**1.2 Availability of amino acids**

Traditional models of nutrient cycling assume that organic N matter must be decomposed by soil microorganisms to release inorganic N, before that N becomes available for plant uptake. But, there are growing evidences that plant can absorb organic N directly. Earlier studies of nutrient absorption demonstrated that higher plants could take up amino acids (Virtanen and Linkola, 1946). More recent studies of amino acid absorption have further focused on the characteristics of the carrier systems and other mechanistic aspects of the uptake process and a wide array of amino acid transporters has been identified in several different plants species (Frommer et al., 1993; Montamat et al., 1999; Neelam et al., 1999).

In the moist tundra of the arctic, inorganic N supplied to plants by mineralization is not sufficient to meet their requirement of N due to low temperatures and anoxic soils. But these soils have large stocks of water–extractable free amino acids (Atkin, 1996). The studies of nitrogen cycling in artic tundra have indicated that some non–mycorrhizal plant species, such as *Eriophorum vaginatum*, could absorb amino acids rapidly, accounting for at least 60% of total the nitrogen absorbed (Chapin et al., 1993). Ectomycorrhizal species have higher amino acid uptake than non–mycorrhizal species (Kielland, 1994). Amino acid uptake was the general ability found widely in plants from boreal forest (Näsholm et al., 1998; Persson and Näsholm, 2001).

## **1.3 Amino acids and nitrate uptake**

Plants can store high levels of nitrate, or they can translocate it from tissue to tissue without deleterious effect. However, hazardous effects may occur when livestocks and humans

in the chloroplast, which is then incorporated into amino acids by the glutamine synthetase−glutamine− 2−oxoglutarate amidotransferase (GS/GOGAT) enzyme system, giving rise to glutamine (Gln) and ultimately other amino acids and their metabolites (Fig. 1; Taiz and Zeiger, 2002). Therefore, NR, NiR and GS constitute the first three enzymes of the

amino acid synthesis (Campbell, 1999). In most plant species only a proportion of the absorbed nitrate is assimilated in the root, the remainder being transported upwards through the xylem for assimilation in the shoot where it is reduced and incorporated into

Traditional models of nutrient cycling assume that organic N matter must be decomposed by soil microorganisms to release inorganic N, before that N becomes available for plant uptake. But, there are growing evidences that plant can absorb organic N directly. Earlier studies of nutrient absorption demonstrated that higher plants could take up amino acids (Virtanen and Linkola, 1946). More recent studies of amino acid absorption have further focused on the characteristics of the carrier systems and other mechanistic aspects of the uptake process and a wide array of amino acid transporters has been identified in several different plants species (Frommer et al., 1993; Montamat et al., 1999; Neelam et al., 1999). In the moist tundra of the arctic, inorganic N supplied to plants by mineralization is not sufficient to meet their requirement of N due to low temperatures and anoxic soils. But these soils have large stocks of water–extractable free amino acids (Atkin, 1996). The studies of nitrogen cycling in artic tundra have indicated that some non–mycorrhizal plant species, such as *Eriophorum vaginatum*, could absorb amino acids rapidly, accounting for at least 60% of total the nitrogen absorbed (Chapin et al., 1993). Ectomycorrhizal species have higher amino acid uptake than non–mycorrhizal species (Kielland, 1994). Amino acid uptake was the general ability found widely in plants from boreal forest (Näsholm et al., 1998; Persson

Plants can store high levels of nitrate, or they can translocate it from tissue to tissue without deleterious effect. However, hazardous effects may occur when livestocks and humans

<sup>−</sup>–N conversion to

nitrate assimilatory pathway. The NR activity is the limiting step of NO3

amino acids (Forde, 2000).

Fig. 1. The main process of nitrate assimilation.

**1.2 Availability of amino acids** 

and Näsholm, 2001).

**1.3 Amino acids and nitrate uptake** 

consume plant material with rich nitrate, they may suffer from methemoglobinemia or carcinoma by converting nitrate to nitrite of nitrosamines. Some countries limit the nitrate content in plant material sold for human consumption.

