Abstract
Plant physiologists set about comprehending the genesis of the C4 photosynthetic pathway after its discovery by Hatch and Slack. They discovered that a sophisticated combination of morphological and biochemical adaptations allowed the plant to concentrate CO2 around RuBisCO to achieve maximum efficiency. We categorize the evolutionary events leading to C4 photosynthesis, beginning with anoxygenic photosynthesis and the evolution of RuBisCO to the cooling of Earth by the Great Oxygenation Event that led to the oxygenic photosynthesis. The evolutionary descent of the C4 plants is a phenomenon that occurred around 30 million years ago. Due to industrialization and population growth, improved photosynthetic efficiency and carbon fixation of C4 plants could contest the current global scenario of rising CO2 concentration. C3 crops engineered with C4 traits, implemented on a large scale, could impact the climate globally. Here we discuss the various strategies used to introduce C4 traits in the C3 plants and the potential techniques to be considered for successful hybridization.
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Introduction
In 1950, Melvin Calvin, Andrew Benson, and James Bassham discovered and elucidated the Calvin/C3 cycle (Bassham et al. 1950). The Calvin cycle lies at the epicentre of plant metabolisms by which plants fix CO2. This pathway also led to the discovery of the enzyme Ribulose-1, 5 Bisphosphate Carboxylase Oxygenase (RuBisCO) in 1963. RuBisCO, a widely studied plant enzyme, occupies 50% volume of the soluble protein in leaves. Despite being the most abundant enzyme in the world and fixing nearly 90% of inorganic carbon into biomass, RuBisCO is inefficient. (Ellis 1979; Sharkey 1988; Hayes 1994). Due to its dual activity as a carboxylase and oxygenase, the enzyme experiences substrate competition resulting in low specific activity and bifunctionality. This leads to photorespiration, an energy-intensive process that causes a loss of CO2.
A more efficient form of photosynthesis evolved as the warming climate exacerbated the competition between the RuBisCO substrates. This pathway, known as the Hatch and Slack/C4 cycle, concentrates carbon around RuBisCO by utilizing pre-existing anatomy and enzymes with biochemical and anatomical variations. To optimize CO2 assimilation, the Phosphoenolpyruvate Carboxylase (PEPC) enzyme with carboxylase activity was localized to the mesophyll cells, while RuBisCO was localized deep in the anatomy within the bundle sheath cells. This enables direct delivery of CO2 to bundle sheath cells, maintaining high concentrations around the RuBisCO active site and eliminating photorespiration.
While only a small percentage of plant species (3%) utilize the C4 pathway, it plays a significant role in the planet’s overall primary productivity (Sage et al. 2012). This is made possible by the efficient resource use of C4 plants, which is achieved through their unique anatomical and biochemical properties that help to suppress photorespiration. This overall improvement in photosynthetic efficiency of the C4 plants propelled the idea of introducing C4 traits to improve C3 crops. After years of research, scientists have made progress in understanding the process of photosynthesis and how it can be genetically modified in C3 plants to include a C4 pathway. This review examines the physiology and evolution of photosynthetic pathways, with the goal of exploring how the C4 route can be introduced into C3 crops and what impact this could have in the future.
Evolution of C3 photosynthetic group: the plant pioneers
Photosynthesis is an incredible process that dates back to the early days of our planet. While it has transformed over time, the basic premise of harnessing the sun’s energy to sustain life has remained constant. Around 3.9 billion years ago, the atmosphere lacked oxygen, which caused the evolution of anoxygenic prokaryotes (as shown in Fig. 1A). It relied on methane and sulphur for survival (Farquhar et al. 2007). Evidence such as stromatolites, microbial ecosystems, and biofilms that depend on anoxygenic photosynthesis suggests that this type of life evolved first (Tice and Lowe 2004; Westall et al. 2006a, b; Westall et al. 2006a, b; Allwood et al. 2007; Westall et al. 2011). Organisms that used oxygen began to emerge when Earth’s temperature stabilized roughly 3.5 billion years ago, as noted by Planavsky et al. in 2014. Over time, the level of atmospheric oxygen gradually rose, with geological features such as sedimentary deposits providing evidence of this increase from around 2.4 billion years ago (Arndt and Nisbet 2012).
Anoxygenic to oxygen-rich green Earth transition A Anoxygenic Earth with microbial flora and their subsequent transformation to a stromatolite (limestone deposits forming reefs due to precipitation activity of microbial community) ecosystem with deposits of trapped oxygen B The Great Oxygenation Event saw the emergence of cyanobacteria as its forerunners. Autotrophic forms then developed through endosymbiosis to give rise to photosynthetic eukaryotes
Atmospheric Oxygen levels rose to nearly 2% after the evolution of cyanobacteria, supplemented by various metabolic processes that produced oxygen as a by-product (Holland 2006; Rasmussen et al. 2008). The evolution of cyanobacteria coincided with ‘The Great Oxygenation Event’, driving the oxygen levels to almost 20%, nearing present-day levels (Holland 2006). Ancestral heterotrophic eukaryotes engulfed cyanobacteria through endosymbiosis (Fig. 0.1B). Because the preserved cyanobacteria acted as a functional organelle, these eukaryotes evolved the capacity for photosynthesis. Due to this, cyanobacteria is considered the origin of chloroplasts and is credited for the oxygenic atmosphere we see today (Yoon et al. 2004).
