Journal of Plant Biology & Soil Health

Review Article

Molecular and Genomic Tools for Improving Photosynthetic Efficiency of C3 Species in the Changing Climates

Tsegaye Babege1* and Temesgen Magule2

  • 1Mizan-Tepi University, College of Agriculture and Natural Resources, Mizan Teferi, Ethiopia, Africa
  • 2Hawassa University, College of Agriculture, Hawassa, Ethiopia, Africa

*Address for Correspondence: Tsegaye Babege, Mizan-Tepi University, College of Agriculture and Natural resources, P.O Box 260, Mizan Teferi, Ethiopia, Africa, E-mail: babege2010@gmail.com
Citation: Babege T, Magule T. Molecular and Genomic Tools for Improving Photosynthetic Efficiency of C3 Species in the Changing Climates. J Plant Biol Soil Health. 2017;4(1): 4.
Copyright: © 2017 Babege T, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Journal of Plant Biology & Soil Health | ISSN: 2331-8996 | Volume: 4, Issue: 1
Submission: 02 February, 2017 | Accepted: 10 March, 2017 | Published: 15 March, 2017

Summary

There is an urgent need to increase crop productivity beyond the existing yield potentials to tackle the challenge of food security in the near future. One of the areas of plant physiology that promises hope in overcoming this problem is increasing photosynthetic efficiency of C3 plants. In C3 plants photosynthesis is limited by features of the carbon-fixing enzyme called Rubisco because it promotes a parallel reaction that captures oxygen, thereby reducing photosynthesis by initiating photorespiration. Here, we review recent information’s about the potential blockages of C3 photosynthesis in a changing climate, with attention to future targets to overcome this problem. Lastly, emerging approaches to improving C3 photosynthetic efficiency are discussed, and recent advances in molecular and genomic techniques needed to realize these improvements are presented.

Introduction

Today seven crop species in the Poaceae family coupled with other specialty crops feed over 7 billion people in the world. But the current rate of increase in crop yield does not sufficient to feed the ever increasing population in the future. This can be explained by the fact that the actual crop yield in most areas of the globe is approaching the ceiling of maximal yielding potential. For example, yield of rice per hectare in Africa was increased only by 18% between 1979 and 1998 and only by 9% between 2007 and 2013 [1]. Similar yield stagnation is evident in other areas of the globe. Moreover, improvement in some staple food crops in Africa such as cassava and yam are even slower than the major cereal crops. For example, the global average increase in yield per hectare of yam, a major staple for West Africa, between 1979 and 2013 was 41%. This is less than half of the 108% increase for maize over the same period; which became a big worry especially for the poorest countries in the future.
The discrepancy observed especially in the first half of 21st century, makes us to review: why actual yield of a crop reach its maximum yielding potential? In the second half of the 20th century, packages such as high-yielding varieties and management techniques developed by some agricultural scientists brought about an unprecedented boom (more than double) in world grain yields. This is the so-called green revolution, one of the 20th century’s greatest technological achievements. The achievement of the first green revolution is great in increasing crop yield. But it seems to be inadequate to feed the exponentially increasing global population, due to the fact that the measures adopted in the first green revolution are now no longer important in increasing crop yield. Due to this fact one may ask: what are the possible ways of dealing with it? The realization of future global food and energy needs will require improvements in crop productivity and carbon fixation. One fundamental component of plant productivity that has yet to be utilized to potential for increasing yield is photosynthesis. Photosynthesis is the most important metabolic process relative to crop productivity, in which the maximum dry matter yield of crop plants is derived from photosynthetic CO2 assimilation [2-5].
Improvement in photosynthetic efficiency of a crop can be possible mainly by way of DNA transfers through genetic engineering. Recombinant DNA technique is a recent technological development which provides us with the means to engineer changes to photosynthesis that would not have been possible previously. Gene editing technique as a sharp tool in improving crop photosynthetic efficiency has been reported by several earlier authors [6]. These approaches are valuable, especially as long-term adaptation strategy to alleviate climate change impacts. In this review, it has been examined the potential blockage of C3 photosynthesis and an important part of future strategy to overcome the obstruction of C3 photosynthesis. Lastly, the recent genetic engineering tools needed to realize these improvements are overviewed.

What are the Potential Blockages of C3 Photosynthesis?

