Ammonium In Nitrogen Uptake Of Cereals Biology Essay

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2 Project summary

This project aims to understand the contribution of ammonium (NH4+) in the nitrogen nutrition and overall nutrition of plants. Although NH4+ and nitrate (NO3-) are the mineral forms of nitrogen that can be absorbed by the plants easily, the interactions between these two ions are often overlooked. In this study, the interactions between these two ions are studied using the cereal maize. Moreover NH4+ in small quantities can improve the plant growth. This research will also look at the contribution this small quantity of NH4+ in the total nutrition of plants. This study has the potential to understand the contribution of NH4+ in improving the nitrogen nutrition of plants thereby help in the nitrogen use efficiency (NUE) of plants.

3 Project details

3.1 Introductory background/Literature Review

3.1.1 Introduction

Nitrogen (N) is one of the major mineral nutrients that are required by plant for its growth and development. It is a major component of chlorophyll (the major pigment responsible for photosynthesis in plants) and amino acids which form the basis of proteins. Proteins are either enzymes or are the building blocks of plant cells. Nitrogen deficiency in plants leads to decreased growth and yellowing of leaves. Although nitrogen is present in all soils, it is often not in sufficient quantities to achieve maximal growth and yield. Nitrate (NO3-) and ammonium (NH4+) ions forms of nitrogen available to plants in agricultural soils (Wolt 1994). NO3- concentrations are generally 10 times that of NH4+ but this is a constant pool of N available to plants. Even though most plants prefer a combination of NH4+ and NO3- plant's preference to NH4+ and NO3- appears to vary depending on environmental factors such as soil pH, soil temperature of where they usually grow. The reasons behind this preference are not well known but it may be related to the energetic costs and pH effects of uptake and assimilation of either NH4+ or NO3-. It may also be related to other aspects of plant nutrition, given that in some cases NH4+ can improve the micronutrient nutrition of plants. The issue is also complicated by research into the effects of NH4+ on NO3- uptake, where many have shown a reduction in NO3-uptake when NH4+ is present.

With soil solution concentrations of NH4+ being so much less than NO3-, the contribution of NH4+ to the overall N budget of crop plants is often overlooked. This research will focus on the contribution of this NH4+- in the nitrogen uptake of corn plants. The study will also investigate whether ammonium has any effect on uptake and utilization of other nutrients and also investigate the inhibition of possible inhibition of NO3- uptake by NH4+.

3.1.2 Literature Review Nitrogen is essential to plants

Nitrogen is one of the major nutrients required by plants in greater amounts for its growth and development and its deficiency often results in stunted growth which in turn leads to low productivity. It is the major constituent of chlorophyll, proteins, nucleic acids, nucleotides and nucleosides. It constitutes about 1.5 to 5% of the dry weight of the plants (Haynes, 1986). The nitrogen limitation in the soil restricts the productivity of all plants especially cereals. Rice, Wheat, maize and to a lesser extent barley are the most important cereals cultivated in the world and account for majority of end products used for food and it is mostly likely that they will contribute to the majority of human diets in the next century. In his studies, (Cassman 1999) reviewed that since 1967, high yield in rice, wheat and maize largely contributed to the high increase in global food production. Cereals are fast growing crops and they demand high levels of nitrogen, which are supplied in the form of nitrogenous fertilizers. Nitrogen Fixing in the Soil

The atmosphere constitutes about 78% of nitrogen in its gaseous form which is unavailable to plants other than legumes. Most nitrogen in the environment that is available to plants has been fixed by lightning or by bacteria in legume nodules. Non-legumes can utilize this nitrogen only if it is fixed in the soil either in inorganic forms (NH4+ or NO3-) or organic forms (amino acids). Industrial fixations of N by the addition of fertilizers also enhance the availability of N in agricultural soils. Although NH4+ or NO3-are the major forms in which plants take up nitrogen, studies have showed that some plants in the arctic tundra regions where organic N is the main form of N, can absorb simples amino acids in the absence of inorganic nitrogen (Chapin, Moilanen & Kielland 1993). Plants can also absorb urea from the soil. Figure 1 represents the nitrogen cycle in the soil.

