Expression of cyanobacterial genes enhanced CO₂ assimilation and biomass production in transgenic Arabidopsis thaliana

Introduction

Atmospheric CO₂ (C_a) concentration has increased due to human activities from 280 ppm in preindustrial times to 400 ppm today (Thompson et al., 2017) and this is considered the major driver of climatic changes (Jovanović et al., 2015). Associated increases in global temperature and reductions in soil water availability are associated with rising C_a, posing significant threats to ecosystem sustainability and crop productivity (Sperry et al., 2019). Plants are the natural carbon sequesters and rely on photosynthesis to sequester and store large quantities of C_a in the form of living plant biomass, both above and belowground (Prior et al., 2011; Sperry et al., 2019).

During photosynthesis, ribulose-1,5-bisphosphate-carboxylase/oxygenase (Rubisco) fixes atmospheric CO₂ (Parry et al., 2013). Rubisco is a bifunctional enzyme which can add CO₂ (carboxylation) as well as O₂ (oxygenation) to the substrate ribulose-1,5-bisphosphate (RuBP) (Janssen et al., 2014). The carboxylation reaction yields two molecules of phosphoglycerate (3-PGA) which is then reduced to sugar molecules in the Calvin Benson cycle (CBC) (Carmo-Silva et al., 2015). However, the oxygenation reaction results in the production of one molecule each of 3-PGA and phosphoglycolate (2-PG) (Carmo-Silva et al., 2015). 2-PG and its immediate product glycolate (GCA) are toxic molecules and need to be metabolized into 3-PGA (Maurino & Peterhansel, 2010). Plants employ photorespiration to catabolize 2-PG and GCA through series of enzymatic reactions that are distributed over chloroplast, peroxisome, and mitochondrion (Hagemann & Bauwe, 2016). Moreover, during photorespiration, hydrogen peroxide (H₂O₂) and ammonia (NH₃) are produced, along with the release of one molecule of already fixed CO₂ (Maurino & Peterhansel, 2010). Photorespiration considerably influences crop productivity by reducing yields in C₃ crops by as much as 50% under severe conditions (Basler et al., 2016; South et al., 2019).

In a rising CO₂ and increasing global temperature scenario in combination with increased abiotic stresses such as salinity and drought, photosynthesis becomes constrained due to decreased stomatal conductance (g_s) which limits CO₂ supply, resulting in an increase in photorespiration (Maiti & Satya, 2014; South et al., 2019). However, under these conditions, photorespiration may have some benefits, by dissipating energy generated during the light reactions and regenerating oxidized nicotinamide adenine dinucleotide (NADP) (Betti et al., 2016). Despite the positive role of photorespiration when photosynthesis is limited by abiotic stresses-mediated enhanced photorespiration this leads to the overproduction of reactive oxygen species (ROS) (Bose, Rodrigo-Moreno & Shabala, 2014; Sharma et al., 2019; Yadav et al., 2020). Interestingly, salinity can lead to the onset of a multitude of responses including osmotic effects, sodium toxicity, and enzyme denaturation, which can further hamper plant metabolic process (Munns & Tester, 2008; Carillo et al., 2011; Negrão, Schmöckel & Tester, 2017). In addition to this, salinity can also inhibit the translocation of photosynthate in plants leading to accumulation of sugars in leaves which may repress the genes encoding the Rubsico enzyme (Moghaieb et al., 2011).

Plants being natural carbon sequesters, can be modified through genetic engineering to enhance CO₂ assimilation and reduce photorespiratory losses (Ruan, Shao & Teixeira Da Silva, 2012). Overexpression of glycolate oxidase (GO), a photorespiratory pathway gene, in rice has been reported to mitigate photorespiration and improve photosynthesis under stress conditions (Cui et al., 2016). Reports on synthetic metabolic pathways that bypass photorespiration, but do not completely eliminate it, have been shown to reduce photorespiratory carbon loss and thereby enhance photosynthetic efficiency and biomass yield (Kebeish et al., 2007; Maier et al., 2012; Dalal et al., 2015; South et al., 2019). In this context, complete photorespiratory bypasses have been engineered in plants by Kebeish et al. (2007), Maier et al. (2012), Dalal et al. (2015), Shen et al. (2019), and South et al. (2019), all of which resulted in an increased plant biomass (Kebeish et al., 2007; Maier et al., 2012; Dalal et al., 2015; Shen et al., 2019; South et al., 2019). Interestingly, engineering of individual genes from these engineered photorespiratory bypasses have also resulted in an enhancement in photosynthetic efficiency (Kebeish et al., 2007; Nölke et al., 2014; Dalal et al., 2015; Ahmad et al., 2016). The premier study on photorespiratory bypasses was reported by Kebeish et al. (2007) in which three enzymes i.e. glycolate dehydrogenase (GDH) comprising of three subunits (D, E and F), glyoxylate carboligase (GCL) and tartronic semialdehyde reductase (TSR) from the bacterium E. coli were utilized to catabolize GCA in the chloroplast. The transgenic Arabidopsis thaliana showed enhanced growth and photosynthetic rate (Kebeish et al., 2007). In addition to the use of a complete bypass, Kebeish et al. (2007) also reported an enhancement in photosynthesis and biomass accumulation by engineering only GDH in Arabidopsis thaliana (Kebeish et al., 2007). Later on, Nölke et al. (2014) also reported an increase in potato yield by engineering GDH alone. Engineering of single gene in these studies has also improved biomass accumulation, however, the exact mechanism behind the increase in biomass is not very much clear. Subsequently, Dalal et al. (2015) utilized only GDH to improve net photosynthesis in the biofuel crop Camelina Sativa (Dalal et al., 2015). The transgenic plants with a single gene substitution exhibited enhanced photosynthesis and biomass accumulation. To elucidate the underlying mechanism associated with the single gene modification, Dalal et al. (2015) also carried out transcriptomic profiling. Transcriptomic analysis revealed that the GDH expressing lines showed upregulation in genes related to sugar transporters, plastidic fatty acid biosynthesis, calcium transporting ATPases, and auxin response proteins (Dalal et al., 2015). This upregulation in different genes suggested an indirect role for the single gene transformation in regulating the plant transcriptome, which improved the biomass accumulation (Dalal et al., 2015).