Photosynthesis in C3 plants is limited by features of the carbon-fixing enzyme Rubisco, which exhibits a low turnover rate. Rubisco typically represents 50% of the soluble protein in the leaf and is the most abundant protein on the world [5,7]. It is not only the world’s most abundant protein but also the world’s most incompetent enzyme. Why such an abundant enzyme is incompetent? This is because Rubisco is an enzyme having two functions, catalyzing both carboxylation and oxygenation. The oxygenation reaction of Rubisco puts an extra cost on the cell in re-assimilating the carbon lost in this process. The process of oxygenation reaction induces photorespiration. Photorespiration imposes a large penalty on net photosynthetic efficiency, which increases with temperature [3,5]. In fact temperature has a significant effect on the solubility of both gases but the relative solubility of O2 is greater than that of CO2 as the temperature increases. In addition, oxygenation increases relative to carboxylation due to the fact that the reaction between the 2,3-enediol intermediate of Rubisco and O2 has a higher free energy of activation than its reaction with CO2. Therefore, loss due to photorespiration rises from up to 30% in cool climates to more than 50% in hot climates. Lowering photorespiration therefore would reduce the inhibition of net photosynthesis of C3 plants. To avoid such loss, it appears that Rubisco in plants has evolved to become more specific for CO2.

Emerging Strategies to Improving Photosynthetic Efficiency in C3 Plants

In view of this, more recent researchers have been identified different alternative strategies to increasing photosynthetic efficiency in C3 plants. Some of the identified targets include; engineering of a full C4pathway into C3 plants, the introduction of improved Rubisco from other species and introducing carbon-concentrating mechanisms.
Introducing C4 metabolism into C3 species
Carbon dioxide is a competitive inhibitor of the oxygenation reaction of Rubisco. Evolution has exploited this in some photosynthetic organisms by the addition of structures to compartmentalize Rubisco and pathways that concentrate CO2 in that compartment. C4 photosynthesis is one solution that has a carbon concentrating mechanism involving two cell types, the mesophyll and bundle sheath cells [5,8]. In C4 plants, which include the crops maize, sorghum, sugarcane, and grain amaranth, Rubisco is isolated to an inner green bundle sheath surrounding the leaf veins. In these plants, carbon dioxide is first captured by carboxylation of phosphoenolpyruvate (PEP) to form a C4 dicarboxylate in an outer photosynthetic tissue or mesophyll and is then transferred to the inner tissue that it surrounds the bundle sheath. Here, it is decarboxylated, releasing pyruvate that is then recycled back to the outer tissue, where it is phosphorylated back to PEP to complete the cycle [5].
Indeed, one approach to increasing photosynthetic efficiency in C3 crops such as wheat and rice is to convert them to C4 plants. But it needs various structural and functional changes. The first manipulation that is necessary is the structural change. This includes the differentiation of the two cell types, increases in bundle sheath size and in their complement of organelles, such as chloroplasts, mitochondria and peroxisomes. Typically, a C3 leaf might have 9-10 mesophyll cells between adjacent bundle sheath cells, whereas in C4 leaves, this reduces to 2-3 mesophyll cells, so that mesophyll and bundle sheath cells are in close proximity [8]. The second manipulation that is necessary is functional change. This involves expressing proteins, whether they are enzymes or transporters, in different cells and organelles, in different amounts and certainly, in some cases, isoforms that are more suited to their role in C4 rather than C3 photosynthesis, such as PEP carboxylase.
With genetic engineering, ability to transform plants at the nuclear and plastid (chloroplast) level opened up a new world of possibilities for novel C4 crops [9,10]. The main method has been to target PEP carboxylase and over-express this enzyme by genetic engineering in tobacco, potato, rice and Arabidopsis. In most of these endeavors, the maize version of PEP carboxylase was used, and its gene was driven with a 35-S promoter, so the enzyme was produced constantly. Effects varied: more efficient use of CO2, excess malate production, less oxygen inhibition of photosynthesis, improved dark respiration, a neutral state of no effect, negative effects such as chlorosis, less sugars and more acids, reduced phosphorylated metabolites, and even stunted growth. The enzyme NADP-malic enzyme has also been over expressed to remove the malate bottle neck and it did improve efficiency. When both enzymes together have been over expressed in the same cell, results were promising in one species and neutral (no improved photosynthesis) in another. PEP carboxykinase (PCK) from the C4 Urochloa has also been expressed in rice, with and without PEP carboxylase, but overall photosynthesis has not been higher. Most attempts to shift C4 have used two-cell systems, meaning classical C4 fixation which is split in space in mesophyll and bundle sheath cells [11]. A single-cell version of C4 metabolism may produce a functioning C4 pathway in C3 plants, but using four enzymes (PEP carboxylase, pyruvate orthophosphate dikinase, NADP-malate dehydrogenase, NADP-malic enzyme) has not yet produced greater plant growth [9,10,12].
Since most of the C3 plants lack the typical Kranz features, they possess a sub cellular separation that enables a concentrating of CO2 at the active site of Rubisco. The genes involved in the development of this peculiar sub cellular anatomy are unknown [13]. Considering the scarcity of sequence information for single cell C4 species, it is difficult to judge if single cell C4 metabolism can be bio-engineered into C3 crops. Today, the coexistence of C3 and C4 plants in the same environment brought up a controversial debate among agricultural scientists. For instance, rice is a C3 plant grown in warm climates where the C4 pathway should be superior. Using this argument, different researchers challenged the scientific community to consider the consequences of engineering the C4 pathway into rice.
