Nitrogen is one of the most essential macro elements

Nitrogen is one of the most essential macro elements, which is significant in the survival of all life forms, including plants. Plants take up nitrogen through their whole existence and their requirement increases with the increase in the size of the plant. Nitrogen is available to plants in three forms: organic nitrogen compounds, nitrate ions (NO3-) and ammonium ions (NH4+). Nitrogen sources in soil or derived from applied N fertilizers are mostly inorganic forms (Yang et al. 2017), which also act as signals for important developmental processes in the plants. It is a major component of chlorophyll and is also a critical component required for the synthesis of amino acids, proteins and nucleic acids. Nitrogen is a major factor controlling the growth of roots and is also known to promote the uptake and utilization of other nutrients, including potassium, phosphorus and thus controls the overall growth and development of the plant. Oftentimes, even though it is abundantly present in the atmosphere, due to the requirement of nitrogen in numerous functions of the plants and presence of insufficient sources, it sometimes becomes a limiting factor. This deficiency is exhibited in the plant in the form of slow plant development because of the plant’s inability to manufacture the essential genetic and structural materials required for its growth. Nitrogen deficient plants can appear pale green or yellowish due to lack of adequate chlorophyll. Necrosis is also seen in the older leaves due to the reallocation of the available nitrogen towards the growth of the newer plant parts and oftentimes the older parts become prematurely senescent and thus drops off. Nitrogen deficiency causes plant stunting and discoloration and adversely affects the fruit quality and quantity. On the contrary, overuse of nitrogen can also cause excessive vegetative growth, especially in the tropical countries. A lower shoot to root ratio is also observed in many nitrogen deficient plants. Therefore, an optimum regulation of available nitrogen to the plants is very essential for maintenance of the plant’s vigor and growth. Cases of nitrogen deficit in the soil is a common phenomenon in all countries across the globe, and this shortcoming is overcome by means of artificial application of nitrogen fertilizers for the improvement of crop yield.

1.1. RICE
The monocotyledonous rice plant plays an important role in food security in many parts all across the globe, especially in the continents of Asia and Africa.
Rice is a semi aquatic annual grass plant that belongs to the family Poaceae. It is grown in over 100 countries in all the continents, except Antarctica, extending from 53°N to 35°S and from sea level to 3000 m altitude (Lu and Chang, 1980; Mikkelsen and Datta ,1991). The commonly grown rice grows half meter to 2 meters long, however there are some species, which can attain a height of 6-9 meters. It can be grown in a wide range of climatic, geographic, soil and water conditions and is mainly grown in the humid tropics and subtropics in China, Philippines and the subcontinent of India. Irrigated and rainfed lowland rice systems account for 92% of the total rice production (Dobermann and Fairhurst, 2000). Rice includes 22 species of genus Oryza, among which 20 are wild. The life cycle of rice plant is generally between 100 to 210 days (Vergara 1970). The two most cultivated forms of rice are Oryza sativa, subspecies indica and japonica, with cultivation mainly centered in Asia and Oryza glaberrima.

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The population of the world is rapidly increasing and so is the dependence and requirement for the major crops production, including rice. By 2050, we would need to produce 87% more of the major food crops than what we are producing today (Kromdijk and Long, 2016). However, the main challenge in the crop production remains the availability of nutrients in the soil, challenging the yield of the crop; nitrogen in the case of rice. There is, therefore, a need for more study of the physiology of the crops for the development of new and improved crop varieties with improved tolerance to the biotic and abiotic stresses and superior and steadier yield potential across different geographical or climatic conditions.

The uptake of ammonium ions has been comprehensively studied in the case of bacteria, fungi and algae, where it has been seen that the ammonium ions can be accumulated against its concentration and electrochemical potential gradients, which results in significant accumulation of ammonium within the plant cells (Smith and Walker, 1978, Kleiner, 1981, Boussiba et al. 1984). In higher plants, there is little knowledge of the ammonium transportation. The uptake of ammonium ions into the plant cells follows a concentration dependent kinetics and a consequent depolarization of the membrane potential. The ammonium is either taken up by the plant roots by active transport and/or by facilitated diffusion, both being coupled to an energy source (Macfarlane and Smith,1982, Kleiner,1985, Deane-Drummond, 1986). Also, important in the process of ammonium intake by the plant roots is the fact that it is dependent on energy. Macklon et al.1990, demonstrated that in the excised roots of Allium cepa, ammonium ions absorption is an active process. It has also been found that uptake of ammonium at higher temperatures (25-30 ?) is related to metabolism (Sasakawa and Yamamoto, 1978) and at lower temperatures or decreased carbohydrates, the uptake is decreased.

