Metabolic Engineering of C3 Plants to Improve Crop Yield
Authors: Dr. Bunty Sharma and Ajay Kumar

Global demand and consumption of agricultural crops for food, feed, and fuel is increasing at a rapid pace. Improving crop yield to meet these demands of an increasing world population is a central challenge for plant biology. This goal must be achieved in a sustainable manner (i.e. with minimal agricultural inputs and environmental impacts) in the face of elevated levels of CO₂ and more extreme conditions of water availability and temperature. To satisfy the growing, worldwide demand for grain, two broad options are available: (1) The area under production can be increased or (2) productivity can be improved on existing farmland. These two options are not mutually exclusive and both will be employed to produce the additional 200 million tonnes/year of corn (Zea mays) and wheat (Triticum aestivum) estimated to be needed by 2017. Both options will alter the environmental footprint of farming. Of the two options, increasing productivity on existing agricultural land is preferable as it avoids greenhouse gas emissions and the large-scale disruption of existing ecosystems associated with bringing new land into production. Through application of new technologies together with novel modeling approaches, increased yield through improved photosynthetic carbon fixation should be an attainable goal in the near to midterm.

In the plant kingdom, there are three pathways of photosynthetic, atmospheric CO₂ fixation. Most of the crops, such as wheat, rice, soybean or potato are classified as C3 plants, fix CO₂ using the enzyme Ribulose1,5 bisphosphate carboxylase/oxygenase (RubisCO) in the Calvin-Benson cycle or C3 cycle [as first stable product is a three-carbon compound, phosphoglycerate (3-PGA)], which plays a central role in plant metabolism, providing intermediates not only for starch and sucrose biosynthesis, but also for isoprenoid metabolism and shikimic acid biosynthesis. However, competition of O₂ with CO₂ at the active site of RubisCO results in a loss of up to 50% of the carbon fixed in a process known as photorespiration. Environmental variables, such as high temperature and drought, can result in an increase in the oxygenase reaction. Two metabolic pathways additional to the C3 cycle have evolved to overcome this, the C4 (first stable compound synthesized is a C4 acid, oxaloacetate) and crassulacean acid metabolism (CAM) pathways. In C4 and CAM plants, PEPcarboxylase (PEPC) plays an important role in the mechanism of CO₂ assimilation. The primary product of PEP carboxylation, oxaloacetate (OAA), is either reduced to malate or transaminated to aspartate in the mesophyll cells of C4 plants. Malate or aspartate is then transported to the bundle sheath cells where CO₂ is released at high rates. Since, bundle sheath cells have a low gas permeability, the CO₂ concentration in solution is drastically increased, which causes a suppression of the RubisCO oxygenase activity and consequently, photorespiration. CAM plants attain increased concentrations at the site of RubisCO by a temporal separation of carboxylation and decarboxylation. In comparison to C3 crops, C4 crops have higher yields, reduced water loss and increased nitrogen use efficiency, particularly when grown in hot and dry environments.

What Limits C3 Photosynthesis

RubisCO enzyme:

A large proportion of the limitation to carbon assimilation in plants using the C3 cycle is due to the catalytic properties of the enzyme RubisCO (Portis and Parry, 2007). As RubisCO is not only an inefficient enzyme but also has a low turnover number. Transgenic plants with reductions in RubisCO protein levels demonstrated clearly that the limitation imposed by RubisCO on C3 carbon fixation is greatest in high light and temperature conditions, which reduce in plants grown in elevated CO_2.

RubisCO activation:

RubisCO is active only when the ε-amino group of its lysine (at position 201) residue reacts with CO₂ to form a carbamate (carbonic acid amide), to which an Mg ion is bound. The activation state of RubisCO is dependent on the enzyme RubisCO activase, which is found to be responsible for the reversible temperature-sensitive reduction in the activation state of RubisCO, leading to inhibition of photosynthesis when plants are subjected to mild heat stress (≥30^oC).

Regenerative capacity:

The regenerative phase of the cycle also plays a role in determining the rate of photosynthesis. Antisense plants with reduced levels of individual enzymes, involved in regeneration of CO₂ acceptor molecule RuBP, revealed that a small reductions in the enzyme sedoheptulose-1,7-bisphosphatase (SBPase), transketolase (TK) and aldolase, resulted in a decrease in CO₂ fixation and growth, identifying these enzymes as a major control point in the C3 cycle.

Past Successes to Improve the C3 Cycle:

Understanding the responses of the calvin cycle to altered demands for photosynthate within the plant and to external environmental conditions is essential for attempts to increase yield and to redirect carbon into important products.

