**2. The role of ion homeostasis in the mechanisms of plant responses to nitrate deficiency**

#### **2.1 Nitrate deficiency and its effects on plants**

Nitrogen is not only the most abundant mineral element in plants [2], but it also contributes signaling [26] and often plays the role of the limiting nutrient for plant growth [27]. However, in aerobic soils, nitrate (NO3 <sup>−</sup>) often represents the predominant source of inorganic nitrogen [28, 29]. However, due to its high solubility in soil water, NO3 <sup>−</sup> is a highly mobile ion [30, 31]. Moreover, its distribution in soils is heterogeneous; thereby in temperate regions the concentrations of nitrate in soil solution can vary from a few micromoles in nonagricultural soils to several millimoles after fertilization [32, 33].

**63**

to sugar metabolism [9].

*Ion Homeostasis Response to Nutrient-Deficiency Stress in Plants*

Nitrogen is known to be one of the major elements contributing to the structure of organic molecules, including proteins, nucleic acids, cofactors and

cally important for plant survival. Indeed, any limitation of nitrate availability affects plant function at different levels—from cellular metabolism to resource allocation, growth and development [2, 13, 34, 35]. The most pronounced visual sign of nitrogen deficiency is chlorosis of older leaves. Since more than half of the total leaf nitrogen is allocated to the photosynthetic apparatus, nitrate shortage results in a marked decrease in the plant photosynthesis [36]. Due to the activity of nitrogen-fixing and nitrifying bacteria, the complete absence of nitrate in soil is rather unlikely. However, this scenario can be simulated in aqueous culture experiments by exclusion of nitrate or other nitrogen sources transformable to nitrate (e.g. urea and ammonium) from the nutrient solution for several days with subsequent re-supplementation [33, 34]. Such experiments proved to be useful to reveal a wide range of responses and multiple mechanisms of plant adaptation to

Adaptation of plants to the prevailing soil nitrate conditions rely on systemic response mechanisms, finely tuned during millions years of terrestrial plant evolution [37]. According to Miller and Cramer [38], plants respond to nitrate stimuli by adjusting expression of the genes, involved in nitrate transport and assimilation—that is, nitrate transporters and assimilatory enzymes, in parallel with posttranslational modifications of these proteins. The simplest model, explaining nitrate sensing in plant root cells, assumes alteration of cytosolic nitrate concentrations in agreement with its external supply [33]. The authors propose that the cytosol represents a finely regulated ionic environment with precise nitrate homeostasis, that is, even minor changes in cytosolic nitrate levels in response to external alterations in

It is well known that nitrate deficiency is closely related to the plant primary metabolism. For example, in *Arabidopsis* leaves, the plant response to nitrate starvation is manifested with the accumulation of sugars and allocation of carbon to roots. This, in turn, results in enhancement of root growth and triggering branching of the lateral roots [8, 13, 39]. This response is important for establishment of root morphology and is accompanied with an increase of root to shoot biomass ratio and in root absorption capacity [40, 41]. As was shown by the RNA microarray techniques, the observed metabolic shifts were underlied by clearly detectable transcriptional changes, which could be mapped to pathways resulting in accumulation of mono- and oligosaccharides, and starch in shoots [8]. For example, enhanced translocation of sucrose from shoots to the roots might indicate an increase in plant root to shoot biomass ratio [8]. Along with the above mentioned fast transcriptional changes, associated with mineral imbalance, the relationship between the cell genome, metabolome and ionome has also become a subject of a special interest [13, 42]. The ionome is usually defined as the mineral nutrient and trace element composition of an organism and represents the inorganic component of cells and organisms [42]. Significant progress has been made in the field of ionomics in the last decade, in which high-throughput elemental profiling is combined with genetics to identify the genes that control the ionome [43]. Since sugars exert metabolite feedback regulation and affect multiple genes involved in photosynthesis, it can be assumed that photosynthesis suppression in nitrogen-deficient plants represents a direct consequence of sugar accumulation in leaves [8]. On the other hand, it needs to be taken into account that primary metabolites play a crucial role in expression regulation of several genes involved in nitrogen consumption and metabolism [44]. Not less important, nitrate itself is involved in regulation of multiple genes assigned

nutrient supply might essentially impact on cell signaling [33].