Nitrogen is the most important nutrient in irrigated rice production (Cassman et al. 1998). Nitrogen is required by the rice plants mainly at two stages. At early and mid tillering stage to maximize the panicle number and reproductive and ripening stages to produce optimum spikelets per panicle (De Datta, 1986). Higher plants absorb nitrogen in several forms from the soil, most notably, in its inorganic forms, viz, ammonium ions or nitrate ions. In nitrogen deficient soil and in waterlogged conditions, ammonium is the predominant form of nitrogen supply to the plants, as the energy required for assimilation of ammonium is less than nitrate. This ammonium is provided to the plants by means of ammonium transporters (AMTs) that are present in the root plasma membrane. AMT1s transfer either the charged (NH4+) ammonium ions or co transfer the NH3 with a proton. However, since excessive ammonium uptake can prove to be toxic for the plants, the regulation of ammonium uptake needs to be modulated (Britto and Kronzucker, 2001).

As ammonium is the major source of inorganic nitrogen for the paddy under hypoxic or anaerobic conditions, the delicate maintenance of its balance is also very important, as high concentrations of ammonium can be toxic to the rice plants. Therefore, the transport of ammonium ions and its efficient assimilation in and by the roots is very critical. Assimilation of ammonium in the plant roots is controlled primarily if not exclusively, by glutamine synthetase (Tobin and Yamaya, 2001, Britto and Kronzucker, 2002). Ammonium that is transported into the cell cytosol or possibly even mitochondria or produced in the root plastids by means of nitrite reduction, is primarily assimilated into the amino group of glutamine, by the conversion of ATP to ADP and Pi, and the glutamine is further on used for transamination reactions to produce other amino acids (Bothe et al. 2007). The reactions of ammonium assimilation and re-assimilation of photosynthetic ammonium are regulated by the GS/GOGAT enzymes (Glutamine synthetase/glutamate-oxoglutarate aminotransferase). Two classes of GS have been identified till date- cytosolic and chloroplast (Lam et al, 1996) and also two types of GOGAT-one utilizing NADP as the electron donor, with possible role in the root and nodules and the other using ferredoxin as the electron donor, with activities prominent in the root tips (Miller and Cramer, 2004). There is also a third enzyme, glutamate dehydrogenase (GDH), present in the mitochondria, with a very low affinity for ammonium, that also plays a role in the ammonium assimilation in plants especially during the photorespiration in the leaf cells. It is believed that most of the ammonium produced in the mitochondria is transported to the chloroplasts, where the enzyme, GS, assimilates it. (Lam et al. 1996, Howitt et al. 2000).