Molecular evidence suggests that land fungi first appeared around 1.6 billion years ago and land plants 680 million years ago (Heckman et al. 2001). These organisms used an ancestral form of RuBisCO, which evolved before the Great Oxygenation Event, for carbon fixation (Erb and Zarzycki 2018). Its carbon concentrating mechanism transported CO2 to RuBisCO in the form of HCO3− with the negative charge restricting its diffusion across membranes. Early photosynthesis utilized only the C3 pathway for its carbon fixing. RuBisCO had fewer competition and errors because the CO2 concentration was significantly higher than O2 (Fig. 2).
Evolution of RuBisCO. A Non-carboxylase ancestral RuBisCO evolved in three forms. B Form III Heterotrophic Proto-RuBisCO evolved first in Archaebacteria followed by C Form II Homoligomeric RuBisCO with non-autotrophic carboxylation and Form II Autotrophic RuBisCO with autotrophic carboxylation. D Form I RuBisCO the advanced form where the green type has higher affinity towards oxygen, and red type has an equal affinity towards oxygen and carbon dioxide. E The functional variations among RuBisCO’s ascending evolutionary forms. F Bifunctional RuBisCO with carboxylation and oxygenation activity with the involvement of three organelles, viz., chloroplast, peroxisome and mitochondrion
The carboniferous era (340 million years ago) saw unfavorable atmospheric conditions for photosynthesis (Sage 1999; von Caemmerer 2000). As the O2 levels rose to 21% and CO2 fell to 0.04%, the oxygenase activity of RuBisCO started reducing the efficiency of photosynthesis. Since C3 plants rely on diffused CO2, it had relatively low CO2 in the vicinity of RuBisCO than its surroundings. With increasing O2, the C3 cycle loses CO2 through Photorespiration, increasing temperature, and excessive competition between the substrates. By then, RuBisCO had integrated itself well as the primary photosynthetic catalyst. Despite evolving early and playing a dominant role in carbon fixation, it had slow catalytic and high error rates. When present in equal concentrations, RuBisCO had a higher affinity toward O2, resulting in photorespiration (van Lun et al. 2014). Metabolism of its toxic by-product, phosphoglycolate, causes a loss of nearly 25% carbon (Ogren 1984; Andrews and Lorimer 1987; Douce and Heldt 2000).
Evolution of C4 photosynthesis
The C4 pathway has evolved over 61 times in 19 families consisting of grasses, sedges, and eudicots (Sage 2016). Multiple independent lineages and their auxiliary function to the C3 pathway suggest genetic preconditioning for the evolution of the C4 pathway. It is disputed that the C4 pathway originated due to falling CO2 levels. CO2 concentration reduced to 370 ppm around 25 million years ago, but C4 plant expansion happened 5–10 million years ago when there was no drastic reduction in CO2 concentration. Though most C4 plants growing in arid regions require precipitation to complete their life cycle, many C4 wetland species have very low drought tolerance.
Some studies suggest that the C4 pathway evolved to adapt to increasing temperature (Osmond et al. 1982; Hattersley 1983; Sage et al. 2018). Temperatures greater than 30 °C can inhibit photosynthesis by up to 30% (Jordan and Ogren 1984; Sage and Sharkey 1987; Ehleringer et al. 1991; Sage 2003). Elevated temperatures lead to carbon limitation by excessive photorespiration. Reduced stomatal conductance lowers CO2 levels and triggers Photorespiration (Guy et al. 1980; Schulze and Hall 1982; Sage 2004). Since high temperature triggers photorespiration and dark respiration, it compensates for the losses incurred by carbon deficiency. (Brooks and Farquhar 1985; Sage and Sharkey 1987).
Several studies show the over-expression of some Calvin cycle genes and a gradual evolutionary trajectory of biochemical and anatomical stages during the transitionary stages of some C3 plants (Emms et al. 2016; Moreno-Villena et al. 2018). Anatomical preconditioning required for a functioning C4 pathway includes dimorphic chloroplast, spatially separated enzymes, closely arranged Mesophyll and Bundle sheath cells (Fig. 3A), reduced interveinal distance, increased bundle sheath size, etc. (Raghavendra 1980; Ehleringer et al. 1997). Recruiting housekeeping genes and re-routing them to perform carbon concentrating mechanisms without comprising their original task suggest gene duplication (Monson 2003). Since the loss of function of genes has a lethal or debilitating effect, having multiple copies of a gene that can be modified and assigned different tasks seems prudent (Lynch and Conery 2000).
Anatomy and physiology of C3 and C4 plants A Histology of NAD-ME dorsiventral C4 leaf showing the mestome (a non-chlorophyllous layer of tissue between the bundle sheath and vascular bundle) along with Kranz features B Histology of isobilateral C4 leaf showing the Kranz features C C3 pathway ( Calvin cycle) showing the enzyme, substrate and carbon assimilation D C4 pathway ( Hatch and Slack pathway) showing the enzyme, substrate and carbon assimilation along with decarboxylation enzymes
Evolutionarily, RuBisCO had reached a dead-end. Though some plants evolved RuBisCO with a higher affinity towards CO2, it was detrimental to the enzymes’ s turnover rate (Andrews and Lorimer 1987). Instead of improving RuBisCO, a trait evolution of the existing biochemical pathway occurred (Doebley and Lukens 1998). Within the cell, pre-existing enzymes had their roles reassigned: PEP carboxylase, playing a vital role in Kreb’s cycle and a ubiquitous enzyme in eukaryotes was recruited for the carbon assimilation due to its carboxylase activity (Chollet et al. 1996); housekeeping enzymes like NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEPCK) were re-routed to aid in the decarboxylation process (Wedding 1989; Edwards & Andreo 1992; Drincovich et al. 2001). In addition to the reassigned enzymes assisting in carbon assimilation and transport, C4 plants also had a fully functional C3 pathway for carbon fixation (Fig. 3B). The reassigned enzymes fix the CO2 and release it in the vicinity of RuBisCO for the C3 cycle, thus reducing the competition between the substrates and ensuring optimum RuBisCO efficiency.
