Understanding the role of the fructose-1,6-bisphosphatase gene for enhancing the photosynthetic rate in Arabidopsis thaliana

Introduction

Plant photosynthesis is a critical process underpinning the survival of most organisms on Earth, by capturing solar energy and making it available within ecosystems. Photosynthesis comprises of two processes: light and dark reactions. During dark reactions the major enzyme RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) performs carboxylation, i.e. fixation of carbon dioxide, producing two molecules of 3-phosphoglycerate (3PGA) (Song et al. 2023). In addition to carboxylation, RuBisCO also performs oxygenation (i.e. fixing oxygen) (Bowes et al. 1971) and produces one 3PGA molecule with another two molecules of phosphoglycolate (2PG). The production of phosphoglycolate molecules is toxic for plants and blocks the functions of RuBisCO and triosephosphate isomerase. To circumvent this toxic effect, the glycolate catabolic reaction takes place during photorespiration, producing one molecule of PGA, ammonia and carbon dioxide (Heyduk 2022). In this process, C₃ type plants waste up to 25% of fixed carbon dioxide and 6.1% of energy (Mizokami et al. 2019).

In contrast, C₄ photosynthetic plants are provided with carbon concentrating mechanisms (CCM) that involve minimal photorespiration, resulting in maximum energy conservation (Long et al. 2019). In comparison to plants with the C₃ mechanism, C₄ plants exhibit 6% higher solar energy conversion (Ferrari et al. 2022). However, it is also established that photorespiration is vital for plants’ existence and its mechanism cannot be completely avoided (Ainsworth and Rogers 2007). Therefore, CO₂ and energy losses should be reduced through setting up the shorter and more efficient 2PG metabolic pathway in C₃ plants (Tan and Chen 2023). Numerous efforts have been made for the modification of C₃ plants to minimise the effect of photorespiration, but RuBisCO modification is a challenging task (Tan and Chen 2023). However, advanced biotechnological methods can serve the purpose, like genetic engineering of potential genes that can modify the photosynthetic pathway to minimise the energy loss and enhance the photosynthesis rate (Dalal et al. 2015). Such genes are found to be present in cyanobacteria that contain different 2PG metabolising pathways and can minimise the losses created by photorespiration (Arrivault et al. 2019).

One such gene is fructose-1,6 B-bisphosphatase (FBPase). It is present in cyanobacteria and has the potential to elevate the levels of photosynthesis in C₃, as it plays a vital role between the regenerative phase of the Calvin cycle and starch biosynthesis and also activates irreversible reactions (Mizokami et al. 2019). Cyanobacterial genes have previously been used to transform the photosynthetic mechanism, with promising results (Mitchell et al. 2020).

The current study reports the successful transformation of the FBPase cyanobacterial gene in model plant Arabidopsis thaliana through floral dip Agrobacterium mediated transformation. The transgenic plants showed significantly higher gene expression, with enhanced plant growth, increased stomatal conductance, improved chlorophyll contents and higher water retaining capacity; resulting in improved photosynthesis when compared to the wild plants. This study will open new avenues to genetically transform the economically important crops that are driven by C₃ photosynthesis mechanism.

Materials and methods

Vector construction

The gene sequence of Synechocystis sp. strain PCC 680 was retrieved from https://www.kazusa.or.jp/database/ and specific primers were generated for constructing an expression vector of FBPase (see Supplementary material S1). The cloned gene was sequenced and transferred into a shuttle vector provided with 35 cauliflower mosaic virus (CaMV) 35S dual promoter, tobacco etch virus 5′-untranslated region (5′-UTR) translation enhancer (TEV), chloroplast transit peptide sequence from tomato smaller subunit of RuBisCO (chlTP) and 35S terminator. The cassette of gene expression was then loaded onto pCAMBIA2300 and was named pSFB (Fig. 1) (Sowjanya et al. 2019). Finally, the expression vector was transformed to GV3101strain of Agrobacterium tumefaciens using the freeze–thaw method.

Fig. 1.

Representation of vector pSFB utilised for transformation of Arabidopsis thaliana plants expressing FBPase. 35SPro, cauliflower mosaic virus promoter; TEV, transcriptional enhancer; chlTP, transit peptide from the sequence of the small subunit of RuBisCO (tomato); FBPase, fructose-1,6-bisphosphatase; 35S Ter, the terminating sequence from the cauliflower mosaic virus.