A major effort is underway to achieve this in rice; however, it requires many changes in both anatomy and inserting and expressing the genes of C4 photosynthesis. In this regard, most of the earlier endeavors have got some sort of challenges. The first challenge is in choosing which type of C4 to shift, with its several pathways and enzymes involved so photorespiration is taken into account. The key enzymes like PEP carboxylase and Rubisco are large and complex with numerous subunits, they are repeatedly turned over in the cell, and they require numerous other proteins to assemble them. This means those enzymes plus their assembly mechanisms all need genes encoded into the DNA. And not all the DNA is nuclear; some of this is chloroplastic DNA, although the control of everything is inherited from nuclear DNA. The second difficulty is ensuring sufficient C4 activity in the absence C4 anatomical features. The third difficulty is placing the required C4 enzymes where they should be functioning in a target cell compartment, and with the original C3 enzymes elsewhere in the cell now switched off. For greatest efficiency, no Rubisco should be present or active in the same cells containing PEP carboxylase. Nevertheless, the fact that this process has successfully and independently achievable, although it will require further understanding of the genetic basis of the dimorphic photosynthetic tissue and localization of components of the C4 and Calvin cycles. Cognizant of the difficulty described above, recent works are focused on the following two novel approaches.
Introducing improved Rubisco from other species into plants
One means of alleviating the deficiencies of Rubisco is to introduce improved Rubisco from other species into plants. For example, thermophilic red algae have a Rubisco that has an improved specificity factor [8]. Increasing photosynthetic efficiency in C3 plants through introduction of improved Rubisco specificity factor from other species was also reviewed by Long et al. and Singh et al. [3,5]. A further opportunity to address the cost of photorespiration is to engineer a more efficient pathway for metabolism of the first product of the oxygenase reaction, phosphoglycolate. Plants and green algae use a single energy-consuming and protracted pathway involving the chloroplast, peroxisome, and mitochondrion, with the release of both carbon dioxide and ammonia in order to recover PGA that is then re-assimilated into the Calvin cycle.
The preceding authors summarized three different approaches for manipulating photorespiration in C3 plants. The first pathway has been engineered using Escherichia coli encoded genes from the glycerate pathway that convert glycolate to glycerate and release CO2 within the chloroplast. This pathway bypass photorespiration by 32% and plants engineered with this pathway resulted in reduced photorespiration and enhanced photosynthesis [3]. In the second approach, transgenic Arabidopsis plants engineered with a pathway to fully oxidise glycollate to CO2 in the chloroplasts. This pathway is found to be beneficial for plants and can enhance photosynthesis. Both bypasses should avoid ammonia release in the mitochondria, which is quite expensive to re-assimilate in terms of the ATP and reducing equivalents required [13]. A third bypass to photorespiration has been engineered by introducing the E. coli enzymes glyoxylate carboligase and hydroxypyruvate isomerase into tobacco for the conversion of glyoxylate into hydroxypyruvate directly in the peroxisome, thus once again avoiding ammonia release in the mitochondria. While this alternative pathway may potentially reduce the cost of 2PG recycling.
In recent reports, the potential of photorespiratory bypasses for the improvement of plants photosynthetic efficiency has been demonstrated. It was shown that introduction of the first bypass in the oilseed crop Camelina sativa greatly increased seed yield, which may be used for the production of biofuels [14]. Another photorespiratory bypass in potato was reported by Betti et al. [13]. They reported that potato (Solanum tuberosum) plants were transformed with the three genes that encode for E. coli glycolate dehydrogenase subunits and the corresponding poly-protein was successfully expressed in the chloroplast, where it was able to catalyze the conversion of glycolate to glyoxylate.
Direct insertion of inorganic carbon transporters into leaf cells
The other alternative approach is direct insertions of bicarbonate and carbon dioxide pumps to the chloroplast membrane and production of carboxysomes and pyrenoids within the chloroplast.
Carboxysomes and pyrenoids are structures within cyanobacteria and algae that provide a compartment that facilitates the concentration of CO2 around Rubisco, thereby reducing photorespiration [15]. Cyanobacteria suppress the oxygenating reaction of Rubisco by concentrating CO2 inside a proteinanous microcompartment called carboxysomes. The carboxysome is a protein micro body in cyanobacteria, comprising a protein coat, and an interior soluble protein phase containing most, possibly all, of the cellular Rubisco [8]. Cyanobacteria, the ancestors of modern day crop chloroplasts, use a different method of concentrating CO2 at Rubisco. These prokaryotes are actively up taking bicarbonate into their cells. Within the cells, both Rubisco and carbonic anhydrase are localized within isoanhydral protein shell bodies termed carboxysomes. Here, carbonic anhydrase catalyzes the formation of CO2, serving to concentrate CO2 around Rubisco to a sufficient level to minimize oxygenation and photorespiration.
To realize the full potential of these bicarbonate pumps, carboxysomes would need to be introduced into chloroplasts to enable CO2 to be concentrated around Rubisco [8]. The feasibility of introducing carboxyzomes in to higher plants was boosted by Betti et al. [13]. They reported that a functional cyanobacterial rubisco was successfully introduced in to tobacco choloroplast together with an internal carboxysomal protein [16]. Similar study was reported by Hay and he reported that soybean transgenic lines expressing ictB gene exhibited large increase in photosynthesis and productivity at ambient as well as elevated CO2 environments under both green house and field agricultural condition [17]. Recent developments in chloroplast transformation technology and the successful assembly of a functional carboxysome in E. coli have lined the foundations for synergizing a functional carbon concentrating mechanism in higher plants [16]. Many eukaryotic algae also include inorganic carbon concentrating mechanisms to suppress the oxygenase activity of Rubisco and photorespiration. These also require bicarbonate transporters in the cell and chloroplast membrane. Within the chloroplasts of these algae, Rubisco concentrates in a region, typically surrounded by starch, and is termed the pyrenoid [5]. Pyrenoids play a major role in global aquatic CO2 fixation. However, their mode of operation is much less well understood than that of carboxysomes.