Several authors reported that free amino acids could down regulate nitrate uptake and nitrate content in plant. It was found that exogenously supplied amino acids and amides could decrease the uptake of nitrate by soybean (Muller and Touraine, 1992); wheat (Rodgers and Barneix, 1993); maize (Ivashikina and Sokolov, 1997; Padgett and Leonard, 1993, 1996; Sivasankar et al., 1997); barley (Aslam et al., 2001)(Table 1). Plants appear to have multiple mechanisms for regulating nitrate uptake in addition to amino acids or Nstatus (Padgett and Leonard, 1993).

Work by Breteler and Arnozis (1985) determined that pretreatment of dwarf bean roots with many different individual amino acids inhibited nitrate uptake to varying degrees dependent upon prior exposure of the plants to nitrogen and the specific amino acid treatment. No significant effect of amino acids on nitrate transport was detected when both NO3− and amino acids were present in the bathing solution, and no correlation emerged between inhibition of nitrate uptake and inhibition of nitrate reductase relative to specific amino acids. A more detailed study, presented by Muller and Touraine (1992), demonstrated inhibition of uptake by 50% or greater by alanine, glutamine, asparagines, arginine, β–alanine and serine when soybean seedlings were pretreated for 18 h prior to exposure to NO3−. The mechanisms of inhibition by arginine and alanine appeared to differ, however. Arginine stopped NO3− uptake immediately upon introduction to the uptake solution, kinetically similar to NH4+ inhibition. The authors suggested that this may be the result of a non-metabolic response such as alteration of membrane potentials. Inhibition by alanine was slower to develop, suggesting a metabolic component to the regulation rather than a physical or chemical interference.


Table 1. Effect of amino acid on NO3<sup>−</sup> uptake in several plants

The N status of the plants could also affect the inhibitory effect of amino acids on nitrate uptake. Rodger and Barneix (1993) had supplied amino acids exogenously to N starved or non−starved wheat seedlings. Exogenously supplied amino acids and amides had no effect on the wheat seedlings under well nourishment. However, some of the amino acids and amides supplied seedlings starved of N for 3 days inhibited up to 50% of the nitrate uptake rate.

Effect of Mixed Amino Acids on Crop Growth 123

amino acids during the induction (Radin, 1977). However when glutamine and asparagine were included along with the "corn amino acid mixture", the inhibition on the induction of NR in corn roots was more severe (Oaks et al., 1977). Chen and Gao (2002) have applied different mixture of glycine, isoleucine and proline replacing nitrate of solution partially (20%) to Chinese cabbage and lettuce in hydroponics. Amino acids enhanced the NR activity

L−tryptophan, considered as a physiological precursor of auxins in higher plants, was applied to soil to evaluate its influence on yield of several crops. Kucharski and Nowak (1994) found that L−tryptophan did not affect the yield of above ground part and roots of field bean. On the other hand, positive effects on corn and cabbage growth were reported

Amino acids were used to partially replace NO3− or foliar spray in many plants. In most case, the application of amino acids led to decreased nitrate content and increased total nitrogen content in lettuce, Chinese cabbage, onion, pakchoi or other leafy crops (Gunes et al., 1994, 1996; Chen and Gao, 2002; Wang et al., 2004). Some authors suggested that plants probably preferred amino acids as sources of reduced nitrogen, and nitrate uptake was inhibited by amino acids. In fact, there was little evidence or data to support the conclusions. It has not been distinguished that increased total nitrogen came of nitrate or

has been attributed to feed−back inhibition (Pal'ove−Balang, 2002). It was found that nitrate uptake rate follows a biphasic relationship with external nitrate concentration, suggesting the existence of at least two different uptake systems (Cerezo et al., 2000). At high external nitrate concentration (> 0.5 mM), a low affinity transport system (LATS), which shows linear kinetics, contributes significantly to the uptake rate and appears to be constitutively expressed and essentially unregulated. At low external concentrations (< 0.5 mM), two high affinity transport systems (HATS) operate, one of these being constitutive whereas the other is induced by nitrate. The HATS for nitrate uptake is sensitive to metabolic inhibitors and