Fig. 1. Nitrogen cycle in soil. (Subbarao et al. 2006)

The decomposition of organic matter plays an important role in the availability of NH4+ and NO3-.(Haynes 1986), in his studies demonstrated that climate, vegetation and topography are some of the major factors that influence the availability of organic matter in the soil. Organic N is mineralized to NH4+ and it is then nitrified to NO3- forming NO2- as the intermediate form. NH4+ and nitrate NO3- behave quite differently in soils. NH4+ is positively charged and it binds with negatively charged clay particles thus making it available to plants and not easily leachable. NO3-on the other hand is present as free ions and easily lost in the runoff water. NH4+ is also available in the soil as fixed NH4+ which is unavailable to plants. Studies have reported that surface soil constitutes about 5% and subsoil contains about 60% of fixed NH4+ (Stevenson & Dhariwal 1959). Under submerged conditions in wetland soils, where the main crop is rice, mineral N is available in the form of NH4+ as it is stable under anaerobic conditions (Islam & Islam 1973). Ammonium and Nitrate in Different Ecosystems

Although plants can also take up organic form from the soil based on their availability, NH4+ or NO3- are the most importance form of N in agriculture. As NH4+ is nitrified to NO3- rapidly; nitrate is the most predominant form of N present in most of the soils (Haynes 1986; Tills & Alloway 1981). In well aerated, less acidic soils the activity of nitrifying bacteria ensures the availability of N in NO3- form and this form is preferred by most higher plants and crops cultivated in the field. On the other hand in areas of the world where soil conditions are unsuitable for the growth of nitrifying bacteria, NH4+ is the major source of N as demonstrated in arctic tundra regions and some temperate grassland. In tundra and boreal ecosystems, NH4+ is the major form of nitrogen in soils because of low nitrification potential (Keeney 1980).

About 90% of the totals N in the most soils are present in the organic matter produced by the microbial decomposition of plants and animal residues (Rosswall 1976). In agricultural soils available N is present in soluble inorganic (NO3-, NH4+, NO2-) and organic N forms. The concentration of NH4+ or NO3- in the soil varies according to the season and also the management practices such as fertilization and grazing. In winter the nitrification is reduced resulting in the reduction of NO3- in the soil. On the other hand NH4+ is nitrified to nitrate during summer months contributing to the low concentration of NH4+ in soils. Plant Preferences/Adaptability to Different Nitrogen Sources

The adaptability of plants to the different environment conditions plays major role in their affinity to NH4+ or NO3- uptake. Although plants can absorb both mineral forms of N (NH4+ and NO3-), because of the abundance of a particular form in the soil, plants get adapted to that form of N. Mineralization and immobilization are the two main factors that influence the availability of a particular form of |N in the soils. Mineralization includes both ammonification and nitrification. Of these nitrification plays a major role in the availability of NO3- ions in the soils. In arable and calcareous soils, nitrification facilitates the formation of NO3- and this form is generally considered to be the major form of N used by higher plants. Most of the agricultural crops prefer NO3- as their N source because of their adaptability to the NO3- which is available in abundance. Similarly, in forest soils NH4+ is found to be the most abundant form of N. In contrast, the study with four species of coniferous trees (Krajina, Madoc-Jones & Mellor 1973) showed that two species grew well with NO3-, one preferred NH4+ nutrition and one showed better growth when grown in a combination of NH4+ and NO3-. White spruce plants preferred NH4+ to NO3- as their nitrogen source (Kronzucker, Siddiqi & Glass 1997) whereas loblolly pine showed an affinity towards NH4+ (BassiriRad et al. 1996).