<sup>−</sup> is criti-

metabolites [2, 13]. Therefore, a sustainable supply of plants with NO3

*DOI: http://dx.doi.org/10.5772/intechopen.89398*

nitrate stress.

*Cell Growth*

source and sink tissues [6–8].

response to NO3

responses to NO3

their development.

ity in soil water, NO3

millimoles after fertilization [32, 33].

**to nitrate deficiency**

ion-transporting systems to deficiencies of K<sup>+</sup>

a pronounced increase in abundance of K<sup>+</sup>

<sup>−</sup> starvation.

soils (especially in the form of NO3

<sup>−</sup> and K+

**2.1 Nitrate deficiency and its effects on plants**

plant growth [27]. However, in aerobic soils, nitrate (NO3

of soils, it might be the case in the modern agricultural practice [3]. In particular, deficiency of nitrogen (N) and potassium (K) is quite common in developing and least developed countries, especially in rice and wheat production in Asia, Africa and Central and South America [4]. Such nutrient deficiency eventually leads to decrease of plant productivity and losses of crop yields. Visual manifestations of stress caused by a deficiency of individual macro- and microelements are well documented [5]. The underlying key physiological processes, affected by mineral deficiencies, are well characterized and include photosynthesis, protein synthesis, primary and secondary metabolism and carbohydrate distribution between

The methods of biochemistry and molecular biology proved to be efficient in disclosing the fine regulatory mechanisms behind ion homeostasis in plants [8]. Thus, the emergence of RNA microarray technology tremendously contributed to the investigation of rapid transcriptional changes associated with mineral imbalance [9–13]. Most of the studies addressing plant responses of

*thaliana* [9, 11–13]. The same is true for phosphorus [14, 15], not further detailed here. However, experiments with such crop plants as wheat [16], tomato [17], rice [18], barley, pepper as well as *Mesembryanthemum crystallinum* [19] revealed

potassium starvation. Similarly, expression of nitrate transporters in wheat roots and leaves [20, 21], sorghum [22] and rice [23, 24] seedlings was enhanced in

The fact, that all mineral nutrients enter the plant in ionic form and, along with involvement in metabolic processes, are crucial for maintaining the cell ion homeostasis seems to be underestimated. Thereby, the existence of cytosolic and vacuolar ion pools needs to be taken into account. These pools are maintained by numerous membrane ion transporters, representing an integrated part of a complex cellular regulatory network [25]. Hence, the specificity of plant responses to nutritional stress may imply a relevant adjustment of systems involved in absorption, trans-

port, distribution, accumulation and remobilization of mineral ions.

Nitrogen and potassium are the most abundant mineral elements in plant nutrition. The principal features of their reception and distribution in plant organs and cells are well studied to date [2]. On the other hand, a lack of these nutrients in

<sup>−</sup> and K+

**2. The role of ion homeostasis in the mechanisms of plant responses** 

Nitrogen is not only the most abundant mineral element in plants [2], but it also contributes signaling [26] and often plays the role of the limiting nutrient for

predominant source of inorganic nitrogen [28, 29]. However, due to its high solubil-

soils is heterogeneous; thereby in temperate regions the concentrations of nitrate in soil solution can vary from a few micromoles in nonagricultural soils to several

that appears to be an appropriate argument for a more thorough analysis of plant

and NO3

<sup>−</sup> rely on *Arabidopsis* 

transporter transcripts in response to

) is quite a common phenomenon [8]

<sup>−</sup>) often represents the

deficiency given the role of ion homeostasis system in

<sup>−</sup> is a highly mobile ion [30, 31]. Moreover, its distribution in

**62**

Nitrogen is known to be one of the major elements contributing to the structure of organic molecules, including proteins, nucleic acids, cofactors and metabolites [2, 13]. Therefore, a sustainable supply of plants with NO3 <sup>−</sup> is critically important for plant survival. Indeed, any limitation of nitrate availability affects plant function at different levels—from cellular metabolism to resource allocation, growth and development [2, 13, 34, 35]. The most pronounced visual sign of nitrogen deficiency is chlorosis of older leaves. Since more than half of the total leaf nitrogen is allocated to the photosynthetic apparatus, nitrate shortage results in a marked decrease in the plant photosynthesis [36]. Due to the activity of nitrogen-fixing and nitrifying bacteria, the complete absence of nitrate in soil is rather unlikely. However, this scenario can be simulated in aqueous culture experiments by exclusion of nitrate or other nitrogen sources transformable to nitrate (e.g. urea and ammonium) from the nutrient solution for several days with subsequent re-supplementation [33, 34]. Such experiments proved to be useful to reveal a wide range of responses and multiple mechanisms of plant adaptation to nitrate stress.