Although the regulatory effect of amino acids on nitrate uptake and NR has been examined extensively, its effect on GS has not been examined in detail. Otherwise, a lot of amino acids were investigated about their regulation on nitrate uptake and assimilation, but very little

In fact, there are two possible reasons for the increase of total N content in the plants: preference for amino acids as sources of reduced nitrogen and regulation of amino acids on

The solution experiments were carried out to investigate the regulation of the induction of NO3– uptake, NRA, NiRA and GSA in radish and red pepper by applying mixed amino

appears to be an active transport system (Daniel−Vedele et al., 1998).

information has been reported about effect of mixed amino acids (MAA).

<sup>−</sup> uptake and reduction systems by nitrogen metabolites

in Chinese cabbage, while it decreased in lettuce.

(Sarwar and Frankenberger, 1994; Chen et al., 1997).

**1.5 Influence on yield and N assimilation** 

amino acids.

**1.6 Objectives** 

Regulation of induction of the NO3

inorganic nitrogen uptake and assimilation.

Aslam et al. (2001) had conducted study on differential effect of amino acids (Glu, Asp, Gln and Asn) on nitrate uptake and reduction systems in barley roots. Similar results were observed i.e. 50–60% inhibition in the NO3− uptake when the roots were supplied with 0.1 mM NO3−. However, no inhibition occurred at 10 mM NO3−. In contrast, Kim (2002) had conducted study on effect of mixed amino acids on nitrate uptake in rice, pea, cucumber and red pepper. The result showed that the effect of mixed amino acids (MAA) on nitrate uptake in nutrient solution was unaffected in low MAA concentration and accelerated in high MAA concentration. The results indicated that external MAA could regulate nitrate uptake.

#### **1.4 Amino acids and enzyme regulation**

Nitrate reductase (NR) is a substrate inducible enzyme involved in the nitrate assimilation in higher plant, and the enzyme occupying a control point in the pathway of nitrate assimilation. Activity of the NR fluctuates widely in response to many environmental or physiological factors, such as the presence of NH4+ or amino acids in the growth medium. In studies of the possible regulation of NR activity by amino acids in higher plants, the results have often been conflicting. For example, Radin (1975, 1977) had shown that the reduction of nitrate to nitrite in cotton roots is inhibited by specific amino acids. On the other hand, Oaks (1977) had found using an *in vitro* assay those amino acids results in enhanced levels of NR and also cause only minor inhibitions in both intact and excised corn roots. Aslam et al. (2001) reported that the amino acids partially inhibited (35%) the induction of nitrate reductase activity (NRA) in barley roots supplied with 0.1 mM NO3−, but no inhibition occurred at 10 mM NO3−. He has concluded that the inhibition of induction of NRA by the amino acids is a result of the lack of substrate availability due to inhibition of the NO3<sup>−</sup> uptake system at low NO3− supply. It has been suggested that glutamate inhibited NRA in roots, but not in shoots (Ivashikina and Sokolov, 1997). This inhibition seems be dependent on plant materials, age of plants, growth conditions, nitrate concentration, amino acid kinds, amino acids concentration and other factors.

Effect of amino acids on the regulation of NR gene expression has been studied at the molecular level. Deng et al. (1991) reported that the addition of 5 mM glutamine to the nutrient solution of tobacco plants grown in 1 mM NO3− resulted in a pronounced inhibition of NR mRNA accumulation in the roots. Vincentz et al. (1993) showed, under low light conditions (limiting photo synthetic conditions), the supply of glutamine or glutamate led to a drop in the level of NR mRNA, while glutamine and glutamate were less efficient at decreasing NiR mRNA than NR mRNA levels. Li et al. (1995) also demonstrated that 5 mM glutamine added together with NO3− resulted in reduced levels of NR mRNA in both root and shoot of maize. Sivasankar et al. (1997) observed that Gln and asparagine (Asn) inhibited the induction of NR activity (NRA) in corn roots at an external supply of 250 M and 5 mM NO3−. They concluded that inhibition was not the result of altered NO3− uptake, and tissue nitrate accumulation was reduced at 250 M external nitrate in the presence of 1mM Asn, but not at 5mM Asn.