Interestingly, cyanobacteria also possess three photorespiratory pathways i.e. plant like (photorespiratory pathway), E. coli-like glycerate and a unique glycolate decarboxylation pathway (Eisenhut et al., 2008). The cyanobacterial glycolate decarboxylation pathway decarboxylates GCA into glyoxylate (GOA) through the activity of GDH, which is then converted into oxalate through hydroxyacid dehydrogenase (HDH). Oxalate is catabolized by oxalate decarboxylase (ODC) into formate and one molecule of CO₂ is released, and lastly, a second molecule of CO₂ is released by the catabolism of formate through formate dehydrogenase (FDH) (Fig. 1) (Eisenhut et al., 2008). Interestingly, the cyanobacterial glycolate decarboxylation pathway yields two molecules of CO₂ during GCA catabolism and offers an opportunity to utilize it in plants to improve photosynthesis by reducing photorespiration. The released CO₂ molecules from the decarboxylation pathway can increase the CO₂ concentration in the vicinity of Rubisco, which can improve photosynthesis.

Recently, we have reported transgenic Arabidopsis thaliana plants independently expressing novel cyanobacterial decarboxylation pathway genes i.e. GDH, HDH, and ODC (GD, HD, and OX transgenic plants) (Bilal et al., 2019). The resultant transgenic plants showed improved growth and chlorophyll content (Bilal et al., 2019). These positive results led us to further analyze these transgenic plants to elucidate the underlying mechanism of single gene transformations in regulating photosynthesis, as well as other plant metabolic processes, which can improve plant growth and biomass accumulation. To investigate this, the photosynthetic and growth-related parameters of transgenic plants i.e. GD, HD, and OX, were evaluated. Furthermore, transcriptomic profiling of the GD transgenic plants was carried out to provide an insight into the effect of a single gene transformation on the transcriptome.

These results prompted us to further analyze several photosynthetic parameters, along with seed and plant biomass accumulation and seed germination under salt stress mediated photorespiratory conditions in GDH, HDH, and ODC expressing transgenic plants. The transgenic plants showed variable but general enhancements in gas exchange and fluorescence-related assessments of photosystem II activity. Interestingly, the OX plants also displayed a significant reduction in their relative photorespiration rate. Alongside this, an improvement was also observed in seed and plant biomass as compared to wild type (WT) plants. Moreover, soluble sugars and starch level was also observed to increase in transgenic lines as compared to WT. These results highlight the potential of engineering individual cyanobacterial decarboxylation photorespiratory pathway genes in C₃ plants that can enhance CO₂ assimilation and crop productivity.

Material and Methods

Results

Independent expression of cyanobacterial genes enhanced photosynthetic efficiency of transgenic plants

To identify the effect of transformations with individual bypass genes on photosynthesis, gas exchange measurements were made on 8-week-old soil-grown plants. Different photosynthetic and related parameters including stomatal conductance (g_s), intercellular CO₂ (C_i), net photosynthetic rate (A), water use efficiency (WUE), and transpiration rate (E) were measured in eight-week-old transgenic (GD, HD, and OX) and wild type (WT) plants. An increase of 24% in GD (P = 0.011), 23% in HD (P = 0.037), and 21% in OX (P = 0.084) plants in g_s was observed, respectively as compared to WT plants (Fig. 2A). Despite increase in g_s, no change in C_i was observed among transgenic plants (GD, HD, and OX) as compared to WT plants (Fig. 2B). Among transgenic plants, GD, HD, and OX exhibited 16% (P = 0.035), 11% (P = 0.066), and 11.5% (P = 0.17) increase in A, respectively as compared to WT plants (Fig. 2C). A non-significant decrease was observed for WUE among transgenic plants (GD: −9%, HD: −8%, OX: −7%) as compared to WT plants (Fig. 2D). However, an enhancement of 22.7% in GD (P = 0.002), 19% in HD (P = 0.026), and 16.3% in OX (p = 0.075) plants was observed in E in comparison to WT plants (Fig. 2E). Our results indicated that independent expression of cyanobacterial glycolate decarboxylation pathway genes improved g_s and an associated enhancement of A.