RubisCO-related bottlenecks:

Three approaches to improve the C3 cycle have targeted RubisCO-associated bottlenecks. The first is to increase the carboxylation efficiency of RubisCO by reducing the oxygenase reaction. Expression of the cyanobacterial ictB protein (involved in HCO₃^- accumulation) in higher plants to reduce the oxygenation reaction of RubisCO resulted in the production of transgenic tobacco (Nicotiana tabacum) with higher photosynthetic rates under limiting, but not saturating, CO₂ levels. A second approach introduced the enzymes of the bacterial glycolate pathway into plants, creating a photorespiratory bypass in the chloroplast. Escherichia coli posses a glycolate catabolic pathway that use glycolate as a sole carbon source. Expression of the pathway associated enzymes viz. glycolate dehydrogenase (GDH), glyoxylate carboligase (GCL) and tartronic semialdehyde reductase (TSR) in Arabidopsis, resulting in reduced photorespiration and low CO₂ compensation point. The introduction of a thermostable version of the RubisCO activase enzyme produced by gene shuffling into C3 plants implies the third approach, showing increased CO₂ assimilation and biomass yield. A new opportunity has also arisen from interspecies comparison of RubisCO catalytic properties that has revealed variation in the specificity, k_cat, and temperature response of RubisCO from natural vegetation in the Mediterranean. Successful expression of RubisCO large and small subunits as a single fusion protein in chloroplast that assembled into an active holoenzyme provides the opportunity to use expression of foreign RubisCO as a strategy to improve C3 cycle.

Regenerative capacity of the C3 Cycle:

Overexpression of a plant SBPase in tobacco plants resulted in increased photosynthetic CO₂ fixation and growth. Analysis of CO₂ response curves revealed that this increase in photosynthesis could be attributed to an increase in the capacity to regenerate the CO₂ acceptor molecule RuBP. Plastid aldolase and TK were also identified as a potential target to increase the regenerative capacity of the C3 cycle.

Overexpression of C4 cycle enzymes in C3 plants:

In laboratories around the world attempts to introduce single cell C4-like CO₂ concentrating mechanism into terrestrial C3 plants by a transgenic approach are in progress. It is believed that the introduction of an intracellular CO₂ pump might improve the efficiency of C3 photosynthesis by a substantial suppression of photorespiration. There has been progress in single, double and multiple overexpressions of C4-cycle enzymes like PEPC, NADP-malic enzyme, PEP carboxykinase and pyruvate orthophosphate dikinase in C3 crops with improved photosynthesis and yield. Fundamental research aimed at understanding the molecular basis of the anatomical specialization and the regulatory processes determining the location and the level of expression of the C4-specific enzymes is under way. Although this has not yet led to any major breakthroughs, the combination of new sequencing and proteomic technologies together with a large international program give optimism for future success in this area.

Future prospective

Engineering of C3 cycle offers an opportunity to increase photosynthesis and yield. A number of clear targets have been shown to have the potential to impact yield in the 3- to 5-year period but to date have been tested only in model species. It is now important that this knowledge is fully exploited in crops. The range of genetic and molecular techniques that are now available, together with the development and application of rapid in vivo techniques to allow in-field analysis of a wider range of species in their natural environments, will facilitate the wider analysis of natural variation in photosynthetic carbon assimilation. This approach has enormous and unexplored potential for future exploitation to improve yield through manipulation of the C3 cycle.

Conclusion

In modern agriculture, in which water, light and nutrients can be abundant, carbon fixation could become a significant growth limiting factor. Being the primary pathway of carbon assimilation in the majority of photosynthetic organism, manipulation of the C3 cycle offers an opportunity to increase photosynthesis and yield. Several approaches like manupulating RubisCO activity, overexpression of different metabolic enzymes and insertion of thermostable RubisCO activase, improve the C3 cycle, but only with limited success. All these findings suggest existing avenues of exploration in the grand challenge of enhancing food and renewable fuel production via metabolic engineering and synthetic biology.

References :

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Whitney, SM, Houtz, RL and Alonso, H (2011). Advancing our understanding and capacity to engineer nature’s CO₂-sequestering enzyme, Rubisco. Plant Physiol. 155: 27–35.

Hibberd, JM and Covshoff, S (2010). The regulation of gene expression required for C4 photosynthesis.

Annu Rev Plant Biol. 61 : 181–207.



About Author / Additional Info:
I have completed my Ph.D. in Biochemistry. I am currently working as a research fellow for 2.5 years in CCS HAU, Hisar.