In the studies of the possible regulation of NR activity by multiple amino acids in higher plants, the conclusions are again contradictory. The inhibition on NR activity by glycine, asparagines, and glutamine could be partially or wholly prevented by the presence of other

Aslam et al. (2001) had conducted study on differential effect of amino acids (Glu, Asp, Gln and Asn) on nitrate uptake and reduction systems in barley roots. Similar results were observed i.e. 50–60% inhibition in the NO3− uptake when the roots were supplied with 0.1 mM NO3−. However, no inhibition occurred at 10 mM NO3−. In contrast, Kim (2002) had conducted study on effect of mixed amino acids on nitrate uptake in rice, pea, cucumber and red pepper. The result showed that the effect of mixed amino acids (MAA) on nitrate uptake in nutrient solution was unaffected in low MAA concentration and accelerated in high MAA concentration. The results indicated that external MAA could

Nitrate reductase (NR) is a substrate inducible enzyme involved in the nitrate assimilation in higher plant, and the enzyme occupying a control point in the pathway of nitrate assimilation. Activity of the NR fluctuates widely in response to many environmental or physiological factors, such as the presence of NH4+ or amino acids in the growth medium. In studies of the possible regulation of NR activity by amino acids in higher plants, the results have often been conflicting. For example, Radin (1975, 1977) had shown that the reduction of nitrate to nitrite in cotton roots is inhibited by specific amino acids. On the other hand, Oaks (1977) had found using an *in vitro* assay those amino acids results in enhanced levels of NR and also cause only minor inhibitions in both intact and excised corn roots. Aslam et al. (2001) reported that the amino acids partially inhibited (35%) the induction of nitrate reductase activity (NRA) in barley roots supplied with 0.1 mM NO3−, but no inhibition occurred at 10 mM NO3−. He has concluded that the inhibition of induction of NRA by the amino acids is a result of the lack of substrate availability due to inhibition of the NO3<sup>−</sup> uptake system at low NO3− supply. It has been suggested that glutamate inhibited NRA in roots, but not in shoots (Ivashikina and Sokolov, 1997). This inhibition seems be dependent on plant materials, age of plants, growth conditions, nitrate concentration, amino acid kinds,

Effect of amino acids on the regulation of NR gene expression has been studied at the molecular level. Deng et al. (1991) reported that the addition of 5 mM glutamine to the nutrient solution of tobacco plants grown in 1 mM NO3− resulted in a pronounced inhibition of NR mRNA accumulation in the roots. Vincentz et al. (1993) showed, under low light conditions (limiting photo synthetic conditions), the supply of glutamine or glutamate led to a drop in the level of NR mRNA, while glutamine and glutamate were less efficient at decreasing NiR mRNA than NR mRNA levels. Li et al. (1995) also demonstrated that 5 mM glutamine added together with NO3− resulted in reduced levels of NR mRNA in both root and shoot of maize. Sivasankar et al. (1997) observed that Gln and asparagine (Asn) inhibited the induction of NR activity (NRA) in corn roots at an external supply of 250 M and 5 mM NO3−. They concluded that inhibition was not the result of altered NO3− uptake, and tissue nitrate accumulation was reduced at 250 M external nitrate in the presence of

In the studies of the possible regulation of NR activity by multiple amino acids in higher plants, the conclusions are again contradictory. The inhibition on NR activity by glycine, asparagines, and glutamine could be partially or wholly prevented by the presence of other

regulate nitrate uptake.

**1.4 Amino acids and enzyme regulation** 

amino acids concentration and other factors.

1mM Asn, but not at 5mM Asn.

amino acids during the induction (Radin, 1977). However when glutamine and asparagine were included along with the "corn amino acid mixture", the inhibition on the induction of NR in corn roots was more severe (Oaks et al., 1977). Chen and Gao (2002) have applied different mixture of glycine, isoleucine and proline replacing nitrate of solution partially (20%) to Chinese cabbage and lettuce in hydroponics. Amino acids enhanced the NR activity in Chinese cabbage, while it decreased in lettuce.