The ammonium transporter (AMT) proteins are an important class of trimeric, intrinsic proteins that are found in every life form. The ammonium as a form of nitrogen is made available to the rice plants by means of ammonium transporters, which belong to the AMT/MEP/Rh (Ammonium Transporter/ Methylamine Permease/ Rhesus) protein family with homologs in bacteria and yeast (Bao et al, 2015). All three have high affinity and high saturation at less than/equal to 1mM ammonium concentration. Plants take up ammonium by both a high-affinity saturable (HATS) component and a low-affinity non- saturable transporter (LATS). The uptake of ammonium from the soil is biphasic and which transport system works depends on the external ammonium concentration. The low affinity system is non-saturable and has its half-maximal transport (Km) in the millimolar concentration range. It plays a part in the transport of ammonium ions or facilitates the diffusion of ammonia through the plasma membrane of the cells. The high affinity system, on the other hand, is saturable with Km in sub millimolar concentrations (Zhen et al.2009) and the driving force for the uptake of ammonium ions at low concentrations is provided by the membrane electric potential. Since the concentration of ammonium in the soil is in quite low concentrations, the HATS predominantly accomplish the uptake of ammonium.
The first ammonium transporters to be isolated were in yeast (MEP1 from Saccharomyces cerevisiae) and Arabidopsis (AMT1) in the case of higher plants. Both were isolated by the functional complementation of a yeast mutant defective in ammonium transport (Ninnemann et al. 1994). The homologs of these proteins have been found in animals (Caenorhabditis elegans) and also in prokaryotes, thus establishing the fact that these belong to an ancient group of proteins (Sohlenkamp et al. 2000). In plants, the AMT family can be subdivided into two subfamilies (Loque and von Wiren, 2004). Members of the AMT1 subfamily are mostly intron-free and those of the AMT2 subfamily mostly contain introns in the gene sequence. In the case of rice, the AMT2 has been further subdivided into two subclades (Suenaga et al. 2003).
Even though the involvement of all the classes of AMT proteins in the transport of ammonia or ammonium ions is universally accepted, their functions are varied. On one hand, prokaryotes and plants are known to take up ammonium from their surroundings with the help of AMT proteins and on the other, mammals use the Rhesus proteins for pH stability and ion homoeostasis in the erythrocytes, liver and kidney cells (Nakhoul et al. 2004, Winkler et al. 2006).
So far all the plant AMTs investigated have been found to be located in the plasma membrane, suggesting their roles in ammonium acquisition (Ludewig et al.2002, Ludewig et al. 2003, Loque et al.2006)

One of the first ammonium transporters isolated was the AMT1;1 of Arabidopsis thaliana (Ninnemann et al,1994). The cDNA of AMT1;1 of Arabidopsis has a length of 1506 bases encoding a highly hydrophobic, 501 amino acids protein with 11 membrane spanning regions. Within the AMT family, the proteins vary in size from 391-622 amino acid residues (Blakey et al, 2002).
Ammonium transporters are homotrimers. Each monomer has central hydrophobic channels thought to be the pathway for the uncharged ammonia. (Khademi et al. 2004, Zheng et al. 2004). The ammonium transporters were assumed to be integral membrane proteins and the computational analyses of the first cloned sequences revealed these to encode highly hydrophobic proteins with a molecular mass of 50-55 kDa (von Wirén and Merrick). The corresponding proteins are estimated to be 400-450 aa in length and have 10-12 transmembrane helices with a C-terminal cytoplasmic extension (Marini et al,1994, Ninnemann et al. 1994). Various analyses have shown the C terminal region of the AMT proteins to constitute a discreet cytoplasmic region with at least 30 aa.

Fig. 1: Structure of the three dimensional fold of AmtB ammonia channel in Escherichia coli with 11 transmembrane helices (M1-M11)
Copied from: Khademi et al 2004

Rice is mainly grown in water-logged conditions, and the uptake of nitrogen by the rice is primarily in the form of ammonium. Therefore, the ammonium transporters play a big role in the uptake of nitrogen in rice. 12 AMT genes, located in the rice root plasma membrane were reported and sub grouped, three genes each in OsAMT1, OsAMT2 and OsAMT3 ,one in OsAMT4 (Suenaga et al. 2003). Furthermore, 2 more AMTs were identified later and grouped in OsAMT5, by Deng et al. The 12 OsAMT genes are distributed on rice chromosomes 1 to 5, 11 and 12. Chromosomes 1 and 2 each contain 3 genes, chromosome 3 contains 2 and chromosomes 4,5,11 and 12 each contain 1 gene and the coded proteins have 10-11 transmembrane domains with an extracellular N-terminus and cytosolic C-terminus. The length of each OsAMT protein in rice ranges from 459 to 502 aa. However, OsAMT4 and OsAMT5.2 have 300 aa to 327 aa, respectively. The OsAMT 1 family of genes have been classified as high-affinity transport system (HATS) and they show a high similarity in the sequence with each other. Low affinity ammonium transporters (LATS) have not been identified as of yet (Sonoda et al. 2003, Loque and von Wiren, 2004).
It has been seen that the bacterial and fungal AMTs act as transceptors with a dual function of both ammonium transporters and receptors, mediating the changes stimulated by the addition of ammonium in both the morphology or transcription of the target genes (Tremblay et al, 2009). Plant AMTs can also have similar functions e.g., AMT1;3 is seen to regulate lateral branching in the roots in response to addition of localized ammonium (Lima et al,2010). Also, studies have revealed that OsAMT 1;3 is specifically expressed in the roots of the plants and its expression is suppressed by the addition of nitrogen, corroborating the theory that OsAMT1;3 is not only responsible for ammonium uptake but is also involved in ammonium sensing in the rice plants (Sonoda et al. 2003).