Adaptation of plants to the prevailing soil nitrate conditions rely on systemic response mechanisms, finely tuned during millions years of terrestrial plant evolution [37]. According to Miller and Cramer [38], plants respond to nitrate stimuli by adjusting expression of the genes, involved in nitrate transport and assimilation—that is, nitrate transporters and assimilatory enzymes, in parallel with posttranslational modifications of these proteins. The simplest model, explaining nitrate sensing in plant root cells, assumes alteration of cytosolic nitrate concentrations in agreement with its external supply [33]. The authors propose that the cytosol represents a finely regulated ionic environment with precise nitrate homeostasis, that is, even minor changes in cytosolic nitrate levels in response to external alterations in nutrient supply might essentially impact on cell signaling [33].

It is well known that nitrate deficiency is closely related to the plant primary metabolism. For example, in *Arabidopsis* leaves, the plant response to nitrate starvation is manifested with the accumulation of sugars and allocation of carbon to roots. This, in turn, results in enhancement of root growth and triggering branching of the lateral roots [8, 13, 39]. This response is important for establishment of root morphology and is accompanied with an increase of root to shoot biomass ratio and in root absorption capacity [40, 41]. As was shown by the RNA microarray techniques, the observed metabolic shifts were underlied by clearly detectable transcriptional changes, which could be mapped to pathways resulting in accumulation of mono- and oligosaccharides, and starch in shoots [8]. For example, enhanced translocation of sucrose from shoots to the roots might indicate an increase in plant root to shoot biomass ratio [8]. Along with the above mentioned fast transcriptional changes, associated with mineral imbalance, the relationship between the cell genome, metabolome and ionome has also become a subject of a special interest [13, 42]. The ionome is usually defined as the mineral nutrient and trace element composition of an organism and represents the inorganic component of cells and organisms [42]. Significant progress has been made in the field of ionomics in the last decade, in which high-throughput elemental profiling is combined with genetics to identify the genes that control the ionome [43]. Since sugars exert metabolite feedback regulation and affect multiple genes involved in photosynthesis, it can be assumed that photosynthesis suppression in nitrogen-deficient plants represents a direct consequence of sugar accumulation in leaves [8]. On the other hand, it needs to be taken into account that primary metabolites play a crucial role in expression regulation of several genes involved in nitrogen consumption and metabolism [44]. Not less important, nitrate itself is involved in regulation of multiple genes assigned to sugar metabolism [9].

#### **2.2 Regulation of NO3 <sup>−</sup> transport and mobilization of NO3 <sup>−</sup> storage pools in plant responses to nitrate deficiency**

Importantly, root growth and branching in response to nitrate deficiency are accompanied with an increase in the number of nitrate transporters in plant roots [31]. It results in enhancement of nitrate influx due to up-regulation of nitrate transporters in the membranes of root cells. Not less important might be improvement of their biochemical properties and increase of nitrogen utilization efficiency within the plant [31, 45].

During the last two decades, multiple nitrate transporters/channels were identified and comprehensively characterized [31, 41, 46]. These proteins belong to at least four different families: nitrate transporter 1/peptide transporter family (NRT1/NPF), nitrate transporter 2 (NRT2) family, chloride channel (CLC) family and slow anion channel-associated homologs (SLAC/SLAH) [31, 46]. Thereby, the complex processes, underlying NO3 <sup>−</sup> uptake, translocation and storage in plants, are controlled by fine regulation and crosstalk between these four main types of transporters [47]. This crosstalk gives plants access to sufficient amounts of bioavailable nitrate under changing levels of environmental nitrogen. Among the listed NO3 − transporters, NRT1.1 (NPF6.3) is one of the best characterized [47]. It was shown to be a dual-affinity transporter [48] with sensing functions [41, 49]. The activity of this protein is regulated via phosphorylation at the threonine T101 by calcineurin B-like (CBL)-interacting protein kinase (CIPK23) [48]. This phosphorylation results in a shift from a low affinity to a high-affinity NO3 <sup>−</sup> uptake kinetics [50] with a Km of approximately 4 mmol/L for the low-affinity phase of uptake and 50 μmol/L for the high-affinity one.