Enzymes for assimilation and fixation are localized to specific cell types or locations within the cells. From studies conducted on C3-C4 intermediate plant species like Flaveria, Panicum, Moricandia, etc., bundle sheath cells showed increased organelle and photosynthetic tissue partition (Brown and Hattersley 1989). Increased bundle sheath size and chloroplast number improve light absorbance. Improved chloroplast numbers within the Bundle sheath cells also act as metabolic sinks for Glycine metabolic pathway. The glycine shuttle helps process the accumulated photorespiratory products, thus increasing the CO2 in the bundle sheath cells and reducing CO2 loss (Brown and Hattersley 1989; Rawsthorn 1992). Marked alterations in metabolite transport and locations are observed during the evolutionary transition from C3 to C3-C4 intermediates to C4 plants (Borghi et al. 2022). Some C3-C4 intermediates also show increased vein density and reduced distance between bundle sheath cells when compared to C3 relative of the plant (Zhu et al. 2022). The increased proximity between bundle sheath and mesophyll cells reduces interveinal distance, improves conductance, and provides structural integrity. The increased proximity also helps retain water by reducing transpiration and improving water use efficiency.
The idiosyncrasy of the C3 and the C4 pathways
Calvin–Benson cycle or the C3 pathway
There are four known CO2 fixing pathways: the reverse citric acid cycle, the reductive acetyl-CoA cycle pathway, the 3-hydroxypropionate pathway, and the Calvin Cycle. The Calvin cycle (the Calvin–Benson–Bassham cycle, the C3 cycle, or the reductive pentose phosphate cycle) is the only pathway seen in plants. Due to the endosymbiotic origin of the C3 cycle, many enzymes and pathways followed by the C3 cycle have a prokaryotic origin.
RuBisCO, central to the plant carbon metabolic pathway, evolved in non-autotrophic conditions. The current autotrophic bases in RuBisCO involved in the Calvin cycle evolved later in bacteria. RuBisCO is the only enzyme that needs to be coded by nuclear and chloroplast DNA. The subunits of RuBisCO that need to be assembled for its function are coded by the nuclear and chloroplast genome. Since RuBisCO evolved before ‘The Great Oxygenation Event’, its two substrates did not face much competition. After the increase in O2 concentration, the enzyme learned to discriminate between CO2 and O2 but at the cost of its catalytic rate.
The Calvin cycle uses ATP and NADPH from the light reaction to fix atmospheric CO2. The enzymes required for CO2 fixation are present in the stroma of the chloroplast, and it functions in a light-dependent manner. The fixed carbon gets converted into simple carbon skeletons that combine to form starch or sucrose for storage (Woodrow and Berry 1988; Geiger and Servaites 1994; Quick and Neuhaus 1997). This cycle involves 13 reactions and 11 different enzymes to catalyze those reactions.
RuBisCO catalyzes the carboxylation reaction of the carbon-accepting molecule Ribulose-1, 5-bisphosphate (RuBP). Carboxylated RuBP consumes ATP and NADPH to release a molecule of glyceraldehyde-3-phosphate (G-3-P) and a molecule of dihydroxyacetone phosphate (DHAP). The majority of the triose phosphate is recycled to regenerate RuBP; the rest goes into storage for the growth and development of the plant (Fig. 3B). Calvin cycle also helps supply intermediate metabolites to various other pathways, cementing its role quite central to plant carbon metabolism (Lichtentahler 1999).
The bifunctionality of RuBisCO results in an oxygenase reaction where the active site is taken up by O2 instead of CO2. This process is called photorespiration or the C2 oxidative photosynthetic carbon cycle. The Calvin cycle and photorespiration occur alongside each other (Ogren 1984; Leegood et al. 1995). RuBP undergoes oxygenation in a reaction catalyzed by RuBisCO. The oxygenated RuBP produces a 3-carbon 3-Phosphoglyceric acid (3-PGA) and a 2-carbon 2-Phosphoglycolate (2-PG) in the chloroplast. 2-PG is hydrolyzed to glycolate by two other organelles: peroxisome and mitochondria (Tolbert 1981).
The transporters in the envelope membrane transport the glycolate into the peroxisome via diffusion. Glycolate oxidase oxidizes it to produce glyoxylate and copious amounts of hydrogen peroxide (H2O2). Transamination of glyoxylate with glutamate eliminates H2O2 and produces glycine. Glycine enters the mitochondria, where Glycine decarboxylase (GDC) assisted by NAD+ produces serine. It requires two molecules of glycine to produce one molecule of serine. α-ketoglutarate transaminates serine to produce hydroxypyruvate after re-entering the peroxisome from the mitochondria. An NADH-dependent reaction reduces the hydroxypyruvate into Glycerate. Phosphorylation of Glycerate takes place in the chloroplast to yield 3-PGA.