Table 1: List of predicted AMT genes in rice

(Table copied: Bao-zhen et al, in Rice Science,2009, 16(4): 314–322)

OsAMT1;1, OsAMT1;2 and OsAMT1;3 are encoded by 499-, 497- and 498- amino acid residues respectively with molecular mass of 52.6, 52.2 and 53.1 kDa, respectively. The sequences also showed a 74.2%, 73.7% and 70.4% sequence similarity with the ammonium transporter of Arabidopsis thaliana, AtAMT1;1 (Ninnemann et al. 1994) and 74.4%, 75.4% and 70.9% sequence similarity with ammonium transporter in tomato , LeAMT 1;1 (Lauter et al. 1996).
Among all, the most studied for the expression and regulation pattern till date has been the OsAMT1 family because these are believed to play a key role in the uptake of ammonium at low ammonium concentrations.
Ammonium is present in all parts of the plant cells. However, the concentration of ammonium in all the parts varies according to the concentration of ammonium in the neighboring plant cell part(s), the differences in the pH and also the electrical potential across the cells chambers and the form of ammonia (neutral ammonia or the cationic ammonium form) transported across the membranes. Changes in these factors may, therefore, have effects on the ammonium transport. GENETIC REGULATION
The plant’s N nutritional status and the external availability of ammonium/nitrate is the principal factor governing the transcriptional regulation of the AMT genes. Local N status of the roots, rather than the N status of the whole plant decides the ammonium uptake extent, as in nitrate uptake (Gansel et al. 2001). Re-supply of ammonium resulted in an upregulation in some plant AMT mRNA levels. This was first observed in tomato roots and later also in OsAMT1;1 and OsAMT 1;2 in the rice roots (von Wiren et al.2000, Sonoda et al.2003). In rice, ammonium can be replaced with glutamine with the same effects of upregulation of OsAMT 1;1 and OsAMT 1;2 suggesting the capability of glutamine as a trigger in the up- and down-regulation of the AMTs genes (Rawat et al.1999, Sonoda et al.2003). In other AMT homologues, a few days of nitrogen deficiency strongly upregulates the AMT 1;1 in Arabidopsis thaliana (Rawat et al,1999) but the response of AMT 2;1 to such condition is comparatively slower (Loque and von Wiren,2004). Sufficient nitrogen supply, on the contrary, results in reduction of AMT1;1 transcripts in A.thaliana (Rawat et al. 1999). These results imply that the AMTs in different plant species are differentially regulated as a means of adaptation to the nitrogen needs of the plant and its presence in the soil.
Also regulating the transcript levels in the roots during the ammonium uptake, particularly the AMT 1;1 transcript levels in Arabidopsis, is the diurnal changes. Several nitrogen uptake studies conducted with 15N labelled-ammonium showed a continuous increase in ammonium influx from the start of the light period till the end and a sharp decline when the light was turned off (Ourry et al. 1996). Sucrose supply in the dark, acting as photoassimilate also regulates ammonium transport and prevents a decline in the mRNA transcript levels of AMT 1;1, AMT 1;2 and AMT 1;3 in Arabidopsis and thus continues the ongoing ammonium influx (Lejay et al,2003).
The AMTs are also regulated at a post-translational level in the presence of elevated ammonium quantities by its phosphorylation. Since the activity of the AMT1s depend upon the interaction between the cytosolic C terminal and the pore region of the neighbouring subunit, the phosphorylation of the C terminus results in the trans-inactivation of the AMT protein complex (Loqué et al. 2007). This inactivation can also be brought about by the removal of the C-terminus or the introduction of a negatively charged amino acid just adjacent to the last transmembrane helix which results in the loss of contact between the two which is essential for the opening of the ammonia conducting pore (Marini et al.2000, Ludewig et al. 2003; Loqué et al. 2007; Neuhäuser et al. 2007). Most likely, AMT1;1 assembles as a trimer and the phosphorylation signal trans inhibits the two neighboring subunits, representing an example of cooperative transporter regulation (Loqué et al., 2007).
It is believed that the ammonium uptake is regulated both at the translational and post-translational level simultaneously (Rawat et al. 1999).
At the transcriptional level, AMT gene expression in Arabidopsis roots is generally repressed by high nitrogen, most likely by the internal pool of Gln, and derepressed under nitrogen deficiency or supply of sugars (Gazzarrini et al. 1999; Rawat et al. 1999). The nitrogen nutrition status of plants may specifically affect transcript stability, as has been observed in Arabidopsis for AMT1;1 but not for AMT1;3 (Yuan et al. 2007). It has also been observed that elevated ammonium concentrations in the cytoplasm inversely correlated with ammonium influx before transcriptional changes in AMT mRNA levels were observed. However, on the contrary, AtAMT1;1 gene expression negatively correlated with root glutamine concentrations, which was indicative for glutamine being the metabolic trigger downregulating the transcription of AtAMT1;1 (Rawat et al. 1999). At high nitrogen concentrations, the uptake of ammonium is repressed. The possible reasons could be low energy supply to the root, accumulation of nitrogenous compounds in the root tissues that suppresses the transport system or high efflux of endogenous ammonium ions (Morgan and Jackson, 1988). However, it has also been observed that the capacity of nitrogen uptake is increased in nitrogen depleted plants of wheat (Tromp,1962), ryegrass (Lycklama,1963), oats (Morgan and Jackson, 1988), barley (Lee and Rudge, 1986) etc.