New Techniques or Tools Used for Genetic Engineering

Tools that would allow insertion of constructs at the same point in the genome, a point that does not interfere with expression of other genes, would decrease variability between events and increase comparability of transformations with different constructs [5]. Genetic engineering involves changing the genetic material of organisms using artificial laboratory techniques. Changing the genes of a plant can change its properties or traits e.g. how it responds to disease, pesticide products or lack of water. More recently, many new molecular and genomic tools have been developed to change the DNA of plants [19]. Collectively many of these new genetic engineering techniques are referred to as “gene editing” techniques. Gene editing technique such as regularly interspaced short palindromic repeats system (CRISPR/Cas), zinc Finger Nucleases (ZFN) types -1, -2 and -3, transcription activator-like effector nucleases (TALENs), Meganucleases (MN) and Oligonucleotide Directed Mutagenesis (ODM) are widely used in engineering of microbial genome [20]. This provides a means to achieve directed insertions in crops either by deleting, substituting or adding DNA sequences in pre-defined locations. But this has only been achieved in a few species such as herbicide tolerant oilseed rape and male sterile trees. Other new genetic engineering techniques: Cisgenesis and intragenesis; RNA-dependent DNA methylation (RdDM); Grafting of non-GMO graft (scion) on GMO rootstock (and vice versa); Reverse breeding (RB) and Agro-infiltration are also widely applicable. Cisgenesis and intragenesis involve insert of DNA sequences derived from the same or a crossable species. So far it is applicable in disease resistant apple and potato. RNA-dependent DNA methylation (RdDM) provides a change in the properties of plants through silencing specific genes in a way that will usually disappear after several generations. For example it delayed tomato ripening and insecticide production in potatoes. The remaining technological advances would also allow successful reconstruction of parts of the plant genome.