Photorespiration involves the circulation of multiple compounds within these organelles. Thus, two molecules of 2-PG (four carbons) move through the peroxisome and mitochondria to undergo photorespiration. It returns to the chloroplast as one molecule of 3-PGA (three-carbon). Thus, 25% of the carbon assimilated by the plant remains lost, while the rest returns to the Calvin Cycle. Photorespiration of the C3 pathway compensates for the energetically costlier C4 pathway.
Hatch and Slack or the C4 pathway
The C4 pathway is a system of complex anatomical and biochemical modifications to the C3 cycle. These modifications increase CO2 concentration in the vicinity of RuBisCO by spatially separating enzymes within the cells or organelles (Edwards et al. 2004). PEPC does the inorganic carbon fixation, producing a 4-carbon acid called Oxaloacetate (OAA) in all variant forms of the C4 plants. The OAA is transported to RuBisCO for decarboxylation. Decarboxylation maintains saturated levels of CO2 at the RuBisCO active site to avoid competition between the substrates, thus reducing the concentration of RuBisCO utilized by the C4 pathway. Since nitrogen is a primary component of RuBisCO, its reduced requirement also improves nitrogen use efficiency (NUE) (Oaks 1994).
C4 plants require coordination between the cells (mesophyll and bundle sheath) to accommodate various enzymes’ spatial separations (Dengler and Nelson 1999). Though some plants have enzymes compartmentalized within the same cell, most plants have a Kranz Anatomy formation (Offermann et al. 2015). Kranz anatomy comprises a wreath-like structure of cells for spatially separating the enzymes. This layer of cells surrounds the vascular bundle. The outermost layer comprises Mesophyll cells, called the photosynthetic carbon assimilation tissue (PCA), which houses the PEPC enzyme. It is closest to the cell surface for easy diffusion of CO2. The inner layer consists of a layer of parenchyma cells called bundle sheath cells Calvin cycle occurs within this tissue called photosynthetic carbon reduction tissue (PCR), because it contains RuBisCO and the other Calvin cycle enzymes (Dengler and Nelson 1999). These layers separate the primary and secondary carbon fixation reactions.
Depending on the predominant enzymes catalyzing the decarboxylation reactions, C4 photosynthesis is classified into three subtypes. The three decarboxylating enzymes are NADP-ME, NAD-ME, and PEPCK. Most C4 plants and crops, such as Maize, Sugarcane, Sorghum, etc., belong to the NADP-ME. Plants like switchgrass, amaranth, pearl millet etc., belong to the NAD-ME subtype. PEPCK subtype co-exists with either NADP-ME or NAD-ME due to energy constraints between the bundle sheath cells and mesophyll cells (Sage 2004; Meister et al. 1996; Furbank 2011; Wang et al. 2014). The overlap of pathways indicates the presence of both subtypes in an evolutionary C4 ancestor (Gowik et al. 2011; Washburn et al. 2015).
The initial CO2 assimilation by carbonic anhydrase and fixation by PEPC in OAA is common to all subtypes. In NAD-ME plants, the aspartate produced from OAA is reduced by deamination to malate in the mesophyll cytosol. NAD-Me decarboxylates malate in the mitochondria of bundle sheath cells. The malate in NADP-ME is transported from the chloroplasts of mesophyll cells to the mitochondria of bundle sheath cells for decarboxylation. PEPCK plants use both aspartate and malate for transport. The aspartate is produced in mesophyll cells’ cytosol and decarboxylated in bundle sheath cells’ cytosol. These enzymes transport the 3-carbon compound produced back to PEPC, which converts it to pyruvate. Pyruvate orthophosphate dikinase (PPDK) phosphorylates the pyruvate to regenerate the primary CO2 acceptor, Phosphoenolpyruvate (PEP) (Hatch 1987).
Compared to C3 plants, C4 plants have larger and more active bundle sheath cells with more chloroplasts. The mesophyll and bundle sheath cells are highly connected using plasmodesmata to transfer metabolites efficiently. There are anatomical variations within the subtypes as well. NAD-ME has a layer of non-photosynthetic mestome sheath cells between the bundle sheath cells and the vascular bundle. The mestome sheath cells are absent in the NADP-ME subtype. Differences in grana development and chloroplast arrangement can be seen within the subtypes (Gutierrez et al. 1974; Hattersley and Watson 1976; Lundgren et al. 2014). The cellular developmental origins of the same cells from different subtypes vary. In NADP-ME, the procambium and ground meristem develops into bundle sheath and mesophyll cells. But in NAD-ME subtypes, bundle sheath and mesophyll cells develop from ground meristem while mestome sheath develops from procambium.
Evolutionary lineages of certain C4 grasses and sedges show PCR tissues composed of cells other than bundle sheath proper. These cells, called the Mestome sheath cells, form the outer layer of the vascular bundle and are anatomically similar to bundle sheath cells. But they have distinct developmental variations. While Bundle sheath cells develop from the ground meristem, Mestome sheath cells develop from vascular meristem (Soros and Dengler 2001). In some C4 lineages, the Parenchyma cells found between the mesophyll and water storage cells host RuBisCO and decarboxylating enzymes. Many plants show modifications in the PCR tissues to reduce the efflux of CO2, like suberization of the walls, which involves impregnating the outer PCR walls with Suberin. But this does not appear to be the norm for C4 plants (Dengler and Nelson 1999). The anatomic design of some plants restricts the exit of CO2 by strategically placed organelles like chloroplasts and vacuoles. Single-celled aquatic plants like Hydrilla verticillata ha ve enzymes localized into organelles to compartmentalize various reactions. PEPC fixes the carbon in the cytosol, which gets delivered to the chloroplast where RuBisCO and NADP-ME are located (Reiskind et al. 1997ab; Casati et al. 2000; Bowes et al. 2002).