Transgenic plants independently expressing GDH, HDH, and ODC exhibited improved light reaction efficiency

The increased photosynthesis in transgenic lines led us to evaluate the influence of independently expressed cyanobacterial GDH, HDH, and ODC genes on fluorescence related parameters using 8-week-old soil-grown plants to assess any impacts on the operation of PS2. Different light and gas exchange related parameters including light adapted quantum efficiency of photosystem II (ΦPSII), photosynthetic electron transport rate (ETR), quantum efficiency of CO₂ assimilation (ΦCO₂), and respiration rate (Rd) in the dark were measured. Among the transgenic plants examined, GD showed the highest increase in ΦPSII followed by OX and HD (Fig. 3A). A 22% (P = 0.001), 12% (P = 0.020), and 10% (P = 0.084) increase was observed in GD, OX, and HD plants, respectively, in comparison to WT plants (Fig. 3A). For ΦCO_2, 15% (P = 0.019), 10.5% (P = 0.071), and 10.5% (P = 0.162) increase was observed in GD, HD, and OX transgenic plants, respectively, as compared to WT plants (Fig. 3B). Furthermore, the installation of the cyanobacterial genes also led to the enhancement in ETR such that GD, HD and OX plants exhibited a 21.5% (P = 0.001), 10% (P = 0.021), and 12.4% (P = 0.086) increase as compared to WT plants (Fig. 3C). In case of Rd, a non-significant increase was observed at P < 0.05 such that in comparison to WT plants GD, HD, and OX plants showed 5%, 3.5%, and 5% increase in Rd (Fig. 4). These results suggest that the cyanobacterial genes can improve the quantum efficiency in transgenic plants.

Single gene transformation altered the relative photorespiration rate in transgenic plants

Among the transgenic plants, GD plants showed the rate of photorespiration relative to gross photosynthesis (Pr/A_gross) and the ratio to electron rates attributable to carboxylation and oxygenation reactions (J_C/J_O) comparable to WT plants (Fig. 5). Similarly, HD plants showed small, yet non-significant decrease (P = 0.19) in Pr/A_gross and increase in J_C/J_O (P = 0.14) as compared to WT plants (Fig. 5). In contrast, the OX plants displayed significant decrease (P = 0.004) in Pr/A_gross and increase in J_C/J_O (P = 0.01) relative to the WT plants, which indicates a possible partial photorespiratory bypass resulting from the single gene transformation (Fig. 5). These results suggest that the single gene has differently affected photorespiration in the transgenic plants.

Transgenic plants showed improved biomass accumulation

The enhancement observed in photosynthetic and fluorescence parameters prompted us to evaluate the effect of GDH, HDH and ODC genes on seed and plant biomass accumulation. Total biomass of mature plants was harvested and dried for further analysis. All transgenic plants exhibited a significant increase in dry weight as compared to WT plants (Fig. 6A). Among transgenic plants, a 28.5 % (GD), 18.7% (HD), and 26% (OX) increase was observed as compared to WT plants (Fig. 6A). Next, the effect of foreign genes on seed quantity was also determined using seeds from dried siliques (Fig. 6B). In comparison to WT plants, GD, HD, and OX plants exhibited a significant increase of 59%, 55%, and 50% in seed weight, respectively (Fig. 6B). Silique length was also measured, and all the transgenic plants showed a significant increase, GD plants showed a 11% increase followed by HD (6.2%), and OX (2.6%) plants (Fig. 6C). Taken together, these results suggest that the introduction of these genes into C₃ plants can enhance plant biomass.

Introduction of cyanobacterial genes improved the sugars and starch contents in plants

The overall increase in biomass accumulation led us to evaluate the improvement in sugars and starch content. Plants samples were dried, and extracts were made to quantify the sugars and starch. The soluble sugars were significantly increased i.e. 21% (GD plants) and 31% (HD plants), while OX plants showed a 4% decrease in comparison to WT plants (Fig. 7). In the case of starch content, there were significant, 49% (GD plants), 33% (HD plants), and 54% (OX plants) increases compared to WT (Fig. 7). Our results suggest that the improvement in photosynthesis has increased the sugars and starch content which positively influenced the biomass accumulation.

Transgenic plants exhibited improved stress tolerance response

The improvement in photosynthesis and plant biomass accumulation due to the independent expression of GDH and HDH genes under ambient conditions prompted us to evaluate their response under stress conditions by exposing them at different salinities. Growing them on 75 mM NaCl media, GD, HD, and OX exhibited a significant 13%, 15.9%, and 25.5% increase in the germination index (GI) as compared to WT plants (Fig. 8). At a higher salt concentration i.e. 100 mM, there was a significant 20%, 15%, and 36% increase in Gi was observed in GD, HD, and OX plants, respectively (Fig. 8). Our results suggest that the foreign genes have not only enhanced the photosynthetic efficiency but also improve the stress tolerance level in plants.