Experiments on various plant species in arctic tundra region showed that most of them preferred NH4+ and glycine as their N source during their initial growth stages (McKane et al. 2002). He also pointed out that the most productive species Eriophorum preferred a combined N source glycine and NH4+ which were the most available N form as compared to NO3- preference by Carex, although nitrate was the least available form. Species specific preferences to either form of N have been established in some grassland (Weigelt et al. 2003) and in some alpine communities (Miller & Bowman 2003). Experiments on Maize (Schrader et al. 1972) and cranberry (Greidanu.T et al. 1972) showed more affinity towards ammonium nitrogen. Plants in cold climates prefer ammonium as it is the major form of nitrogen in these regions (Keeney 1980). Ecosystem

The ecosystem plays a major role in the dominance of a particular form of N. Research in the past have shown that in the forest ecosystem where the soils are mostly acidic, inhibition of nitrifying bacteria increases the NH4+ compared to NO3- (Cole 1981) . Similarly in tropical and subtropical grasslands, the grasses and sedges grow well when grown in NH4+ (Gigon & Rorison 1972). In the submerged soils, where rice is the main crop grown, the anaerobic condition decreases nitrification and NH4+ is found to be the dominant form of N (Ponnamperuma 1978). Studies have also shown that most of the forest tree species which are originated from the acidic soil environments showed greater responses to NH4+ than NO3-. Similarly, in acidic soils, where nitrification is a problem the main form of available N is NH4+. Research with the mycorrhizal plants in some of the boreal forests have demonstrated that they use organic N as their N source (Nasholm et al. 1998) as this forms the major N reserve in these soils. Similar results were found in Australian high land forests. But most of the agricultural crops respond better when nitrogen is supplied as a combination of ammonium and nitrate. (Cox & Reisenauer 1973), in their studies with wheat demonstrated that maximum dry matter was obtained when plants were grown in a combination of ammonium and nitrate. Similar results were also obtained from maize (Schrader et al. 1972). Climate

Climate forms another important factor that decides the availability of a particular N form in the soils. Studies have indicated that nitrification is inhibited by the cold temperature and this facilitates NH4+ in these soils. But during the summer and autumn when the temperature is more the nitrification of NH4+ increases the NO3- content of the soil. In temperate regions, no nitrification or ammonification occurs in winter, but in summer mineralization results in the production of more NO3- (Gasser 1961). Concentration of NH4+ and NO3-in the soils vary with the change in season and management practices such as fertilization and grazing.(White, Haigh & Macduff 1987) Soil Temperature

Soil temperature also influences the N form in the soil. At low temperature nitrification occurs at a lower rate than ammonification (Harmseng & Kolenbrandger 1965). The optimum temperature for nitrification is usually between 25 and 35oC (Frederick 1956). At low temperature nitrification ceases which results in the accumulation of NH4+ -in the soil. Soil pH

Soil pH otherwise soil acidity is one of the key factor which affects the uptake of either NH4+ or NO3- as the N source for many plants. Activity of microorganisms, mineralization rate and precipitation of nutrients are some of the factors that are affected by the pH in the soil (Brady & Weil 2001). As discussed earlier the soils of boreal coniferous forests and temperate regions the low pH facilitates the availability of NH4+- N and high NH4+: NO3-. But any disturbance can increase the pH and in turn increases the NO3-concentration (Hope, Prescott & Blevins 2003). Nitrate and Ammonium Uptake

Plants acquire their nutrients from the environment surrounding the rhizosphere. The ions diffuse into the epidermal cells or cortical cells in the roots and the active ion absorption occurs in plasmalemma. The ion is then transported into the symplast. The external ion concentration, the internal ion concentration and the potential difference are the three factors that influence the electrochemical gradient of an ion across the plasma membrane. Mechanism of uptake