The different AMTs show distinct expression patterns as has been demonstrated by Sonoda et al in 2003 e.g. OsAMT1;1 is constitutively expressed in the shoots but is stimulated by ammonium supply in the roots. Expression of OsAMT1;2 is root-specific and can be induced by ammonium and OsAMT1;3 is also expressed in the roots but its expression is repressed by nitrogen supply. OsAMT2;1 is constitutively expressed in roots and shoots and is closely related to yeast MEP transporter sequence (Marini et al.1997 , Suenaga et al. 2003). The ammonium uptake characteristics of OsAMT1s have been compiled in Table 2.

Table 2: Characteristics of ammonium transporters in ammonium uptake in rice roots

Ammonium uptake transporter Expression plant parts Tissue localization(roots) Gene expression Reference
OsAMT1;1 Shoots, Roots – Upregulated by + NH4+ Sonoda et al, 2003
OsAMT1;2 Roots Exodermis,Sclerenchyma, Endodermis, Pericycle cells of of primary root tips(- ? + NH4+) Upregulated by + NH4+ Sonoda et al, 2003
OsAMT1;3 Roots Exodermis,Sclerenchyma, Cortex, Stele(-N) Upregulated by -N Sonoda et al, 2003,Ferreira et al,2015

1.4. CBLs-CIPKs
Adverse external conditions like environmental stress situations, pathogen attack or unbalance of the ion homeostasis halt the development of the plants (Sanders et al. 2002, White and Broadley, 2003). In plants, the calcium ion is involved in almost all the biological processes, including a vital role in regulating the plant’s defense responses during unfavorable circumstances. Calcium ions are released to act as a second messenger that gives way to a large cascade of signal transduction processes for the plant to perceive the fluctuations from the normal conditions and thus help the plant generate appropriate measures (Zhai et al. 2013). In plants, the family of CBL-interacting protein kinases (CIPKs) coupled with their activators, the calcineurin B-like (CBLs) proteins, perceive the changes in the cytosolic calcium concentrations (Weinl and Kudla, 2009). The stimuli are distinct, eliciting specific responses and this specific heightening of calcium in the cytosol is known as “calcium signature”.
Studies have identified more than 250 EF-hand proteins in the model plant, Arabidopsis thaliana (Day et al, 2002). Out of these, four main EF hand-calcium sensor protein families have been distinguished: the calmodulin protein family (CaM), calmodulin-like protein family (CML), calcium-dependent protein kinases (CDPK) and the calcineurin B-like protein family (CBL). CaM is highly conserved in the eukaryotes, CML is found in the plants and some eukaryotes and the other two have evolved in the plants (Luan et al. 2002, Harper and Harmon 2005, Batistic and Kudla 2009, Weinl and Kudla 2009).