Results

Molecular characterisation of putative transformants

Plants that showed resistance to the selection media provided with antibiotic resistance gene were considered as putative transformants. Out of twenty plants, ten were selected for PCR analysis, amongst which seven plants were found positive for the presence of FBPase gene (Fig. 2a). Expression analysis revealed that overexpression of the FBPase gene was significantly higher in transgenic as compared to the wild type plants (Fig. 2b).

Fig. 2.

(a) PCR based amplified bands of the FBPase gene in Arabidopsis plants. Molecular weight markers (M): Invitrogen 1 kb ladder, FBPase PCR product (205 kb). Lanes 1–7: FBPase positive plants (transgenics) showing bands of expected size. Lane C: Negative control (wild type plant DNA of Arabidopsis. (b) Relative expression pattern analyses of wild type (WT) and transgenic Arabidopsis plants estimated and normalised using Actin 7 as the internal control by qPCR. Expression analysis revealed that overexpression of the FBPase gene was significantly higher in transgenics, as compared to the wild type plants. * indicates P ≤ 0.05. Error bar denotes standard error.

Plant height

Transgenic plants showed early bolting as compared to the wild plants (Fig. 3a). After 35 days of sowing transgenic plants showed significantly greater heights, with a maximum of 34.5 cm in the T3 line; the minimum was recorded as 24.4 cm in the T6 line. In comparison, amongst wild type plants the maximum recorded height was only 17.5 cm (Fig. 3b).

Fig. 3.

Comparison of plant height between transgenic and wild plants at maturity. (a) Representative photograph of transgenic and wild Arabidopsis showing early bolting and increased height. (b) Comparison of plant heights between transgenic (T1–T7) and wild (WT) plants. Data are shown as the mean ± s.e. of seven plants. Vertical bars represent the plant height and the asterisk sign indicates significant difference (P = 0.05) according to Student’s t-test.

Number of leaves and chlorophyll contents

After 32 days of growth, the number of leaves were counted for both transgenic and wild plants. It was observed that transgenic plants gained maximum of 25 more leaves than wild plants that grew with a maximum of 12 leaves (Fig. 4a). Data indicated that transgenic plants accumulated more chlorophyll content as compared to wild plants. The highest record of chlorophyll content was 30.5 μmol m⁻² in as compared to the highest recorded in wild plants, 19 μmol m⁻² (Fig. 4b).

Fig. 4.

Comparison of leaf numbers and chlorophyll contents between transgenic and wild plants. (a) Comparison of leaf numbers between transgenic (T1–T7) and wild (WT) plants. (b) Chlorophyll content analysis in both transgenic (T1–T7) and wild (WT) plants. Data presented as the mean ± s.e. of seven plants. Vertical bars represent the leaf numbers and chlorophyll contents. The asterisk sign indicates significant difference (P = 0.05) according to Student’s t-test.

Stomatal conductance and water use efficiency

The maximum level of stomatal conductance in the transgenic plants was 22 mmol m⁻² s⁻¹ as compared to 12 mmol m⁻² s⁻¹ in wild plants (Fig. 5a). Water use efficiency was increased to a maximum of 2.6 μmol CO₂ μmol⁻¹ H₂O in comparison to 1.5 μmol CO₂ μmol⁻¹ H₂O in wild plants (Fig. 5b).

Fig. 5.

Stomatal conductance and water use efficiency analysis between transgenic and wild plants. (a) Graphical representation for stomatal conductance rate in both (T1–T7) and wild (WT) plants. (b) Water use efficiency analysis in both transgenic (T1–T7) and wild (WT) plants. Data presented as the mean ± s.e. of seven plants. Vertical bars represent chlorophyll content, the asterisk sign indicates significant difference (P = 0.05) according to Student’s t-test.

Photosynthesis rate

Transgenic plants exhibited enhanced photosynthetic rate. Among all the seven tested lines, 2.7 μmol m⁻² s⁻¹ was recorded as the highest rate; whereas the highest photosynthetic rate in wild plants was 1.3 μmol m⁻² s⁻¹ in wild plants (Fig. 6).

Fig. 6.

Analysis of net photosynthetic rate between transgenic (T1–T7) and wild (WT) plants. Data presented as the mean ± s.e. of seven plants. Vertical bars represent photosynthesis rate, the asterisk sign indicates significant difference (P = 0.05) according to Student’s t-test.