The preponderant pathway
C4 plants have an immediate physiological advantage in the present scenario concerning carbon concentration mechanisms to overcome the loss faced during photorespiration. Though they only account for 3% of all the plants, they contribute nearly 23% of the gross primary productivity. It is a complex assimilation of anatomical and biochemical modification to counter the various fallibility of the ancestral C3 pathway. To compensate for the losses incurred by the competition between substrates, mesophyll cells of C3 plants encompass more RuBisCO. Production of RuBisCO, in turn, requires a considerable amount of nitrogen. C4 plants, on the other hand, operate at saturated CO2 levels due to the efficient carbon concentrating mechanism. Hence the amount of RuBisCO needed is relatively less, and nitrogen use efficiency (NUE) is better.
An increase in CO2 levels in the future might level the playing field in favor of C3 plants since they have more room for improvement. The photosynthetic rates might be the same in both plant types. But C4 plants will be energetically costlier than C3 plants due to reduced photorespiration, thus making C4 plants less efficient. An increase in CO2 will reduce Photorespiration in C3 plants. Increment in global warming and greenhouse gases is going to increase the CO2. Once the CO2 crosses the threshold beyond which C4 plants might have no competitive advantage, C3 and C4 will have similar photosynthetic rates.
Some studies show improved yield and WUE in C4 plants in elevated CO2 conditions (Li et al. 2019). Since summers and winters might get warmer, both C4 and C3 plants have advantages. The increasing CO2 favors woody C3 plants, where C4 plants predominantly exist since they encroach and shade over the grass. (Bond et al. 2003; Davis et al. 2007; Bond and Midgley 2012). Many grasslands worldwide have seen invasions by woody C3 species, which could result from elevation in atmospheric CO2 (Polley et al. 1996; Bond et al. 2003; Bond and Midgley 2012). This invasion could result in a reduction in C4 species over the coming years as the shade from tree canopies restrict the sunlight needed for their growth. But they could flourish in regions with nutrient deficiency due to their efficiency (Sage and Kubien 2003). C4 plants also show a higher tolerance to nanomaterial toxicity (Bai et al. 2021).
Engineering C4 traits into C3 plants—retrospection
The discovery of the C4 photosynthetic pathway in the 1960s made us aware of the refinements that could be made to the C3 route. The polyphyletic nature of its origin led to the understanding that both C3 and C4 plants could be within the same genus. Closely related plants of the same genus were used for hybridization to understand the genetic traits and segregation and to enhance the prospect of producing fertile progeny of higher quality. Atriplex rosea (C4) and Atriplex triangularis (C3) were the first plants to be crossed to obtain a hybrid. The experiment failed to produce seeds or a functioning C4 pathway (Bjorkman et al. 1969).
Various other hybridization experiments were carried out between other Atriplex C3 and C4 plants (Osmond et al. 1980; Kadereit et al. 2010). Efforts to produce C3 × C4 hybrids shifted to C3, C4, and C3-C4 intermediate plants of Flavaria and Panicum species. But the hybrids produced were not viable, and the ones that survived had various genetic aberrations. (Brown et al. 1986; Apel et al. 1988; Brown and Bouton 1993). These studies demonstrated the necessity of Kranz anatomy and the C4 biochemical pathway for a completely operational C4 photosynthetic pathway (Björkman 1976; Brown and Bouton 1993). Though some hybrids were viable, they did not significantly improve their photosynthetic efficiency. Some hybrids had compensation points (Γ), carboxylation efficiency, and NUE values intermediate to the C3 and C4 values. Although some hybrids displayed certain overlapping characteristics with its C4 parent, due to independent segregation of the traits, inheriting all the traits of C4 parents seemed unlikely. (Brown and Bouton 1993; Oakley et al. 2014). Eventually, all the hybridization studies and experiments were abandoned by the late 1990s.
By the 2010s, hybridization studies increased due to the emergence of the bioinformatics and sequencing era. This helps in getting intricate genetic variations between the hybrids. The hybrids’ genetic, anatomical, and biochemical shortcomings can be identified accurately and rectified. Proto-Kranz-like traits were induced in C3 Rice by expressing development and organelle volume-increasing genes from Maize (Wang et al. 2017). Recently five photosynthetic enzymes were introduced into C3 rice as a single construct for the introduction of C4 characters (Ermakova et al. 2021). A photorespiration bypass inspired by the CO2 concentrating mechanism in the C4 pathway was engineered in Arabidopsis thaliana, a C3 plant (Roell et al. 2021). Engineering rice has been a priority among various studies to address the overpopulation concern (von Caemmerer et al. 2012; Lin et al. 2016, 2020; Giuliani et al. 2019; Ermakova et al. 2020). Extensive research is glacially inching toward a C4 rice prototype. The information gained by these works is being evaluated for furthering the research on C4 engineering into C3 crops.