Principal component analysis revealed influence of photosynthetic and fluorescence parameters towards enhancing biomass accumulation

After evaluating the photosynthetic and fluorescence parameters independently, we set out to elucidate the relative influence of these parameters on the enhancement of photosynthesis and biomass accumulation. For that, principal component analysis (PCA) was performed to analyze the weightage of each parameter (Fig. 9). A total of 98% of the variation was found for the first two principal components i.e. PC1 and PC2. Among the different photosynthetic and fluorescence traits studied, the most significant for the PC1 were A, g_s, ΦPSII, ΦCO₂, ETR, and E. While C_i was the significant positive contributor for the PC2. Finally, the WUE and Rd was found to be least significant contributor for both the PC1 and PC2. Furthermore, the contribution of different parameters towards enhancing photosynthesis also led to the categorization of transgenic plants indicating the influence of each cyanobacterial gene (Fig. 9B). Among transgenic lines, GD plants ranked higher followed by OX, and then HD in comparison to WT (Fig. 9B). All the variables measured and their contribution to the PCA are given in Table 1.

Table 1:

Principal component analysis of plant growth, photosynthetic and fluorescence related parameters.

Variables	PC1	PC2
Net Photosynthesis Rate (A)	0.312	−0.071
Stomatal Conductance (gs)	0.3	0.363
Water Use Efficiency (WUE)	−0.306	−0.169
Intercellular CO2 (Ci)	0.283	0.545
φPSII	0.291	−0.502
Dark Respiration (Rd)	−0.303	0.027
φCO2	0.312	−0.075
Electron Transport Rate (Jmax)	0.291	−0.498
Transpiration Rate (E)	0.309	0.062
Dry weight	0.301	−0.098
Seed Weight	0.308	0.131

DOI: 10.7717/peerj.11860/table-1

Transcriptome analysis of GD plants showed differentially expressed genes in different plant metabolic pathways

The improvement in photosynthetic parameters in GD plants prompted us to evaluate the effect of GDH on other genes which have contributed towards enhancement in biomass accumulation. Transcriptomic profiling of GD plants was carried out and the differentially expressed genes (DEG) were evaluated based on the calculation of FPKM (fragment per kilobases per million). The transcriptomic data was validated through real time RT-PCR of selected genes (data not shown). In total, 1,425 genes were differentially expressed in GD plants, out of which 650 genes were up regulated while 775 genes were down regulated as shown in the volcano map (Fig. 10). The DEGs were categorized into twenty gene ontology (GO) terms for both up regulated and down regulated genes (Figs. 11, 12). Maximum number of genes were over expressed related to abiotic stresses while the overexpression level was high in genes related to photosynthesis and photosystems (Fig. 11). In case of down regulation, maximum number of genes related to single-organism processes were downregulated, however, the highest down regulation in expression was observed in cytoskeleton and microtubules (Fig. 12). The KEGG analysis exhibited upregulation of light harvesting complex (Lhc, a, and b) from photosystem 1 and II (Fig. 13). The genes related to the light harvesting chlorophyll protein complex a (Lhca) were AT3G54890 (Lhca1), AT3G61470 (Lhca2), AT1G61520 (Lhca3), and AT3G47470 (Lhca4) while the genes related to Lhcb were AT2G34430 (Lhcb1), AT2G05100 (Lhcb2), AT5G54270 (Lhcb3), AT3G08940 (Lhcb4), AT4G10340 (Lhcb5), and AT1G05207 (Lhcb6), respectively (Fig. 13). The transcriptomic profiling of GD plants also revealed that genes related to glyoxylate and related metabolic processes, chlorophyll biosynthesis, sugar and starch metabolism, and plant antioxidative defense mechanism were also upregulated (Table 2). Taken together, these results indicate that the engineering of GDH has also affected the plant transcriptome, which improved plant growth and stress tolerance.

Table 2:

Description of important up regulated genes in GD plants and their functions.

