PhysiologyHigh irradiance improves ammonium tolerance in wheat plants by increasing N assimilation
Introduction
Soil-derived nitrate (NO3−) and ammonium (NH4+) are the main forms of inorganic nitrogen (N) taken up by plants (Lopes and Araus, 2006), but most available crop varieties have been genetically selected for nitrate or mixed nutrition. However, in contrast to the regulation of nitrate acquisition and its assimilation mechanism in crop plants, which are relatively well established (Forde, 2002), much less is known about the regulation of ammonium assimilation when it is the main N source available (Canales et al., 2010). Although the redox state of ammonium eliminates the need for its reduction in the plant cell and it is an intermediate in many metabolic reactions, it can result in toxicity symptoms in many, if not all, plants when cultured on NH4+ as the exclusive N source (Britto and Kronzucker, 2002). Ammonium toxicity syndrome in plants includes lower plant yield, net photosynthesis and shoot:root ratio, leaf chlorosis (Konnerup and Brix, 2010), disorders in pH regulation (Walch-Liu et al., 2000), imbalance of essential cations, such as decreased concentrations of K+, Ca2+ and Mg2+ or increased concentrations of SO42− or PO32− (Britto and Kronzucker, 2002) and carbohydrate limitation for growth due to excessive consumption of soluble sugars for NH4+ assimilation (Gerendás et al., 1997, Schortemeyer et al., 1997). Presently, no single mechanism can provide an adequate explanation for ammonium toxicity (Kotsiras et al., 2005). There is no consensus as to which traits confer NH4+ tolerance to a plant (Cruz et al., 2011), as this tolerance seems to arise from physiologically complex processes, or may even be reached by convergent mechanisms (Ariz et al., 2011b). However, ammonium tolerance in plants has been associated with some metabolic mechanism, such as the capacity of the plant to accumulate a large amount of ammonium in the root (Belastegui-Macadam et al., 2007), possibly to protect the photosynthetic parts of the plant against ammonium toxicity (Aarnes et al., 2007), and the capacity to maintain high levels of inorganic nitrogen assimilation in the roots (Cruz et al., 2006, El Omari et al., 2010).
It is widely accepted that NH4+ coming from the reduction of nitrate and coming directly from the external nutrient solution is assimilated primarily into organic compounds via glutamine synthetase/glutamate synthase (GS/GOGAT; GS, EC 6.3.1.2; GOGAT, EC 1.4.1.14) (Lea and Miflin, 1974). Plants have two types of GS isoenzymes that localize in different compartments: one located in the cytosol (GS1), and the other in the plastid/chloroplast (GS2) (Lam et al., 1996, Caputo et al., 2009). In most plant species, a single gene for GS2 and a small gene family for GS1 have been identified, suggesting that the different gene members are differentially regulated (Simon and Sengupta-Gopalan, 2010). In addition, it has been shown that, in leaves, chloroplastic GS2 functions to assimilate the ammonium that is produced from the reduction of nitrate and also to re-assimilate ammonium that is released during photorespiration, while ammonium coming from the soil and derived from the fixation of dinitrogen in legumes is assimilated into glutamine (Gln) by cytosolic GS1 in roots (Sakakibara et al., 1996). Thus, the overall understanding of their physiological functions is complicated (Ishiyama et al., 2004).
In addition to the GS/GOGAT pathway, glutamate dehydrogenase (GDH EC 1.4.1.2) catalyzes the reversible amination of α-ketoglutarate to glutamate (Glu). Therefore, it can theoretically either assimilate or liberate ammonium. The precise role of GDH activity in higher plants has been under continuous debate (Dubois et al., 2003). Some authors have proposed that GDH could operate in the direction of ammonium assimilation (Lacuesta et al., 1989, Oaks, 1995, Melo-Oliveira et al., 1996), particularly under stress conditions favoring ammonium accumulation (Skopelitis et al., 2006). However, others have argued strongly that under standard growth conditions, GDH operates in the direction of Glu deamination to form ammonium (Robinson et al., 1992, Fox et al., 1995, Stewart et al., 1995, Glévarec et al., 2004).