The Chenopodiaceae family had plants with a single-celled C4 system. Since earlier research indicated that C4 photosynthesis required anatomical segregation (Kranz anatomy), scientists reacted skeptically to the discovery of a single-celled C4 terrestrial system. The single-celled system worked in two novel pathways (Freitag and Stichler 2000; Voznesenskaya et al. 2001). The spatially separated cytoplasmic domains had dimorphic chloroplasts, each type constrained to each cytoplasmic domain. Either the chloroplasts are localized to either end of the cell, which is elongated in Suaeda aralocaspica, or they are restricted to the periphery and center of the cell, as witnessed in the Bienertia species (von Caemmerer et al. 2014). The cells are analogous to the cells in C4 plants. The cells where carboxylation of CO2 takes place are identical to the mesophyll cells, while the RuBisCO-containing cells act as the bundle sheath cell analog. In the case of the peripheral arrangement of the cells, only the cells in the center could do the Carbon reduction step, while the outer cells handled the assimilation. These cells showed high carbon assimilation rates, at par with C4 plants, evidencing little photosynthetic disadvantage due to the lack of Kranz anatomy (Edwards et al. 2007).
Deciphering these single-celled C4-like plants can provide valuable insights to support ongoing efforts of engineering the C4 pathway into C3 plants. Since it is challenging to replicate a functional Kranz anatomy, understanding the mechanisms of single cells that perform the C4 pathway as efficiently as real C4 plants can be beneficial (Offermann et al. 2011). Han et al. 2023 explored the complex genetic and metabolic pathways that enables Bienertia sinuspersici to perform efficient photosynthesis within individual cells without Kranz anatomy. Functional enrichment analyses indicated an upregulation of genes involved in energy metabolism particularly those linked to cyclic electron flow. This is essential for adapting the photosynthetic machinery to the demands of single-celled photosynthesis. Comparative analysis of chloroplast genomes of C3 and C4 plants (including the single-celled C4 type) revealed the underlying genetic changes might not be as extensive as previously assumed (Sharpe et al. 2020). Further studies involving genome sequencing of Suaeda aralocaspica were also performed to get an understanding of these plants. The study underscored the importance of redox homeostasis and energy metabolism regulation in establishing and maintaining a single-celled pathway.
In aquatic settings, show several carbon-concentrating mechanisms. There is low inorganic carbon accessibility due to inadequate diffusion in aquatic environments. The increasing O2 concentration resulted in high photorespiration rates. This lead to high RuBisCO’s oxygenase activity. Freshwater plants like Hydrilla verticillata, Egeria densa, Elodia canadensis Sagittaria subulata, and Orcuttia viscida perform facultative C4 photosynthesis within a single cell (de Groote and Kennedy 1977; Keeley 1998; Casati et al. 2000; Bowes et al. 2002). Marine macroalgae and diatom Udotea flabellum and Thalassiosira weissfloggi also show C4 metabolism (Reiskind & Bowes 1991; Reinfelder et al. 2000; Johnston et al. 2001). Extensive studies were done on H. verticillata to characterize the single-cell system (Rao et al. 2002; Estavillo et al. 2007; Miyao et al. 2011). It is a freshwater angiosperm, performing the C3 pathway during the winters when it grows on open water and the C4 pathway during the summer when it is submerged. The low CO2 levels and high O2 levels in the water, warm temperature, and high irradiance induce the C4 pathway in these plants. There is no structural modification taking place to accommodate this phenomenon. Since the plant lacks the presence of Kranz anatomy, a characteristic feature of terrestrial C4 plants, the compartmentalization of enzymes such as PEP carboxylase and RuBisCO into separate cells is not perceived (Reiskind et al. 1997a, b). They include two morphologically distinct chloroplast-containing cells encasing PEP carboxylase and RuBisCO in their cytosol and chloroplast, respectively (Magnin et al. 1997).
These findings suggest that we can improve the efficiency of C3 plants by adopting the intracellular compartmentalization and metabolic routing observed in single-celled C4-like plants. Gnanasekaran et al. 2016 engineered the dhurrin pathway into tobacco chloroplasts to enhance metabolic processes using photosynthetic reducing power. Similar techniques could be utilised to direct C4 photosynthetic traits into C3 chloroplasts. The intracellular organization observed in single-celled C4-like plants can be replicated using techniques like CRISPR-Cas9. Existing genes can be adjusted to enhance the segregation of photosynthetic functions within chloroplasts of C3 plants. This approach could bypass some of the cellular energy transfer limitations inherent in conventional photosynthesis, thereby enhancing carbon fixation efficiency.
The C4 isoenzymes present in the plant, such as PEP carboxylase, PPDK, and NADP-ME, are upregulated during the summer to accommodate environmental fluctuations. The plant also demonstrates typical C4 qualities viz., low Γ rates, minimal RuBisCO oxygenase reaction, and increased net photosynthetic rates (Salvucci and Bowes 1981, 1983). This inducible C4 pathway was also showcased by Eleocharis vivipara, an amphibious leafless sedge. E. vivipara exhibits C3 biochemical characteristics when submerged underwater but develops Kranz anatomy and the accompanying C4 biochemical traits under aerial conditions. This complete process appears to be regulated by abscisic acid (Ueno 1998). Experiments trying to replicate the Hydrilla-like C4 pathway in Rice were attempted by overexpressing PEP carboxylase, PPDK, NADP- Malate dehydrogenase and NAD-malic enzyme (Ku et al. 1999; Fukayama, et al. 2001; Tsuchida, et al. 2001; Taniguchi et al. 2008).