Gene ID	Name	Function	Log Fold Change	Reference (related to function)
AT5G13630	Magnesium chelatase	Such enzymes are involved in Mg+2 insertion in protoporphyrin during chlorophyll biosynthesis.	1.32	Mochizuki et al., 2001
AT3G56940	Putative dicarboxylate diiron	Involved in chlorophyll biosynthesis.	1.4	Kauss et al., 2012
AT1G44446	Chlorophyllide a oxygenase	This enzyme is also involved in chlorophyll biosynthesis and performs committed step of conversion of chlorophyllide a to chlorophyllide b. Lack of function mutants are deficient in chlorophyll b, thus hampering light harvesting by photosystem II.	1.62	Yang et al., 2016
AT1G74470	Geranylgeranyl reductase activity	It is also involved in chlorophyll biosynthesis, which converts geranylgeranyl-chlorophyll to chlorophyll a.	1.21	Lange & Ghassemian, 2003
AT3G13790	Cell wall invertase/Glycosyl hydrolase	These proteins are involved in carbon partitioning and cleave sucrose into glucose.	1.47	Minic, 2008
AT3G60130	Beta glucosidase 16	It is involved to provide intermediates during cell wall development such as lignification.	1.1	Sing, Verma & Kumar, 2016
AT2G18700	Trehalose phosphatase/synthase	This gene encodes a protein which is putatively involved in trehalose biosynthesis, which is also involved in osmotic regulation during stress.	1.93	Ponnu, Wahl & Schmid, 2011
AT1G60140	Trehalose phosphate synthase	This gene encodes a protein which is putatively involved in trehalose biosynthesis, which is also involved in osmotic regulation during stress	1.44	Ponnu, Wahl & Schmid, 2011
AT1G23870	Trehalose -6-phosphatase synthase	This gene encodes a protein which is putatively involved in trehalose biosynthesis, which is also involved in osmotic regulation during stress	1.41	Ponnu, Wahl & Schmid, 2011
AT5G55700	Beta amylase 4	This gene encodes a protein which is involved in starch breakdown and localized in chloroplasts.	1.1	Thalmann et al., 2019
AT2G13360	Alanine: glyoxylate aminotransferase	Involved in photorespiration that performs transamination of glyoxylate.	1.03	Liepman et al., 2019
AT1G23310	Glyoxylate aminotransferase	It is involved in glycine biosynthesis and may have localization in peroxisomes and chloroplasts.	1.4	Liepman et al., 2019
AT1G68010	Hydroxy pyruvate reductase	It is involved in oxidative photosynthetic pathway, specifically synthesis of glycerate biosynthesis.	1.03	Timm et al., 2011
AT1G55920	Serine acetyltransferase	Involved in cysteine biosynthesis in chloroplasts and cytosol.	1.1	Tavares et al., 2015
AT5G50950	Fumarase	It is reportedly located in chloroplasts, cytosol and mitochondria. It is involved in fumarate synthesis from malate in leaves and help to promote nitrogen assimilation.	1.4	Nunes-Nesi et al., 2007
AT5G18170	Glutamate dehydrogenase 1	It has mitochondrial localization and supposedly involved in ammonia assimilation.	1.54	Labboun et al., 2009
AT1G13700	6-Phosphogluconolactonase-1	Involved in pentose phosphate pathway. Important source of NADPH-a reducing equivalent.	1.52	Xiong et al., 2009
AT4G39640	Gamma-glutamyl trans peptidase 1	It is present in leaves and degrades glutathione. It is also involved to relieve oxidative stress by metabolizing GSH-glutathione.	1.2	Gong et al., 2005
AT2G02930	Glutathione S-Transferase 16	Member of ubiquitous large gene family involved in mitigation of wide range of stresses predominately oxidative stress.	1.35	Gong et al., 2005
AT1G10360	Glutathione S-Transferase 29	Member of ubiquitous large gene family involved in mitigation of wide range of stresses predominately oxidative stress.	1.1	Gong et al., 2005
AT2G25080	Glutathione peroxidase 1	It is involved in alleviation of oxidative stress through peroxidase activity.	1.84	Hasanuzzaman et al., 2017
AT4G35090	Peroxisomal catalase	Also named as cat2-1, which is involved in H2O2 detoxification in peroxisomes. The loss of function mutant showed elevated H2O2 levels.	1.91	Mhamdi et al., 2010

DOI: 10.7717/peerj.11860/table-2

Discussion

Plants being the natural carbon sequesters, employ photosynthesis to capture atmospheric CO₂ and convert it into aboveground and belowground biomass (Ruan, Shao & Teixeira Da Silva, 2012). It is well known that the process of photosynthesis can be compromised by photorespiratory activity in C₃ plants, which is exacerbated under stress conditions (Dusenge, Duarte & Way, 2019).

In addition to installing a complete photorespiratory bypass, transformations involving a single gene has positively contributed to an enhancement of plant biomass in different C₃ plants including Solanum tuberosum, Camelina sativa and Arabidopsis thaliana (Kebeish et al., 2007; Nölke et al., 2014; Dalal et al., 2015; Ahmad et al., 2016; Cui et al., 2016; Hagemann & Bauwe, 2016). Although the single gene transformation resulted in an improvement in plant biomass the underlying mechanism(s) contributing to this enhancement is largely unknown. Therefore, the role of single gene-associated photorespiratory bypasses in the enhancement of plant growth needs to be elucidated. We recently reported Arabidopsis thaliana independently expressing cyanobacterial glycolate decarboxylation pathway (Fig. 1) genes i.e. glycolate dehydrogenase (GDH: GD plants), hydroxyacid dehydrogenase (HDH: HD plants), and oxalate decarboxylase (ODC: OX plants) (Bilal et al., 2019). The transgenic lines exhibited the expression of the introduced transgenes, increasing growth, and chlorophyll concentration. These results led us to further evaluate these lines for different photosynthetic, fluorescence and growth-related responses, as well as investigating their transcriptomic profile (GD plants), in order to understand the effect of a single gene on the plant transcriptome.