The tricarboxylic acid (TCA) cycle is a universal feature of the metabolism of aerobic organisms. This flux lies at the heart of mitochondrial respiratory metabolism, oxidizing organic carbon substrates to generate the reducing equivalents (NADH and FADH2) that fuel ATP synthesis by oxidative phosphorylation (Sweetlove et al., 2010). In plants, this is not the only function of the cycle, since the reactions of TCA metabolites are intertwined with large metabolic networks, such as nitrogen metabolism and metabolization of organic acids generated from other pathways (Lancien et al., 2000, Plaxton and Podesta, 2006). In fact, recent evidence suggests that in plants, carboxylic acid metabolism (TCA) is organized in a manner highly dependent on the metabolic and physiological demands of the cell (Sweetlove et al., 2010). In this way, TCA cycle provides carbon skeletons for nitrogen assimilation (Oaks, 1992), mainly as α-ketoglutarate. As a first step to ammonium assimilation by the plant, plants fed with ammonium as the sole N source demand higher levels of C skeletons (Magalhaes et al., 1992), and roots of ammonium-fed plants may be a stronger sink for carbohydrates compared to the roots of nitrate-fed plants (Schortemeyer et al., 1997).
The importance of carbon skeletons to assimilate ammonium in the root has been reported by several authors. Roosta and Schjoerring (2008) provided extra C as CaCO3, Bialczyk et al. (2005) as HCO3− and Ikeda et al. (2004) as glucose in the root zone, and showed that the carbon enrichment alleviated the ammonium toxicity effects. Thus, carbon enrichment enhanced the synthesis of carbon skeletons and, accordingly, increased the capacity for ammonium assimilation and the N export to the shoots. Another experimental approach has increased the light energy input. Some authors have proposed that higher photosynthetic photon flux density (PPFD) could improve the tolerance to ammonium nutrition (Gerendás et al., 1997, Ariz et al., 2011b). In light of these results, it can be hypothesized that an input of light energy, which would favor photosynthetic carbohydrate biosynthesis, could also favor, in turn, the amount of carbohydrates translocated to the root, thereby improving the tolerance to ammonium nutrition (Gerendás et al., 1997, Zhu et al., 2000, Ariz et al., 2011b). In this work, we tested the hypothesis that increasing the PPFD could provide higher carbon skeletons to fuel the TCA cycle and increase the plant ammonium assimilation. With this purpose in mind, we determined the carbon and nitrogen metabolites as well as the main enzymatic activities and their polypeptide presence involved in N assimilation.
Section snippets
Growth conditions and experimental design
Seeds of wheat (Triticum aestivum L. var. Cezanne) were germinated in perlite:vermiculite (1:1) in 1-L pots in a phytotron (Servicio Fitotrón e Invernadero SGIKer, UPV/EHU). The controlled environmental conditions were set at a 14-h photoperiod with two different light intensity conditions, 300 μmol photon m−2 s−1 (low irradiance, LI) and 700 μmol photon m−2 s−1 (high irradiance, HI), and an atmospheric CO2 level of 400 ppm CO2. Temperatures were 24 °C in the light period and 18 °C in the dark period,
High irradiance reduces the difference in biomass between ammonium and nitrate nutrition
Biomass accumulation in wheat plants was approximately 3–4 times higher under HI than under LI conditions (Table 1). Under LI conditions, ammonium-fed plants presented 25% lower shoot biomass and 52% lower root biomass than nitrate-fed plants, while under HI, the decrease in the biomass of ammonium-fed plants with regard to nitrate-fed plants was only 19% in shoots and 30% in roots (Table 1). Therefore, the total plant biomass decrease in ammonium-fed plants at LI conditions with respect to
High irradiance improves ammonium tolerance and enhances ammonium assimilation
Some of the toxicity symptoms caused by ammonium, such as a lower plant growth (Lu et al., 2005), a decrease in net photosynthesis (Claussen and Lenz, 1999) or an enhancement of the shoot:root ratio, were observed in our experimental conditions in wheat plants in response to the ammonium provision as the sole N source compared to nitrate nutrition. However, the extent of these symptoms depended on the environmental light conditions. Thus, under high irradiance (HI) conditions, the CO2
Acknowledgments
I. Setién holds a PhD grant by UPV/EHU. This research was financially supported by the Basque Government (IT526-10, K-EGOKITZEN, ETORTEK 2010–12) and by the Spanish Government (MICINN-AGL 2009-13339-CO2-01, AGL 2012-37815-C05-02, RTA2009-00028-CO3-03). Technical support provided by M. Lema from the Unidade de Técnicas Instrumentais de Análise Servizos de Apoio á Investigación (Universidad de A Coruña) and by G. Garijo from the Departamento de Ciencias del Medio Natural (Universidad Pública de
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