A recent study showed improved photosynthetic efficiency, plant height and grain weight by over-expressing PPDK and NADP-ME genes of model plant Setaria italic in Rice (Swain et al. 2021). Over-expression of NADP-dependent Malate dehydrogenase from Suaeda monoica in C3- Tobacco also enhanced photosynthesis and carboxylation (Haque et al. 2022). Overexpression of cytoplasmic carbonic anhydrase of C4-Flaveria bidentis in C3- Arabidopsis thaliana showed improved photosynthetic capacity, amino acid and WUE (Kandoi et al. 2022). Though they have all been successfully overexpressed individually, only those transgenic Rice which had all four enzymes overexpressed showed improvement in photosynthetic rates (Taniguchi et al. 2008). But that wasn’t contributed by the carbon concentrating mechanism of the introduced pathway.
C3 plants were found to have two sets of genes. One set codes for the housekeeping enzymes of C3 pathway and the codes for the C4 pathway enzymes albeit at insignificant level of expression (Ku et al. 1996). This result produced by comparative studies and the simplicity of single-cell photosynthesis in H. verticillata led to a series of overexpression studies. These studies done using DNA recombinant technology, with enzymes being overproduced at the exact location needed, have aided in understanding its physiological impact. Even though the overproduction of a single enzyme is not improving the photosynthetic efficiency of the plants, positive effects were seen on some physiological characteristics.
Overexpression of chloroplastic PPDK improved the number and weight of the seeds being produced in tobacco (Sheriff et al. 1998). Overproduction of C4-PEP carboxylase in transgenic Rice helped the root elongation by conferring resistance to aluminium (Miyao et al. 2001). Studies also show drought and stress-induced C4 syndrome in plants, which suggests exposure to abiotic stress could induce a C4 pathway (Zhu et al. 2022). Microalgae are also known to have higher photosynthetic efficiency than both C3 and C4 plants due to faster growth in photosynthetic cells and higher RuBP regeneration rate (Treves et al. 2022). This could also be used as a template for improving C3 pathway.
The advent of next-generation sequencing significantly impacted the C4 photosynthetic pathway research. It is a compelling platform for gene discovery, which can be exploited in genetically engineering a C4 pathway into a C3 plant. Using whole-genome sequencing and RNA expression analyses, various insights into the evolutionary relationship between C3 and C4 plants were provided. The genes and transcription factors responsible for the evolution of the C4 pathway have also been identified by comparing sequence information of closely related C3, C4, and C3-C4 intermediate plants.
Few plant species belonging to the Chenopodiaceae family, such as Haloxylon aphyllum and Haloxylon persicum, were found to have different photosynthetic pathways within various photosynthetic organs. The cotyledons were shown to perform the C3 photosynthetic pathway, while the assimilating roots performed the C4 pathway. When the transcriptomes of both the organs were compared, 2959 genes were found to be differentially expressed (Li et al. 2015). Studying the gene expression pattern of developing C4 plants and attempting to replicate them in C3 plants such as Rice is also being attempted (Li et al. 2010). In silico mining of large amounts of data available is being done to check the hypotheses of gene function and to develop DNA constructs for creating transgenic plants.
Forecasting the role of transgenesis in rewiring C3 plants
Since the C4 route was discovered more than 50 years ago, there has been an information explosion about its biology and physiology. But the intricate molecular data flow is often stalled due to various barriers, primarily due to the C4 enzymes’ polymeric nature, as it loses its regulatory function after isolation. The C4 anatomy, spatial separation of enzymes within a single cell or different cells, makes physiological values challenging to decipher. Evaluating metabolite levels becomes tedious since they are compartmentalized into various cellular organelles. The lack of proper model systems has hindered the growth of the information. Plants such as maize and sugarcane are quite tricky to grow in a laboratory environment. Setaria viridis is a potential model C4 grass due to its successful genetic transformation mediated by Agrobacterium (Brutnell et al. 2010). Though the complete sequencing of the model has been done, it is still being developed to become a full-fledged model system.
In 1987, Terada et al. 1987 conducted a groundbreaking study in conventional plant breeding, utilizing somatic hybridization to transfer traits between sexually incompatible species. The experiment involved the fusion of protoplasts from C3 rice and C4 barnyard grass. While many of the outcomes displayed abnormal morphological features, a few stable hybrids exhibited distinctive traits. This research demonstrated the feasibility of transferring both nuclear and cytoplasmic genomes between species with differing photosynthetic capabilities. Unfortunately, the hybrid plants did not thrive for an extended period, leaving uncertainty regarding the cause of their eventual demise. In 1993, Wang et al. 1993 carried out a study involving the fusion of protoplasts from maize and an intergeneric wheat hybrid. This effort yielded stable hybrids, primarily with chromosomes inherited from a single parent, although one hybrid displayed intermixed chromosomes. Notably, these hybrids proved to be more stable compared to hybrids created through traditional sexual hybridization between maize and wheat, as established in earlier studies by Laurie and Bennett 1986 and 1989, as well as Laurie et al. 1990. Chromosome elimination was identified as a hindrance to hybridization, but this study offered promising prospects for gene transfer between species.