Conclusion

Increasing the efficiency of photosynthesis could increase productivity and this is a key research target in the first half of 21st century. In view of this, improvement of the enzyme Rubisco is the main focus of cutting age technology. Photosynthesis in C3 plants is limited by features of the carbon-fixing enzyme Rubisco, which exhibits a low turnover rate and can react with O2 instead of CO2, leading to photorespiration. However, recent technological advance now allow higher plant Rubisco to be engineered and assembled successfully through different approach. Some of the most novel approaches to improve photosynthetic efficiency include; the introduction of improved Rubisco from other species, introducing carbon-concentrating mechanisms and engineering of a full C4 pathway into C3 plants. A simpler approach is to transfer bicarbonate transporters from cyanobacteria into chloroplasts of plants. This is because, in cyanobacteria, the carbooxysomes improve the efficiency photosynthesis by concentrating CO2 near the enzyme Rubisco. It has been realized that gene editing technique is a sharp tool in improving crop photosynthetic efficiency in the changing climate.

References

  1. FAOSTAT (2013) Agricultural database. Food and Agriculture Organization of the United Stations.
  2. DePaoli HC, Borland AM, Tuskan GA, Cushman JC, Yang X (2014) Synthetic biology as it relates to CAM photosynthesis: challenges and opportunities. J Exp Bot 65: 3381-3393.
  3. Singh J, Pandey P, James D, Chandrasekhar K, Achary VM, et al. (2014) Enhancing C3 photosynthesis: an outlook on feasible interventions for crop improvement. Plant Biotechnol J 12: 1217-1230.
  4. Simkin AJ, McAusland L, Headland LR, Lawson T, Raines CA (2015) Multigene manipulation of photosynthetic carbon assimilation increases CO2 fixation and biomass yield in tobacco. J Exp Bot 66: 4075-4090.
  5. Long SP, Marshall-Colon A, Zhu XG (2015) Meeting the global food demand of the future by engineering crop photosynthesis and yield potential. Cell 161: 56-66.
  6. Evans JR (2013) Improving Photosynthesis. Plant Physiol 162: 1780-1793.
  7. Pessarakli M (2001) Handbook of Plant and Crop Physiology (2nd edn.), Marcel Dekker, Inc., Madison Avenue, New York.
  8. Leegood RC (2013) Strategies for engineering C4 photosynthesis. J Plant Physiol 170: 378-388.
  9. Miyao M, Masumoto C, Miyazawa SI, Fukayama H (2011) Lessons from engineering a single-cell C4 photosynthetic pathway into rice. J Exp Bot 62: 3021-3029.
  10. Peterhansel C (2011) Best practice procedures for the establishment of a C4 cycle in transgenic C3 plants. J Exp Bot 62: 3011-3019.
  11. von Caemmerer S, Quick WP, Furbank RT (2012) The development of C4 rice: current progress and future challenges. Sci 336: 1671-1672.
  12. Peterhansel C, Hirsch HJ, Kreuzler F (2008) Overexpression of C4 pathway genes in the C3 dicots potato, tobacco, and Arabidopsis; experiences and future challenges. In: Sheehy JE, Mitchell PL, Hardy B (Eds), Charting new pathways to C4 rice. World Scientific Publishing Company, Singapore, pp. 163-174.
  13. Betti M, Bauwe H, Busch FA, Fernie AR, Keech O, et al. (2016) Manipulating photorespiration to increase plant productivity: recent advances and perspectives for crop improvement. J Exp Bot 67: 2977-2988.
  14. Dalal J, Lopez H, Vasani NB, Hu Z, Swift JE, et al. (2015) A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa. Biotechnol Biofuels 8: 175.
  15. Price GD, Badger MR, von Caemmerer S (2011) The prospect of using cyanobacterial bicarbonate transporters to improve leaf photosynthesis in C3 crop plants. Plant Physiol 155: 20-26.
  16. McGrath JM, Long SP (2014) Can the cyanobacterial carbon-concentrating mechanism increase photosynthesis in crop species? A theoretical analysis. Plant Physiol 164: 2247-2261.
  17. Hay WT (2012) Engineering cyanobacterial genes into Glycine max (soybean) leads to increased photosynthesis and productivity. Dissertation, University of Illinois, Urbana-Champaign, USA.
  18. Hanson MR, Gray BN, Ahner BA (2013) Chloroplast transformation for engineering of photosynthesis. J Exp Bot 64: 731-742.
  19. TACD (2016) New policy resolution on consumer concerns about new genetic engineering techniques. Trans Atlantic Consumer Dialogue, Consumers International, UK.
  20. Jiang W, Zhou H, Bi H, Fromm M, Yang B, et al. (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41: e188.