The transgenic plants exhibited improved photosynthetic parameters, including a higher stomatal conductance (g_s), net photosynthesis (A), and transpiration rate (E). However, the intercellular CO₂ (C_i) concentration and water use efficiency (WUE) of transgenic plants was comparable to wild type (WT) plants (Fig. 2). Transgenic plants also showed improvement in light adapted photosystem II (ΦPSII) quantum efficiency, quantum yield of CO₂ assimilation (ΦCO₂), photosynthetic electron transport rate (ETR), and dark respiration rate (Rd) as compared to WT (Figs. 3, 4). Importantly, the OX plants also displayed a reduced relative rate of photorespiration (Fig. 5B) compared to WT plants which hints at a functional, yet partial, photorespiratory bypass as a result of single gene (ODC) transformation. Furthermore, the transformed genes led to a higher biomass accumulation, increased seed production, and silique length in transgenic plants as compared to the WT (Fig. 6). Additionally, the soluble sugar and starch content were also higher in the transgenic lines (Fig. 7). Interestingly, transgenic plants also exhibited a higher seed germination stress index under salt stress conditions (Fig. 8). Additionally, principal component analysis (PCA) revealed the contribution of the studied parameters, including g_s, C_i, A, E, ΦPSII, ΦCO_2, ETR, and WUE towards the overall variation observed in photosynthesis and biomass accumulation (Fig. 9). Furthermore, the transcriptomic profiling showed differentially expressed genes that were both up and down regulated in GD plants (Figs. 10–13).

Our study revealed that the GD plants exhibited a significant increase in A as well as g_s, while a non-significant increase in A and g_s was observed in HD, and OX plants only showed a significant increase in g_s (Fig. 2). An increase in A can be attributed to the enhanced sequestration of atmospheric CO₂ (C_a) due to a higher g_s (Fig. 2). A positive correlation exists between g_s and A such that a higher g_s, will be associated with a higher A (Faralli, Matthews & Lawson, 2019). Furthermore, stomatal aperture is under the control of external and internal ques which can modulate CO₂ uptake and downstream diffusion towards the chloroplast (Faralli, Matthews & Lawson, 2019). In the transgenic plants, there is a need to further elucidate the contribution of the components of stomatal aperture including pore size and internal cues in causing an increase in g_s which were associated with a higher A. In addition to this, there is a need to examine the nutrient requirements of transgenic plants expressing partial or complete bypasses under ambient atmospheric CO₂ (C_a) levels as modifications in N availability or allocation could lead to an enhancement of A. In a kinetic bypass model study, we evaluated the effect of the complete glycolate decarboxylation pathway on A and reported increase in A at ambient C_i concentrations, but in the A-Ci curve, the bypass model attained steady state even at 380 ppm and further increases in C_i up to 950 ppm had no effect on A (Khurshid et al., 2020). Furthermore, we identified that the higher C_i requires more inorganic phosphate (Pi) to cause any further increase in A, which was shown in both the C₃ and bypass models (Khurshid et al., 2020).

Previous studies reported the catabolization of glyoxylate (GOA) through enzymes such as pyruvate dehydrogenase complex (PDC) within the chloroplast resulted in CO₂ release within the chloroplast (Kearney & Tolbert, 1962; Kisaki & Tolbert, 1969; Blume et al., 2013). Oliver & Zelitch (1977) reported enhancement in photosynthesis in the GOA treated tobacco leaf discs mainly because of enhanced CO₂ fixation and reduced GCA level, which is indicative of lower oxygenative photosynthesis (Oliver & Zelitch, 1977). In a latter study, the soyabean mesophyll cells treated with GOA has also been reported that lower concentrations of CO₂ were required for the half maximal velocity of Rubisco which resulted in improved photosynthesis, thereby decreasing the oxygenative reaction and affected GCA synthesis (Oliver, 1980). In line with these reports, the GDH expressing transgenic potato plants exhibited enhanced GOA contents through breakdown of GCA in the chloroplast (Ahmad et al., 2016). The same gene (GDH) was introduced into Arabidopsis thaliana which could have produced the similar results in GD plants. Dalal et al. (2015) has also reported an increase in GOA content and photosynthesis in GDH-expressing Camelina Sativa plants. Interestingly, HD plants also showed an increase in A (Fig. 2C), which likely suggests the catabolization of GCA by HDH. Though the enzymatic activity of HDH in transgenic Arabidopsis plants was not measured, 2-hydroxyacid dehydrogenases (2-HDH), are ubiquitous enzymes having the ability to catabolize multiple substrates in humans as well as in plants (Baker et al., 2004). The enzyme HDH has been implicated in the catabolism of GCA into GOA, which is then converted into oxalate in humans (Baker et al., 2004). Eisenhut et al. (2008) has also reported the catabolism of GOA by HDH in the cyanobacterial glycolate decarboxylation pathway. Considering the role of HDH reported in the literature, we speculate that HDH has utilized GCA as a substrate and catabolized it, causing an increase in A (Fig. 2C), but this needs to be further evaluated. The other enzyme, ODC, is involved in the cyanobacterial decarboxylation pathway, where it catabolizes oxalate into formate and releases one molecule of CO₂ as well (Eisenhut et al., 2008). Despite the enhancement observed in OX transgenic lines (Fig. 2C), the role of ODC in GCA catabolism and biomass accumulation needs further investigation to identify the role of ODC in plant metabolism and photosynthesis.