In 1997, researchers conducted somatic hybridization between C3 rice and C4 Megathrysus maximus using PEG-mediated fusion. This intricate process involved the deactivation of the C3 protoplasts with iodoacetamide and the C4 protoplasts with X-ray treatment before asymmetric fusion (Hua-wei et al. 1997). Although the fusion process resulted in hybrids, these hybrids exhibited exaggerated sexual characteristics, altered floral morphology, and low fertility. Xu et al. (2003) conducted a remarkable experiment utilized PEG-mediated fusion to combine albino maize, characterized by limited regeneration abilities, with non-dividing wheat mesophyll protoplasts. The outcome of this fusion process was the production of green callus. Microscopic analysis confirmed the successful hybridization, with maize cytoplasm mixing with wheat chloroplast. However, the hybrid plants that could be regenerated only produced sterile male and female flowers.
Understanding the gene regulation and molecular biology of the C4 pathway is incomplete. Well elucidates studies showing the evolution of C4 photosynthesis from C3 pathway in the genus Flaveria can be utilised in developing a C4 plant (Munekage and Taniguchi 2022). With numerous genes being differentially expressed between C3 and C4 plants, it is a formidable task to incorporate the C4 pathway into C3 with incomplete knowledge. Since the anatomical and biochemical parts of the C4 pathway are essential for obtaining a functional C4 system, a lack of expertise in the genes involved in Kranz anatomy makes transgenic C4 plant development difficult.
Localizing various enzymes, which also play housekeeping roles within the cell in different compartments and carbon fixation, is an added challenge and must be regulated strictly. Deconstructing every single aspect of the C4 pathway and trying to replicate it externally without knowing the complete picture has resulted in more failures than successes. A complete sync between multiple factors is necessary for obtaining a functioning C4 pathway.
Conclusion and future perspectives
It’s fascinating to note that the C4 pathway has evolved independently on over 61 occasions, utilizing enzymes and metabolites that already exist in the C3 pathway. The components of the C4 pathway are essentially being redirected towards greater efficiency. Given the lack of novelty involved and the sheer number of times this pathway has evolved independently, it is certainly plausible to engineering the biochemical networks into C3 crops.
Hybridization and transgenesis in plants have not enhanced the photosynthetic efficiency to our expectations. Techniques such as protoplast fusion can be considered alternatives for the randomized incorporation of the C4 pathway in C3 plants (Fig. 4). Complete fusion of the cell as in somatic hybridizations will unravel the causes of independent segregation of traits and identify every key photosynthetically crucial genetic component. As elaborated earlier in this review, some hybrids produced by somatic hybridisation showed genetic stability.
The theoretical workflow of rewiring C3 traits using protoplast fusion A Heterofusion of C3 and C4 protoplast B Multigene natural transgenesis as a consequence of heterofusion C Products of heterofusion and its natural reproduction D Real-time physiological analysis of the offspring E Gene expression analysis from cellular stage to differentiated states F Metabolomic analyses of parental lines and offspring
The use of robotic platforms and AI is being explored to standardize regeneration, trait identification, and selection in modern breeding programs (Ranaware et al. 2023). Yogadasan et al (2023) used machine learning to study plastic genome evolution in the C4 pathway to identify and predict C3 and C4 status. Alloteropsis semialata is an ideal plant system for studying the genomic evolution of C4 photosynthesis due to its unique combination of C3, C3-C4, and C4 members. Its genome has already been elucidated, providing many insights (Pereira et al. 2023). Synthetic biology techniques have the potential to enhance plant photosynthesis by creating new pathways and modifying existing ones to improve the process (Batista-Silva et al. 2020). This can be achieved by targeting specific light reactions, improving electron transport, and optimizing carbon fixation through innovative biochemical routes. Integrating pathways from single-celled C4-like plants or non-plant systems (such as bacterial genes) into C3 plants can also be explored, increase the efficiency of photosynthetic reactions. Although C4 molecular determinants and evolutionary pathway is extensively studied, various gaps need to be addresses.
The growth of the human population shows no signs of stopping, and their accomplishments escalate the climate change issues. The ever-increasing temperatures and CO2 will have a different impact on the growth of plants and their produce. It has been predicted that the current food being produced will not be able to feed the world population by the year 2050. The efficiency of the majority of the crops has been saturated. Until photosynthetic genetic and molecular information can be collected through sequencing and data mining, different engineering strategies, such as protoplast fusion and grafting for increasing photosynthetic efficiency in plant populations can be attempted.
Data availability
The data supporting the conclusions of this study are included within the article.
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Acknowledgements
The authors thank The Manipal Academy of Higher Education (MAHE) providing the infrastructure for carrying out the research towards drafting this review.
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Open access funding provided by Manipal Academy of Higher Education, Manipal. The authors thank The Manipal Academy of Higher Education (MAHE) for providing the prestigious Dr. T. M. A. Pai Ph.D. scholarship to NSM and for providing the infrastructure for carrying out the preliminary research towards drafting this review.
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Nidhi S Mukundan: Conceptualization, Investigation, Writing – original draft, Writing – review & editing. Kapaettu Satyamoorthy: Writing – review & editing. Vidhu Sankar Babu: Conceptualization, Writing – review & editing, Visualisation, Supervision, Project administration.
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Mukundan, N.S., Satyamoorthy, K. & Sankar Babu, V. Investigating photosynthetic evolution and the feasibility of inducing C4 syndrome in C3 plants. Plant Biotechnol Rep (2024). https://doi.org/10.1007/s11816-024-00908-2
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DOI: https://doi.org/10.1007/s11816-024-00908-2