As far as NRT2 is concerned, it functions as a high-affinity nitrate transporter in association with a smaller protein NAR 2 (NRT3), which facilitates targeting of NRT2 to the plasma membrane [31, 32]. Under a limited access to exogenic nitrate, altogether four NRT2 transporters (NRT2.1, NRT2.2, NRT2.4 and NRT2.5) take up approximately 95% of the total amount of this anion, with NRT2.1 and NRT2.2 being the main contributors [51]. In durum wheat plants, NRT2.5 was shown to be mostly up-regulated under the conditions of NO3<sup>−</sup> starvation [20]. In sorghum, N stress caused higher abundance of NRT2.2, NRT2.3, NRT2.5 and NRT2.6 transcripts [22].

It is well known that nitrate, as the principal nitrogen source, can be accumulated in leaves and stems of some crop plants, specifically in the plants belonging to the families Chenopodiaceae, Brassicaceae and Asteraceae where nitrate contents can exceed 300 mmol/kg fresh weight or 17–24% of the plant dry weight [52]. Such a strong accumulation of nitrate can be explained by the existence of a small "metabolic" pool of nitrate accessible to nitrate reductase (NR) in cytosol and a large "storage" pool, compartmentally separated from the sites of its metabolism [53]. Later, the predominant identity of this storage compartment was confirmed as the central vacuole of the leaf cell [54]. Further evaluation of cytosol nitrate concentrations using compartmental analysis and cell fractionation techniques provided a solid evidence for the existence of nitrate homeostasis [55] and supported existence of a variable nitrate pool [56]. It was shown that a nitrate/proton (2NO3 <sup>−</sup>/1H+ ) antiporter AtCLCa, localized in the tonoplast, is expressed in both shoots and roots and mediates nitrate accumulation in the vacuole [57] as the most important requirement for maintaining nitrate homeostasis in cells. The major mechanisms prospectively involved in plant responses to nitrate deficiency are summarized in **Figure 1**.

Due to a pronounced success in isolation of pure fractions of barley leaf vacuole vesicles, the storage nitrate pools were found to range from 58% [58] to 99% of the total cellular nitrate content [54]. Indeed, over 90% volume of a mature plant cell is occupied by the vacuole, while the nitrate concentrations in cytoplasm and vacuole

**65**

taining 4.25 mmol/L NO3

**Figure 1.**

[23, 29]. The reuse of NO3

*Ion Homeostasis Response to Nutrient-Deficiency Stress in Plants*

are generally in the range of 3–5 and 30–70 mmol/L, respectively [52, 59, 60]. For example, when Arabidopsis plants were grown in modified Hoagland solution, con-

*−*

*and branching, sustaining their adsorption capacity and provides young (sink) leaves with NO3*

*cell, root cell-to-cell distribution, root to shoot and source to sink leaves translocations.*

*(source) leaves undergo chlorophyll degradation. Chlorosis of leaves serves as a key indicator of NO3*

*Schematic view of plant responses to nitrate deficiency. (A) The effect of NO3*

*in plants. The carbon flow from shoots to roots is triggered in response to NO3*