1.4.1. CALCINEURIN B-LIKE (CBL) PROTEINS AS CALCIUM SENSORS The adaptations and corresponding response in the plants against the temporary fluctuations in the surroundings are carried out by a distinct molecular machinery. Calcineurin B-like (CBLs) proteins are a family of calcium sensors found in all the known land plants and some chlorophyte green algae (Weinl and Kudla, 2009, Batistic et al.2011). These proteins are named according to their homology to the B regulatory subunit of the phosphatase calcineurin B (CNB) and to neuronal calcium sensors (NCS0 from the animals (Kudla et al. 1999, Luan et al. 2002).
CBLs contain 4 calcium binding EF hands (contains 12 aa residues) with a helix-loop-helix structure and arranged at invariant spacing which bind at most 4 calcium ions and contain a subcellular localization signal at their N terminus (Kolukisaoglu et al. 2004, Batistic et al. 2008,2010, Luan 2009). The CBLs were originally identified in the model plant, Arabidopsis, which contains 10 CBLs in the tonoplast or the plasma membrane (Kim et al. 2007, Batistic et al. 2010). All the CBLs are almost similar in size, at 23-26kD owing to the invariant spacing of the EF-hand domains. The CBLs physically and functionally interact with CBL-Interacting Protein Kinases (CIPKs), which in turn mediates signal transduction by the process of phosphorylation. A motif PFPF was newly discovered, which is thought to play a role in the phosphorylation of CBLs by CIPKs (Du et al. 2011). Arabidopsis genome and the rice genome harbour 10 CBLs each (Kolukisaoglu et al. 2004).
CBLs have been divided into two groups : those with short N-terminal domain with 27-32 aa and those with longer N-terminal domain with 41-43 aa. GFP studies have confirmed the presence of CBL proteins with short N-terminal domain, CBL-1,4,5 and 9 to be present at the plasma membrane and CBL-2,3 and 6 with long N-terminal domains in the tonoplast. CBL7 and CBL8 is localized both in the nucleus and the cytoplasm. CBL10 belonging to neither category because it is unusually big with 74 aa, is found in the tonoplast and in endosomal compartments (D’Angelo et al. 2006, Cheong et al. 2007, Batistic et al 2008).

Fig.2: Structure of CBLs (EF:Elongation Factor hand domain)

The CBLs specifically target a class of SNF-1 (sucrose non-fermenting 1)-related serine/threonine kinases, group3 (SnRK), viz CIPKs (CBL interacting protein kinase) to transmit the perceived calcium signal to the downstream elements (Kudla et al. 1996, Kim et al. 1999).
CIPKs are composed of several domains- a conserved N terminal kinase and a C-terminal regulatory domain, which are separated by a variable junction domain. The calcium bound CBL compound-activates the catalytic activity of the CIPKs through a conserved NAF or FISL motif in the C-terminal regulatory domain (Albrecht et al 2001, Guo et al,2001). The less conserved C-terminal domain are unique to this subgroup of kinases, except the NAF domain, which is an evolutionary conserved region. The NAF domain, being made up of 24 amino acids is named after the principally conserved amino acids: Asn(N)-Ala(A)-Phe(F) (Albrecht et al,2001). The C-terminal domain also harbours the phosphatase interaction motif-PPI, responsible for interaction with 2C-type protein phosphatases (PP2C) (Guo et al, 2001). The conserved N terminal part is composed of a catalytic domain representative of the Ser-Thr-Tyr kinases.
So far, 26 CIPKs have been identified in Arabidopsis. In the rice genome, 30 CIPKs have been discovered which shows specific and selective interactions with the CBLs (Weinl et al. 2009). Almost all the OsCIPK proteins have been found to contain the complete protein kinase domain (with the exception of OsCIPK23) and the NAF domain (with the exception of OsCIP8) (Xiang et al. 2007).