Photosystem II (PSII) is a highly conserved protein complex, which drives the light reaction of photosynthesis by extracting electrons from water (Liu et al., 2019). It is well documented that a linear relationship exists between ΦPSII and ΦCO₂ (Cheng, Fuchigami & Breen, 2001). ETR depends upon PSII quantum yield and has also been reported as a direct determinant of A (Kubota & Yoshimura, 2002). Our results indicate that the increases in A, in GD (significant) and HD and OX plants (non-significant), are associated with an enhancement in ΦPSII, ΦCO₂ and ETR (Fig. 3) (Cheng, Fuchigami & Breen, 2001; Kubota & Yoshimura, 2002).

It has also been reported that bypass genes led to quick floral induction and plants produced more siliques, which resulted in more seeds being produced (Dalal et al., 2015). Transcriptomic analysis also revealed an enhanced expression of sugar transporters which would increase the transport of photosynthate produced during photosynthesis to the sink cells, thereby increasing seed yield (Dalal et al., 2015). In agreement with this report, our transgenic plants also exhibited a significant increase in silique length in GD and HD plants, although the effect was not—significant in OX plants, and this has contributed to a higher seed weight (Fig. 6).The increase in seed weight can also be correlated to an increase in cauline branches of transgenic Arabidopsis thaliana plants expressing cyanobacterial genes, as reported in our previous study (Bilal et al., 2019). Additionally, we also observed an enhancement in seed size based upon the 1,000 seed weight (data not shown). However, further work would need to be carried out on the underlying reason(s) for the increase in seed size and stored contents. The previous studies on photorespiratory complete as well as partial bypass reported an interesting phenomena that biomass accumulation enhanced almost at double rate than A (Kebeish et al., 2007; Dalal et al., 2015). It needs to be noted that the increased biomass/seed production in our transgenic plants clearly indicates an increased capacity for CO₂ fixation, as photosynthesis provides all the carbon necessary for plant growth. This increased capacity appears to be the result of either (a) photosynthetic upregulation with no change in the relative rate of photorespiration (i.e. GD plants), (b) the result of a decrease in the relative rate of photorespiration even without a significant photosynthetic upregulation (i.e. OX plants) or (c) a combination of a small photosynthetic upregulation and a small decrease in the relative rate of photorespiration (i.e. HD plants).

Engineering of photorespiratory bypasses, complete as well as partial, and modifications in other genes, such as phosphophenol pyruvate carboxylase (PEPC), cytochrome c₆ (Cyt c₆), and pyruvate phosphate dikinase (PPDK) have resulted in improved photosynthesis, as well as an enhancement in soluble sugars and starch (Kebeish et al., 2007; Xia & Cao, 2013; Nölke et al., 2014; Dalal et al., 2015; Yadav et al., 2018; Yadav, Rathore & Mishra, 2020). Nölke et al. (2014) have also reported an enhancement in sugar contents by transforming GDH alone in potato. Furthermore, Dalal et al. (2015) has also related the enhancement in A with an increased level of starch production. In our case, soluble sugars were significantly increased in the GD and HD transgenic plants while starch content was significantly enhanced in all the transgenic plants (Fig. 7). Gago et al. (2016) has also reported that sugars and organic acids can influence g_s and positively affect A (Gago et al., 2016). Interestingly, OX showed a decline in sugars, although there was an increase in starch content and the reason for this needs further investigation.

Seed germination is a crucial stage which is required for successful growth of plants (Eastmond & Graham, 2001). Seeds need to be able to utilize stored reserves and convert them into a utilizable form to support plant growth (Bewley & Black, 2013). The glyoxylate cycle has also been reported in seeds and is utilized to generate energy in the form of ATP (Eastmond & Graham, 2001). Stressful environmental conditions cause deleterious effects on seed germination (Duan et al., 2014). Cui et al. (2016) overexpressed glycolate oxidase (GO) in rice at the whole plant level which resulted in an enhanced H₂O₂ concentration that triggered the stress tolerance response in transgenic plants (Cui et al., 2016). Contrary to Cui et al. (2016) our results indicate that at a relatively low salt concentration, the transgenic plants exhibited a significant increase in the seed germination index (Gi) as compared to WT plants, possibly due to the fact that the cyanobacterial genes do not produce H₂O₂ (Fig. 8). Transcriptomic profiling of GD plants also revealed the overexpression of oxidative stress tolerance related genes, which might have improved the stress tolerance response. Interestingly, among the transgenic plants, OX plants exhibited the highest tolerance as compared to GD and HD plants which may be attributed to oxalate metabolism by ODC (Fig. 8). However, this notion gives an indication that ODC expressing plants can be further evaluated for their potential to reduce the impact of other abiotic stresses as well. It has already been reported that the degradation of oxalate results in an improved stress tolerance specifically against fungal diseases and heavy metal stress (Donaldson et al., 2001; Wan et al., 2009; Xian et al., 2020). Ghosh et al. (2016) has also reported that oxalate reduces the oxidative burst during stress and regulates the defense mechanisms involved.