*(B) The major transport systems, involved in plant response to NO3*

*activation of the plant nitrate transport systems, involved in NO3*

*by the mechanisms of metabolic adjustment and NO3*

cells were 2.8 mmol/L, whereas in vacuoles of the same cells, this value reached 31.8 mmol/L [61]. It is important to notice that nitrate, stored in the vacuole, may be available for assimilation, serving thereby as a reservoir supporting growth for longer periods when external nitrogen supply is limited to only several days [62]. Van der Leij et al. [63] were the first to study the remobilization of vacuolar nitrate in the root cells of barley seedlings during 24 h of nitrate deprivation using double-barrelled nitrate-selective microelectrodes. Their measurements showed much slower remobilization from the vacuoles of cortical cells than those in epidermal cells. The cytosolic nitrate concentrations (activities) in both roots and shoots slowly decreased from 3.7 to 2.9 mmol/L during the first 24 h of the nitrate deprivation, whereas the vacuolar and xylem nitrate activities gradually decreased from 69.6 to 40.2 mmol/L in the same period. In this regard, the authors proposed that the remobilization of vacuolar nitrate can be treated as short term, that is, lasting not more than a few days [63]. It was shown in later studies that nitrate, accumulating in the vacuole, cannot be rapidly transported into the cytoplasm and the transport rates depend on genotypes and differ between individual cultivars

from the vacuole to the cytoplasm and vice versa [64, 65]. According to Glass et al.

<sup>−</sup>, nitrate concentrations in the cytosol of leaf mesophyll

*−*

*−*

*−*

 *remobilization. This carbon flow supports root growth* 

*−*

 *uptake, accumulation, remobilization in root* 

*−*

 *deficiency. NO3*

 *deficiency on carbon metabolism* 

 *deficiency in soil, and is underlied* 

*−*

 *deficiency results in* 

*, whereas older* 

 *deficiency.* 

*−*

<sup>−</sup> in plant tissues to a great extent depends on its transport

*DOI: http://dx.doi.org/10.5772/intechopen.89398*

*Ion Homeostasis Response to Nutrient-Deficiency Stress in Plants DOI: http://dx.doi.org/10.5772/intechopen.89398*

#### **Figure 1.**

*Cell Growth*

**2.2 Regulation of NO3**

within the plant [31, 45].

for the high-affinity one.

complex processes, underlying NO3

**in plant responses to nitrate deficiency**

**<sup>−</sup> transport and mobilization of NO3**

Importantly, root growth and branching in response to nitrate deficiency are accompanied with an increase in the number of nitrate transporters in plant roots [31]. It results in enhancement of nitrate influx due to up-regulation of nitrate transporters in the membranes of root cells. Not less important might be improvement of their biochemical properties and increase of nitrogen utilization efficiency

During the last two decades, multiple nitrate transporters/channels were identified and comprehensively characterized [31, 41, 46]. These proteins belong to at least four different families: nitrate transporter 1/peptide transporter family (NRT1/NPF), nitrate transporter 2 (NRT2) family, chloride channel (CLC) family and slow anion channel-associated homologs (SLAC/SLAH) [31, 46]. Thereby, the

controlled by fine regulation and crosstalk between these four main types of transporters [47]. This crosstalk gives plants access to sufficient amounts of bioavailable nitrate under changing levels of environmental nitrogen. Among the listed NO3

transporters, NRT1.1 (NPF6.3) is one of the best characterized [47]. It was shown to be a dual-affinity transporter [48] with sensing functions [41, 49]. The activity of this protein is regulated via phosphorylation at the threonine T101 by calcineurin B-like (CBL)-interacting protein kinase (CIPK23) [48]. This phosphorylation

a Km of approximately 4 mmol/L for the low-affinity phase of uptake and 50 μmol/L

As far as NRT2 is concerned, it functions as a high-affinity nitrate transporter in association with a smaller protein NAR 2 (NRT3), which facilitates targeting of NRT2 to the plasma membrane [31, 32]. Under a limited access to exogenic nitrate, altogether four NRT2 transporters (NRT2.1, NRT2.2, NRT2.4 and NRT2.5) take up approximately 95% of the total amount of this anion, with NRT2.1 and NRT2.2 being the main contributors [51]. In durum wheat plants, NRT2.5 was shown to be mostly up-regulated under the conditions of NO3<sup>−</sup> starvation [20]. In sorghum, N stress caused higher abundance of NRT2.2, NRT2.3, NRT2.5 and NRT2.6 transcripts [22]. It is well known that nitrate, as the principal nitrogen source, can be accumulated in leaves and stems of some crop plants, specifically in the plants belonging to the families Chenopodiaceae, Brassicaceae and Asteraceae where nitrate contents can exceed 300 mmol/kg fresh weight or 17–24% of the plant dry weight [52]. Such a strong accumulation of nitrate can be explained by the existence of a small "metabolic" pool of nitrate accessible to nitrate reductase (NR) in cytosol and a large "storage" pool, compartmentally separated from the sites of its metabolism [53]. Later, the predominant identity of this storage compartment was confirmed as the central vacuole of the leaf cell [54]. Further evaluation of cytosol nitrate concentrations using compartmental analysis and cell fractionation techniques provided a solid evidence for the existence of nitrate homeostasis [55] and supported existence