There are four distinctive transport systems which operate in plants for the uptake of ions. They are induced high affinity transport system (iHATS), constitutive high affinity transport system (cHATS), induced low affinity transport system (iLATS) and constitutive low affinity transport system (cLATS). HATS operates for both NH4+ and NO3- , when the concentration of the ions are in the range of 10-250µM (Crawford, N 1995; Lauter et al. 1996). When the ionic concentration in the medium exceeds 250µM LATS operates for both the ions. When the concentration of the ion is low in the medium a low capacity cHATS and a high capacity - iHATS had been identified for NO3. When the external concentration increases LATS, which shows a linear correlation with external concentration becomes apparent. For NO3- the electrochemical gradient is always energetically uphill at low concentration. NO3- uptake is determined by a regulatory mechanism depending on the nitrogen demand of the plant.

In his experiments with rice Wang et al. (1994) demonstrated that in the case of NH4+, constitutive HATS comes into play when the external ion concentration low and a cLATS operates even when the NH4+ concentration is as high as 40mM in the environment. The nitrogen content in the tissue is considered to be a major element for regulating the uptake of NH4+. Ryan & Walker (1994) showed that the N deficiency in the plants increases the NH4+ uptake by roots. For NH4+ the electrochemical gradient is energetically downhill because it is a positively charged ion and the potential difference is negative. Rhizosphere pH

Rhizosphere pH has many influences on the nitrogen nutrition in plants. But the results of experiments investigating this influence vary because of its complexity. Glass and his coworkers (Glass, ADM et al. 1990) in their studies demonstrated that NO3- influx increased between a pH range of 4.5-7 and the maximum influx was observed between a pH of 4.5-6.0. Doddema & Telkamp (1979) in their experiments with Arabidopsis showed that optimum ph for nitrate uptake is 8.0. In contrast to this finding, barley plants showed maximum NO3- uptake at a pH of 4.0 (Rao & Rains 1976). Similarly, the optimum pH for NO3- uptake was found to be 5.5 in Picea abies (Peuke & Tischner 1991), 4.5-5.0 in Typha latifolia (Brix, Dyhr-Jensen & Lorenzen 2002) and 4.5-5.0 in soybean (Vessey et al. 1990). In Eucalyptus nitens no difference in uptake was seen between a pH range of 4.0 and 6.0 (Garnett & Smethurst 1999).

In the cell suspension of Vigna radiata ammonium uptake was higher at an acidic pH of 4.5.(Afzal, Zia & Chaudhary 2006) and this supported the studies by Steiner & Dougall (1995), who observed that there is 25% increase in NH4+ uptake at pH 4.5 than at 5.5. Optimum pH for NH4+ uptake for Typha latifolia and soybean is found to be 6.5 and 6.0 respectively, whereas, Garnett & Smethurst (1999) showed that there was 200% more NH4+ uptake at pH 4.0 than at 6.0 in E. nitens. In E. oblique higher NH4+uptake rate was observed at pH 6.0 than at pH 4.0. Ruan et al. (2007) in his experiments with tea plants showed that these plants take up NH4+ or at any pH range. Rhizosphere temperature

Soil temperature has a great influence on the uptake of NH4+ and NO3- by plants. Many studies in the past have demonstrated that NO3- uptake is sensitive to low temperature as in Lolium perenne (Clarkson, Hopper & Jones 1986) where at low temperature 85% on N absorbed was in the form of NH4+. Similar results were observed in E. nitens where at temperature 10oC the NO3- uptake is decreased and a subsequent increase in NH4+ uptake (Garnett & Smethurst 1999). Many other studies have also shown that absorption of NH4+ was higher at low temperature than those of NO3- (Clarkson, Jones & Purves 1992; Macduff & Jackson 1991). The experiments in rose plants showed that the uptake rate of NO3- was similar at temperatures 22oC and 14oC (Calatayud et al. 2008). This experiment even showed that even though NO3- uptake was higher at 14oC, the water uptake was lower at this temperature. Thus these plants need to improve their nitrate uptake mechanism to absorb more nutrients with less water uptake. Concentration of NO3- and NH4+