The contribution of different traits towards enhancing photosynthesis and biomass accumulation among transgenic plants can vary and few traits have more influence in causing overall variability than the others (Fig. 9). Dissection of traits as per their contribution in causing overall variability among the plants was carried out using PCA analysis (Afsar et al., 2020). In our study the PCA analysis identified A, g_s, ΦPSII, ΦCO₂, ETR, and E as significant traits positively associated with the enhancement of plant productivity (Table 1, Fig. 9A). Furthermore, the contribution of the traits identified through the PCA analysis has also shown that the contribution of genes to the improvement in biomass accumulation, followed the order GD > HD > OX (Fig. 9B).

The transcriptomic analysis provides an opportunity to identify other targets which can be manipulated to further enhance photosynthesis as well as biomass accumulation (Luo et al., 2019; Peng Yuan et al., 2020). Dalal et al. (2015) has reported 587 DEGs related to the amino acid biosynthesis pathway, cytochrome b, tryptophan biosynthesis, and superoxide dismutase, through transcriptomic profiling of E. coli GDH expressing transgenic plants. Similarly, in our study, transcriptomic profiling of GD plants not only showed that the genes related to photosynthesis were mainly overexpressed but also revealed the highest number of genes over expressed in response to abiotic stimulus (Figs. 10, 11). In the GD plants, ETR was significantly higher than WT plants, and the transcriptomic analysis also revealed an enhancement in genes expression related to the light harvesting complex (Lhc, a, and b) (Fig. 13) which further supports the role of GDH in enhancing photosynthesis. Concomitantly, overexpression of chlorophyll biosynthesis genes, such as magnesium chelatase (Mochizuki et al., 2001), putative dicarboxylate diiron (Kauss et al., 2012), chlorophyllide a oxygenase (Yang et al., 2016), and geranylgeranyl reductase (Lange & Ghassemian, 2003) was also observed in GD plants (Table 2). The mutants lacking the chlorophyllide a oxygenase gene in rice has been reported to impair the light harvesting complex, thereby affecting plant yield (Yang et al., 2016). Moreover, genes related to sugar and starch metabolism, such as cell wall invertases/Glycosyl hydrolase (Minic, 2008), β-glucosidase 1-6 (Singh, Verma & Kumar, 2016), trehalose phosphatase/synthase (Ponnu, Wahl & Schmid, 2011) and beta amylase 4 (Thalmann et al., 2019) were also upregulated (Table 2). Glycosyl hydrolases are involved in cell wall polysaccharide metabolism, modifications in sugars and secondary metabolites (Minic, 2008). β-amylases have diverse roles in plants, such as starch degradation in plastids, modulation of stomatal opening and transcriptional regulation in association with brassinosteroid signaling (Thalmann et al., 2019). In addition to these genes, glyoxylate metabolism related genes were also observed to be upregulated, including glyoxylate aminotransferase (Liepman et al., 2019), hydroxypyruvate reductase (Timm et al., 2011), serine acetyltransferase (Tavares et al., 2015), fumarase (Nunes-Nesi et al., 2007) glutamate dehydrogenase (Labboun et al., 2009), and 6-phosphogluconolactonase-1 (Xiong et al., 2009) (Table 2). Interestingly, these upregulated genes have a role in the biosynthesis of different metabolites involved in downstream reactions of the photorespiratory pathway involving glycine and glycerate metabolism, ammonia synthesis, and the production of reducing equivalents (Labboun et al., 2009; Xiong et al., 2009; Timm et al., 2011; Liepman et al., 2019). Consequently, the enhanced expression of these genes suggests an influence of GDH transformations on the regulation of the photorespiratory pathway. Furthermore, the transcriptomic analysis also revealed the overexpression of genes related to oxidative stress mitigation, such as glutathione S-Transferase (Gong et al., 2005), glutathione peroxidase 1 (Hasanuzzaman et al., 2017), and peroxisomal catalase (Mhamdi et al., 2010) (Table 2). These genes are thought to have a key role in reducing the oxidative burst during stress conditions (Gong et al., 2005; Mhamdi et al., 2010; Hasanuzzaman et al., 2017). Therefore, their upregulation resulting from the engineering of GDH indicates that they might have contributed to the increased salt stress tolerance of seed germination in GD plants.

Conclusions

The current study revealed that the transformation of a single gene from the cyanobacterial glycolate decarboxylation pathway into Arabidopsis thaliana has the potential to enhance different photosynthetic, fluorescence and growth-related parameters. Elucidation of the underlying mechanism(s) involved via transcriptomic profiling of GD plants revealed an upregulation of the photorespiratory pathway, sugar and starch metabolism, and chlorophyll biosynthesis pathway genes. Based on these results the transformation of a single gene, has the potential to not only influence photorespiration but also modify genes in different metabolic pathways, with a consequent improvement in plant productivity. However, there is a need to carry out transcriptomic (HD and OX plants) and metabolomic profiling of GD, HD, and OX plants to enhance our understanding of the indirect effects of engineered cyanobacterial genes on improvements in plant biomass.

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