Fig. 3: Schematic representation of CBL-interacting protein kinases (CIPKs)-CIPK are comprised of an N terminal catalytic (shown in blue) and a C terminal regulatory (shown in pink) part. The catalytic domain harbors an activation loop between the conserved amino acids DFG and APE (shown by diagonal lines). The regulatory domain is unique and harbors the NAF motif (shown with check box). The PPI domain is adjacent to the NAF motif. Catalytic and regulatory domains are connected by a junction domain, which is also responsible for kinase activation (shown by red colour).
Copied from Mahajan et al, 2006

The CBLs, as opposed to other proteins, lack enzymatic activity. CBL is the group of calcium sensors with only the calcium binding domain and no effector domain because of which these proteins need to interact with another downstream target protein to regulate an reaction. The combinatorial effect of CBL-CIPK complex shows a complete bimolecular reaction by the CBL binding with the calcium ions, exhibiting sensor relay function, thereby specifically interacting with the corresponding CIPK proteins, which shows kinase activity as a response function, which contributes to the temporal and spatial specificity of the calcium ions (Albrecht et al. 2001, Batistic and Kudla 2004, Kolukisaoglu et al. 2004).
In the absence of CBLs, CIPKs in vitro have little kinase activity and the NAF motif of the CIPK covers its activation loop to keep the protein in an auto-inhibited state. The binding of calcium ions to the EF hand of the CBLs leads to changes in its molecular surface properties and helps its attachment with the C-terminal NAF motif of the CIPK proteins. This in turn, brings about conformational changes in the CIPK (C- terminal domain is released from the kinase domain) exposing its activation loop and the auto-inhibition is removed (Guo et al. 2001). The CIPK can also be phosphorylated by an unknown kinase and become active. This in turn phosphorylates the C-terminal tail of the ammonium transporters (Nühse et al. 2004). There are several CBL-CIPK interaction mediated pathways, e.g. CBL1/9-CIPK23 was identified to positively regulate potassium absorption (Cheong et al. 2007).
The CBL-CIPK complex has been seen in regulating the sodium (Na+), potassium (K+) ,nitrate (NO3-) transport across the plasma membrane and auxin, abscisic acid signaling in Arabidopsis (Liu et al. 1998;2000, Xu et al. 2006, Weinl and Kudla 2009, Luan et al. 2009).

Fig. 4: Pictorial representation of the CBL-CIPK network in regulation of cellular ion homoeostasis
Copied from Zhu et al, 2013

Since the invention of PCR (polymerase chain reaction) by Mullis in 1983 (Mullis, 1990), the laboratory technique has gained ground in the amplification of a specific DNA in an exponential manner with the help of a suitable heat-resistant DNA polymerase. Each PCR cycle consists of three steps: (i) denaturation at approximately 95? (ii) annealing at 5? below the Tm of the primer (this is machine and polymerase dependent) and (iii) elongation at 72?.
For understanding the genetic effects and for the quantitative analysis of the gene expression of the ammonium transporters and the associated proteins involved in its modulation, qRT-PCR was employed. It was first demonstrated by Higuchi (Higuchi et al,1992) and has been since then used as a high-throughput transcriptome analysis and quantifying tool for amplification and simultaneous quantification of the low amount of target DNA (Bustin, 2000). RNA is not used as a template because of its instability, therefore, it has to be converted to cDNA for use in qRT-PCR. SYBR green is the simplest and most commonly used dye for this purpose. The quantification is done by means of the Ct (cycle threshold) value where the accumulation of the fluorescent signal detected by the number of cycles exceeds the background level or the threshold and is inversely proportional to the quantity of nucleic acid present in the sample (Manit et al. 2005).