Because of the toxicity of NH4+ at high concentration (Mehrer & Mohr 1989) and the capacity of assimilatory enzymes, glutamate synthetase, to assimilate any free NH4+ (Givan 1979) , the concentration of NH4+ in the cytoplasm is thought to be very low. In contrast to these, some studies have shown that in some plants the cytoplasmic NH4+ is found to be in mM range as demonstrated in rice (Wang et al. 1993).When compared with NH4+, NO3- is not toxic to plant at any concentration and concentration of NO3- is found to be in mM range in the cytoplasm of plants. As vacuoles are the main storage organ of plants both NH4+ and NO3- are found to be stores in the vacuoles.

The concentration of both ions in the soil solution as well as in the roots play major role in the uptake of these ions by the plants. Kafkafi (1990) in his studies showed that the preference for plants to absorb NO3- increases when the concentration of NH4+ is 10% of total N content in the medium. Some studies in the past showed that the uptake of NO3- is not influenced by the internal concentration (Deanedrummond & Glass 1983). On the other hand, Lee & Drew (1986) in their studies demonstrated that there was stimulation in the NO3- uptake by NO3- starved barley plants. Jackson et al. (1976) also demonstrated that there was marked decrease in the uptake of NO3- when the concentration of NO3- in the root increased. Nitrogen transporters in Plants

NH4+ and NO3- enter the root cells with ease but they have very low permeability across the plasma membrane. For this reason, they require transporters to move across the plasma membrane. These transporters facilitate the movement of these ions against the electrochemical gradient. Recent research towards cloning and characterizing the genes involved in plant ion uptake systems have resulted in the identification of two gene families namely NRT1 and NRT2, that play an important role in NO3- uptake, but they have no sequence similarity (Chrispeels, Crawford & Schroeder 1999; Crawford, NM & Glass 1998; Forde 2000). In Arabidopsis thaliana 4 members of NRT1 family and 7 members of NRT2 (Glass, A et al. 2001) family have been identified . The first nitrate transporter was identified in a T-DNA tagged chlorate resistant mutant of Arbidopsis thaliana called CHL1 (Tsay et al. 1993). This is a member of NRT1 family. Initially it was thought that the inactive gene of this mutant was involved in LATS. Later on studies have shown that it is dual affinity nitrate transporter (Liu, Huang & Tsay 1999). The first NRT 2 transporter was identified in Aspergillus nidulans and Chlamydomonas reinhardtii . In Arabidopsis another gene that can encode a second transporter has been identified and is called NRT3. The corresponding protein is found to be a member of NRT1 family.

The first NH4+ transporter AtAMT1 encoding a high affinity transport system was identified in Arabidopsis thaliana (Ninnemann, Jauniaux & Frommer 1994). Since then may studies have isolated homologues of AMT1 from A. thaliana (AtAMT1;1, AtAMT1;2, AtAMT1;3) (Gazzarrini et al. 1999; Rawat et al. 1999), tomato (Lauter et al. 1996) and in rice (Sonoda et al. 2003). Marini et al. (1997) cloned and characterized the NH4+ transporters in yeast (MEP 1, MEP 2 and MEP 3). Another type of AMT that are more similar to MEP in yeast has been cloned in A. thaliana and they are called as AtAMT2 (Sohlenkamp et al. 2000). Two genes of AMT2 were cloned from rice namely, OsAMT2;1 and OsAMT2;2 (Suenaga et al. 2003). These transporters are seems to be functioning HATS in plants. No gene encoding LATS has been identified yet. Ammonium Inhibition on Nitrate Uptake