of a variable nitrate pool [56]. It was shown that a nitrate/proton (2NO3

porter AtCLCa, localized in the tonoplast, is expressed in both shoots and roots and mediates nitrate accumulation in the vacuole [57] as the most important requirement for maintaining nitrate homeostasis in cells. The major mechanisms prospectively involved in plant responses to nitrate deficiency are summarized in **Figure 1**.

Due to a pronounced success in isolation of pure fractions of barley leaf vacuole vesicles, the storage nitrate pools were found to range from 58% [58] to 99% of the total cellular nitrate content [54]. Indeed, over 90% volume of a mature plant cell is occupied by the vacuole, while the nitrate concentrations in cytoplasm and vacuole

results in a shift from a low affinity to a high-affinity NO3

**<sup>−</sup> storage pools** 

−

<sup>−</sup> uptake kinetics [50] with

<sup>−</sup>/1H+

) anti-

<sup>−</sup> uptake, translocation and storage in plants, are

**64**

*Schematic view of plant responses to nitrate deficiency. (A) The effect of NO3 − deficiency on carbon metabolism in plants. The carbon flow from shoots to roots is triggered in response to NO3 − deficiency in soil, and is underlied by the mechanisms of metabolic adjustment and NO3 − remobilization. This carbon flow supports root growth and branching, sustaining their adsorption capacity and provides young (sink) leaves with NO3 − , whereas older (source) leaves undergo chlorophyll degradation. Chlorosis of leaves serves as a key indicator of NO3 − deficiency. (B) The major transport systems, involved in plant response to NO3 − deficiency. NO3 − deficiency results in activation of the plant nitrate transport systems, involved in NO3 − uptake, accumulation, remobilization in root cell, root cell-to-cell distribution, root to shoot and source to sink leaves translocations.*

are generally in the range of 3–5 and 30–70 mmol/L, respectively [52, 59, 60]. For example, when Arabidopsis plants were grown in modified Hoagland solution, containing 4.25 mmol/L NO3 <sup>−</sup>, nitrate concentrations in the cytosol of leaf mesophyll cells were 2.8 mmol/L, whereas in vacuoles of the same cells, this value reached 31.8 mmol/L [61]. It is important to notice that nitrate, stored in the vacuole, may be available for assimilation, serving thereby as a reservoir supporting growth for longer periods when external nitrogen supply is limited to only several days [62].

Van der Leij et al. [63] were the first to study the remobilization of vacuolar nitrate in the root cells of barley seedlings during 24 h of nitrate deprivation using double-barrelled nitrate-selective microelectrodes. Their measurements showed much slower remobilization from the vacuoles of cortical cells than those in epidermal cells. The cytosolic nitrate concentrations (activities) in both roots and shoots slowly decreased from 3.7 to 2.9 mmol/L during the first 24 h of the nitrate deprivation, whereas the vacuolar and xylem nitrate activities gradually decreased from 69.6 to 40.2 mmol/L in the same period. In this regard, the authors proposed that the remobilization of vacuolar nitrate can be treated as short term, that is, lasting not more than a few days [63]. It was shown in later studies that nitrate, accumulating in the vacuole, cannot be rapidly transported into the cytoplasm and the transport rates depend on genotypes and differ between individual cultivars [23, 29]. The reuse of NO3 <sup>−</sup> in plant tissues to a great extent depends on its transport from the vacuole to the cytoplasm and vice versa [64, 65]. According to Glass et al.

