Heterologous expression of key C and N metabolic enzymes improves re-assimilation of photorespired CO2 and NH3, and growth
Anish Kaachra, Surender Kumar Vats, Sanjay Kumar
Plant Physiology, June 2018
Abstract
We investigated the effect of the heterologous expression of phosphoenolpyruvate carboxylase(ZmPepcase), aspartate aminotransferase (GmAspAT), and glutamine synthetase (NtGS) on carbon (C) and nitrogen (N) metabolism in Arabidopsis (Arabidopsis thaliana). These transgenes were expressed either separately or in different combinations. The highest gains in shoot dry weight were observed in transgenic lines coexpressing all three genes. Tracer experiments using NaH14CO3suggested that the coexpression of ZmPepcase, GmAspAT, and NtGS resulted in a higher flux of assimilated CO2 toward sugars and amino acids. Upon feeding the leaf discs with glycine-1-14C, transgenic lines evolved significantly lower 14CO2 levels than the wild type, suggesting that a higher reassimilation of CO2 evolved during photorespiration. Leaves of transgenic plants accumulated significantly lower ammonium without any significant difference in the levels of photorespiratory ammonium relative to the wild type, suggesting a higher reassimilation of photorespired NH3. Transgenic lines also showed improved photosynthetic rates, higher shoot biomass accumulation, and improved seed yield in comparison with wild-type plants under both optimum and limiting N conditions. This work demonstrates that the heterologous coexpression of ZmPepcase, GmAspAT, and NtGS reduced the photorespiratory loss of C and N with concomitant enhancements in shoot biomass and seed yield.
The global human population is consistently on the rise and requires an additional supply of food (Schmidhuber and Tubiello, 2007). To feed a population of 9 billion people by 2050, agricultural production needs to be increased by 60% to 110% (Tilman et al., 2011). This task calls for the engineering of crops having improved yield potential. Biomass production and yield depend upon a number of genetic and environmental factors wherein assimilated carbon (C) and nitrogen (N) play a central role (Lawlor, 2002). The production of C compounds through photosynthesis is a fundamental process to assimilate atmospheric CO2 into carbohydrates in plants, which, in turn, provide both energy and the C skeletons for various metabolic processes.
The CO2 assimilation efficiency of C3 crops like wheat (Triticum aestivum) and rice (Oryza sativa) suffers from the photorespiratory loss of fixed C due to the oxygenase activity of Rubisco (Andrews et al., 1971). The photorespiratory cycle involves nine consecutive enzymatic steps distributed between chloroplast, peroxisomes, mitochondria, and the cytosol. Gly decarboxylation in the mitochondria is an important step of the cycle and releases both CO2 and NH3, resulting in a net loss of C and N (Sharkey, 2001). C and N also are lost through other metabolic processes such as respiration and amino acid catabolism, respectively (Leegood et al., 1995; Barbour and Hanson, 2009), but their loss through photorespiration is much higher yet. While photorespired CO2 may be reassimilated in the chloroplast to some extent, nearly 30% of fixed C is lost through photorespiration (Sharkey, 1988). Similarly, photorespired NH3 can be reassimilated through Gln synthetase-Glu synthase pathways but needs energy and C skeletons for NH3 assimilation. Inhibiting photorespiration was thought of as a promising approach to improve the photosynthetic efficiency of C3 plants. However, photorespiration interacts with other metabolic pathways (Obata et al., 2016) and has adaptive significance, for example, under conditions of low CO2 availability (Eisenhut et al., 2017). Furthermore, photorespiration can increase the net CO2 assimilation rate by diverting the photorespiratory C toward amino acids (Busch et al., 2018).
Plants with the C4 pathway of photosynthesis or carbon-concentrating mechanism (CCM) have developed mechanisms to minimize photorespiratory losses by building a higher concentration of CO2 around Rubisco, and they exhibit better nitrogen use efficiency (NUE) than C3 plants (Vats et al., 2011). The introduction of C4-like traits into C3 plants, therefore, has been a favored approach to improve the photosynthetic efficiency of C3 plants and enhance crop yield (Hibberd et al., 2008; Raines, 2011). Over the last several decades, several genetic transformations were attempted for the expression of single or multiple enzymes of the C4 pathway in C3 plants (Raines, 2006; Ruan et al., 2012). Despite achieving higher activities of C4 enzymes in transgenic plants, these attempts failed to significantly improve photosynthetic performance or yield. With the high biochemical and anatomical complexities involved, the successful implantation of C4-like traits in C3 plants remains a challenge (Kajala et al., 2011; Miyao et al., 2011).
In addition to the efficient fixation of C via photosynthesis, adequate plant nutrition has a paramount influence on the overall growth and development of plants. Among nutrients, N is an important element for plant growth and largely determines crop success in a majority of agricultural systems. Over the past 50 years, the application of N fertilizers has significantly increased global food production. However, the excessive use of N fertilizers not only raises the farming input cost but also poses a serious threat to the quality of water, soil, and air (Raven and Taylor, 2003). Therefore, efforts have been made to engineer plants for enhanced NUE and higher yields. Key genes from N metabolism, namely nitrate reductase, nitrite reductase, glutamine synthetase (GS), asparagine synthetase, aspartate aminotransferase (AspAT), and alanine aminotransferase, have been utilized for heterologous expression in both model and crop plants (Pathak et al., 2008). However, these attempts offered limited success in improving plant growth or yield through enhanced NUE.
There is strong evidence to suggest a close link and coordination between C and N metabolism for optimal plant growth (Foyer et al., 2001; Lawlor, 2002). A large amount of N is required by the photosynthetic machinery to keep optimum CO2 assimilation rates through photosynthesis. On the other hand, significant amounts of photosynthetically fixed C are essential for the assimilation of inorganic N into amino acids. Additionally, both C and N can act as potent signaling molecules to regulate the expression of several genes participating in C and N metabolism (Coruzzi and Bush, 2001). Despite the close link between C and N metabolism, there are few studies where the simultaneous enhancement of both metabolisms has been targeted to improve plant growth and yield.
We have shown previously that C3 plants grown at low (1,300 m) and high (4,200 m) altitudes did not exhibit a significant difference in net photosynthetic rate (PN), despite the reduced partial pressure of gases, high light intensity, and changes in other environmental parameters observed at high altitude (Kumar et al., 2006). Plants grown at high altitude exhibited a shift in photosynthetic mechanism, and significant increases in the activities of phosphoenolpyruvate carboxylase (PEPCase), AspAT, GS, and Rubisco at high altitude were proposed to have a role in optimizing the photosynthesis rate and minimizing the photorespiratory loss of C and N. An increase in the activities of these enzymes also was attributed to conserving C and N in plants. Therefore, we asked if the coexpression of three genes, Pepcase, AspAT, and GS, would conserve C and N in a C3 species and, if so, via what mechanisms.
![Hypervalent Iodine(III) Mediated Counter Anion Controlled Intramolecular Annulation of Exocyclic-β-Enaminone to Carbazolone and Imidazo[1,2-a]pyridine Synthesis](https://ihbt.res.in/cache/mod_bt_contentslider/d15710ff30468bdc470e1bd7951ee130-pd.png "Hypervalent Iodine(III) Mediated Counter Anion Controlled Intramolecular Annulation of Exocyclic-β-Enaminone to Carbazolone and Imidazo[1,2-a]pyridine Synthesis")