Many studies have demonstrated that NH4+ has an inhibitory effect on the NO3- uptake. This inhibition maybe as result of the inhibition on NO3-efflux (Jackson et al. 1976; Mackown, Jackson & Volk 1982) or it may be by stimulating the efflux (Ayling 2006; Chaillou et al. 1994; Deanedrummond & Glass 1983; Doddema & Telkamp 1979; King et al. 1993). However ,studies have suggested that (Aslam, M., Travis & Huffaker 1994) the internal concentration of NO3-plays an important role in the stimulation of NO3-efflux in the presence of NH4+. NH4+ inhibition of NO3-transport system was also studied by many scientists (Aslam,M. et al. 1996). Aslam, M., Travis & Rains (2001) also concluded that the inhibition of nitrate uptake by ammonium takes place when the internal concentration of NO3-is high. Assimilation of Nitrate and ammonium

NH4+ and NO3- that are absorbed by the roots have to be assimilated in the plants in order to utilize the nitrogen for organic compound synthesis. In most plants NO3- absorbed by the roots can be assimilated both in the roots as well as in the shoots. In herbaceous plants nitrate assimilation takes place mainly in leaves and in woody plants the assimilation mostly occurs in the roots. The first step in the assimilation of nitrate is catalyzed by the enzyme nitrate reductase (NR).


NO3- + NAD(P)H + H+ NO2- + NAD(P) + H2O

2e-This is succeeded by the reduction of NO2- to ammonium by nitrite reductase (NiR).

NO2- + 6 ferredoxinred + 8 H3O + NH4+ + 6 ferredoxin ox + 10 H2O

The energetic cost involved in the assimilation of NO3- is greater than those required for the absorption and assimilation of NH4+ (Bloom, Sukrapanna & Warner 1992). This is mainly due to the fact that NO3- must first be reduced to NO2- and then to NH4+ in the presence of the enzymes nitrate reductase and nitrite reductase respectively.

NH4+ is present in the plants not only by the absorption from the medium but also by the reduction of nitrate, during photorespiration, and catabolism of proteins. About 95% of the NH4+ in the plants was assimilated by the Glutamine synthetase (GS)/ Glutamate synthase (GOGAT) cycle.



The nitrogen in the amide group of glutamine and glutamate can be transferred to other molecules to form various nitrogenous compounds in the cell such as amino acids, polyamines, arginine etc. When NH4+ or NO3- used as sole source of N in plants

Plants can absorb both NH4+ and NO3- as their nitrogen source. In agricultural soils and well aerated soils nitrification increases the availability of NO3- as the major form of N and most crop plants absorb this nitrogen. The absorption of nitrate by the plants also enhance the absorption of cations K+, Mg+ and Ca+, but deficiencies of some trace elements, phosphate and suphate can be seen when plants are grown only with NO3-. On the other hand, ammonium absorption facilitates the uptake of phosphate and sulphate but limits the absorption of cations (Gahoonia et al., 1992). NH4+ also helps in the absorption of iron form the soil solution. NH4+ at high concentration is toxic to plants whereas, NO3- is a non toxic ion at any concentration. NO3- uptake is an energy dependent process as demonstrated by many researchers. NO3- assimilation requires more energy as it has to be converted to nitrite and then to NH4+ during the process of assimilation (Bloom, Sukrapanna & Warner 1992).

Research Questions

Many researches in the past have demonstrated the importance of nitrate in the nitrogen nutrition of plants. But interaction between ammonium and nitrate has often given no importance. I am trying to answer the following research questions in my research

Ammonium as a contribution to total N uptake has been overlooked. Is it important?

Small quantity of Ammonium enhances plant growth. Why?

Most plants prefer NH4+ and NO3- combination. Why?

3.3 Aims/ Objectives

Thus the aim of my study is to understand contribution ammonium in the nitrogen uptake and overall nutrition of plants. The objectives derived out of this aim are

To quantify the effect of ammonium on maize growth

To quantify the changes in ammonium flux capacity across the maize life cycle and its effect on N nutrition

To understand the effect of ammonium on nitrate uptake in maize



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