Discussion

In the current scenario of environmental pollution, temperature and CO₂ levels are elevated to a greater level, which negatively affects crop growth and productivity (Song et al. 2023). Environmental stress has become exacerbated in C₃ type plants, as they must perform photorespiration, which ultimately causes lower photosynthetic activity (Ferrari et al. 2022). There is an immense need to adopt methods that can modify C₃ type plants to minimise the energy loss created by photorespiration. Various methods have been formulated and adopted for mechanising the C₃ photosynthetic pathway, like RuBisCO modification and engineering a C₄ mechanism of photosynthesis, but the success rate is low as it is quite difficult to bypass photorespiration (Tan and Chen 2023). Cyanobacteria are known to have independent routes for the catabolism of toxic molecules of glycolate into products that are non-toxic. Therefore, cyanobacterial genes have become an interesting and valuable tool as they are in wide use in genetic transformation of plants with a C₃ mechanism, for enhancement of biomass and photosynthesis (Satta et al. 2023). Previously, overexpression of the cyanobacterial gene SBPase in tobacco resulted in an elevated photosynthetic rate (Mitchell et al. 2020). In this report, we were successful in achieving an enhanced photosynthesis rate and change in related parameters like chlorophyll contents, stomatal conductance and water use efficiency. In transgenic plants, 2.7 µmol m⁻² s⁻¹ was recorded as maximum rate of photosynthesis as compared to the 1.3 µmol m⁻² s⁻¹ in wild plants (Fig. 6). In addition, a maximum of 30.5% chlorophyll contents, 22% stomatal conductance and 2.6 μmol CO₂ μmol⁻¹ H₂O water use efficiency was recorded as compared to 1.5 μmol CO₂ μmol⁻¹ H₂O in the wild plant. These results give a clear indication that the transformed gene showed its maximum expression (Fig. 2a). Previously, along with photosynthetic rate, increased leaf surface and chlorophyll contents have been enhanced by using antisense cyanobacterial genes for modifying the photosynthetic mechanism in C₃ plants (Sahrawy et al. 2004).

Plant height in the transgenic plants was higher than the wild type plants (Fig. 3). In this respect, our results are in line with previous experimental studies in potato and Cannabis sativa when transformed with glycolate dehydrogenase genes of cyanobacteria (Nölke et al. 2014). The increased vegetative growth is attributed either to activation of metabolic activities within non-photosynthetic tissues by cyanobacterial enzymes like dehydrogenases and fructose diphosphatase, or alternatively to plants having saved energy by minimising photorespiration, with that energy subsequently redirected to carbohydrate synthesis (Long et al. 2015). It is also suggested that FBPase genes control the carbon metabolism and enhance the sugar contents, which ultimately results in the enhanced phenotypic characters like increased plant height, leaf number and multiple branching (Otori et al. 2017).

In addition, such genes also take control to regulate the contents of the carbohydrates in the transgenic plants (Sahrawy et al. 2004). It was later confirmed that mutant lines of cyanobacterial gene produced the plants with two-fold lower photosynthesis (Rojas-González et al. 2015). On the other hand, with the use of mutant FBPase genes, an increased amount of H₂O₂ was noted and decreased amount of organic acid was also reported. This supports the use of the FBPase gene, not only for controlling photosynthetic machinery enhancement, but also for improving resistance against oxidative stress in transgenic plants.

The success rate of transformation is always understood to be dependent upon many factors like the choice of plant, transformation methods and also the candidate gene (Waheed et al. 2016). Arabidopsis has been a great choice for transformation experiments as it is acquiescent to genetic modification due to its simpler genome and shorter life cycle. The method of genetic transformation was robust, economic and gave stable integration. It also raised the chance of successful transformation chances and, last but not least, the candidate gene does not interfere with the regulatory and growth mechanism of the host plants (Fernie and Yan 2019). However, the results of photosynthesis related tests are required to better understand the mechanism at the transcription level.

Conclusions

The current study reports a robust and successful genetic transformation of A. thaliana to express the cyanobacterial gene FBPase, leading to an enhanced photosynthetic rate (up to 2.7 μmol m⁻² s⁻¹) with higher chlorophyll contents, stomatal conductance and water use efficiency. However, a few areas still need to be explored, like differential transcriptomic analysis of transgenic crops, so that this gene can be utilised to enhance photosynthetic rate in economic crops as well.

Supplementary material

Supplementary material is available online.