher gene expression, protein abundance and enzymatic activity of CA, in the transgenic lines. Two independent transgenic lines (L7 and L9) along with WT plants as control (C) were used for functional validation under salinity stress. This was indicated by the presence of higher chlorophyll content in the leaf disks of salinity-stressed transgenic plants, whereas WT plant leaves became yellow. These results indicate that the introduced trait is functional in transgenic plants and that it is also stable (Sahoo et al., 2022). Similar findings have been reported earlier (Amin et al., 2012; Gill et al., 2013; Tuteja​ et al., 2013, 2015, Sahoo et al., 2022).


 
The antioxidant machinery of CA transgenic rice performed better than control plants
 
The salt-induced changes in the catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol peroxidase (GPX), proline, hydrogen peroxide, ion leakage, accumulation of MDA (lipid peroxidation product), relative water content (RWC) were compared with C rice seedlings. Reduced levels of MDA, H₂O₂ and ion leakage and increased level of proline content in transgenic lines as compared to the C plants under salt (200 mM NaCl) stress were observed. The activities of CAT, APX, GPX, GR and RWC were increased in transgenic plants as compared to C plants (Fig 2 a-i). Under salinity stress, plants produce more ROS, which can cause serious damage to plasma membrane, chloroplasts and mitochondria through peroxidation and de-esterification of membrane lipids, as well as damage to nucleic acids and proteins (Hasanuzzaman et al., 2020). In the present study, lipid peroxidation, ion leakage and H₂O₂ production were found to be significantly decreased as compared to control plants under salinity stress. Previous studies have also shown decreased level of MDA, ion leakage and H₂O₂ production under salinity stress in different overexpression studies in rice (Yang et al., 2012; Gill et al., 2013; Tuteja et al., 2013; Sahoo et al., 2022). The damage caused due to salinity to membrane stability is a consequence of generation of H₂O₂ which is an important ROS that can cause oxidative damage to biomolecules including nucleic acids, proteins and lipids (Gill and Tuteja, 2010). To protect the plants from the injurious effects of H₂O₂, plants produce higher levels of APX through the AsA-GSH cycle, where APX uses ascorbate as hydrogen donor. Other antioxidant enzymes, such as GR, catalyse the NADPH-dependent reduction of GSSG (oxidized form) to GSH (reduced form) and maintain high ratio of GSH/ GSSG (Hasanuzzaman et al., 2019). In our investigation, the antioxidant enzymes APX, GPX and GR showed significantly higher activities under salinity stress in transgenic lines compared to control plants, suggesting that this provides better ROS scavenging during the stress. The exact mechanism on how salinity stress tolerance works in plants is still far from being understood. The excessive generations of ROS intermediates, such as superoxide radicals (O₂"), hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH”), are the unfortunate consequences of salinity and other stresses in plants. Since the antioxidant enzyme machinery was found to be stronger in CA transgenic rice plants, hence, the salinity stress tolerance might be due to better detoxification of ROS. Generally, plants adapt to stress by accumulating amino acids and/or amino acid derivatives, sugar alcohols and several other substances. These were found to be accumulated at higher levels in CA transgenic rice plants and may help plants to better adapt to changing environment. The salinity stress tolerance could also be due to the interaction of CA with the components of different signalling pathways (Mishra et al., 2023).


 
Agronomic performance of marker-free PDH45 transgenic plants under stress
 
The agronomic performance of CA transgenic (under 200 mM NaCl stress) was compared with C plants under 0 mM NaCl (water) stress. Better agronomic characteristics were observed in CA transgenic plants as compared to C plants (Table 1). Several phenotypic characteristics of transgenic plants were recorded and found to be almost similar to the C plants grown in water (0 mM NaCl). Similar studies have been reported in rice (Tuteja et al., 2013; Sahoo et al., 2022).


 
Photosynthetic characteristics of marker-free CA transgenic plants under stress
 
The photosynthetic characteristics of transgenic plants were observed and compared to WT and VC plants after 12 d of induction of salt (200 mM NaCl) stress. The photosynthetic rate declined by 33% in C plants as compared to CA transgenic lines. The photosynthetic rate, stomatal conductance and intracellular CO₂, CO₂ release and transpiration rate was also higher in transgenic lines as compared to the C plants (Fig 3a-e). The better photosynthetic activities like photosynthetic yield, photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, CO₂ release and transpiration rate were observed in CA transgenic lines as compared to the control plants.  The retention of chlorophyll content in transgenic lines indicates the better control over photosynthetic apparatus under salinity stress. Our data are in agreement with the earlier reports on PDH45, SUV3 and BAT1 over expressing rice plants in stress (Sahoo et al., 2012; Gill et al., 2013; Tuteja et al., 2013, 2015, Sahoo et al., 2022).

Photosynthesis and CO₂ availability are the ultimate determinants for crop growth and yield. The overall photosynthetic performance was found to be higher in marker free transgenic rice compared to the controlled ones. The yield and yield attributing factors viz; time required for flowering (days), no. of tillers/plant, no. of panicle/plant, no. of filled grain/panicle, no. of chaffy grains/panicle, straw dry weight (g), 100-grain weight, seed weight per plant were also enhanced in transgenic rice compared under salinity stress. Thus the current piece of work provides evidence of a unique function of CA in providing salinity stress tolerance in transgenic rice without affecting yield. It also provides a profound example for the exploitation of C4 components in C3 plants like rice for enhanced photosynthesis as well as agricultural production that have the ability to withstand extreme climatic conditions.

The author thanks Centurion University of Technology and Management, Bhubaneswar, Odisha, India, for the invaluable support in carrying out the research work.

 
The authors declare that they have no conflict of interest.