[66], low concentrations of NO3 <sup>−</sup> in the cytosol are sufficient to ensure NR activity, whereas relatively high amounts of nitrate in the vacuole only minimally influence the NR activity [66].

Based on the ability to sustain nitrate deficiency, tolerant and sensitive genotypes can be distinguished [51]. At least to some extent, this difference might depend on the rates of nitrate transport across the tonoplast from vacuole into cytoplasm during nitrate starvation and on the ability to restore the vacuolar pool under normal nitrate supply afterwards [52]. Thereby, the tonoplast proton pumps (V-ATPase and V-PPase) play a key role in the distribution of NO3 <sup>−</sup> between vacuole and cytoplasm [59] by building an electrochemical transmembrane proton gradient [52, 67]. It was proposed that the NO3 <sup>−</sup> transport from the cytoplasm to the vacuole is mainly mediated by the vacuole H<sup>+</sup> / NO3 <sup>−</sup> antiport system [68]. Later, it was demonstrated that AtCLCa operates as a NO3 <sup>−</sup>/H<sup>+</sup> exchanger with a 2:1 nitrate-proton stoichiometry, building an up to 1:50 nitrate gradient between the two compartments [57]. In this context, the nitrate/proton symport system might be responsible for the remobilization of the vacuolar nitrate [69]. However, the mechanisms behind nitrate efflux from vacuoles to cytoplasm are mostly unknown so far. The tonoplast efflux NO3 <sup>−</sup> transporters were not identified as well [31]. However, three members of the NRT1/NPF family, localized in the tonoplast— NPF5.11, NPF5.12 and NPF5.16—have been proposed to be the vacuolar nitrate efflux transporters in Arabidopsis [67]. They are predominantly expressed in the cells of root pericycle and xylem parenchyma, thus acting as important players in modulation of nitrate allocation between roots and shoots and being crucial for plant adaptation to the changing environment [67]. Generally, the vacuole is not only a storage location for nitrate but also impacts on turgor maintenance and osmotic balance [33, 50, 70].

Usually, the highest concentrations of nitrate are found in stems and petioles, whereas the contents of nitrate are lower in leaf blades [50]. According to our own observations, in lettuce and Chinese cabbage, the corresponding differences in nitrate concentrations can reach two- to eightfold under high nitrate supply [71]. In response to a 7-day long nitrate exclusion from the nutrient solution, the nitrate contents decreased primarily in vascular tissues. Essentially this reduction in nitrate contents was accompanied with a pronounced increase in the levels of chloride and anions of organic acids, obviously acting to compensate for nitrate to maintain the required turgor in the vacuoles.

It is logical to assume that the ability of different plants to generate, maintain and mobilize the reserve nitrate pools in vascular tissues is one of the strategies for their survival under adverse conditions of nitrogen deficiency. In particular, nitrate storage pools enable survival of winter cultivars during the periods of nitrate depletion in soils due to intensive rains [32]. This strategy is in agreement with the plant inherited nitrate mobilization and transport from older (source) to younger (sink) leaves during plant ontogenesis or when the external nitrate availability is decreasing [50, 72]. This was confirmed by the results of gene expression analysis. Thus, it was shown that the expression of the AtNRT1.7 gene is induced by nitrogen starvation in the sieve elements/companion cells of the leaf minor veins and facilitates phloem loading in older leaves [73]. Analogously, AtNRT2.4 and AtNRT2.5 are induced by nitrogen starvation and repressed by nitrogen resupply [74]. NRT2.4 is expressed in the phloem of leaf major veins and might retrieve nitrate under nitrogen starvation towards minor veins [75]. In turn, NRT2.5 is expressed in minor veins and, together with NRT2.4, may affect leaf remobilization and phloem transport of nitrate [51]. Moreover, NRT1.11 and NRT1.12, localized in the companion cells of the leaf major veins, were shown to impact on phloem loading, besides playing a role in xylem-to-phloem transfer [76].

**67**

*Ion Homeostasis Response to Nutrient-Deficiency Stress in Plants*

**3.1 Potassium deficiency and its effects on plants**

patterns and their relative abundances in higher plants [83].

bilization, the concentration of biologically available K<sup>+</sup>

cantly increased upon re-supplementation of soil with water [89].

1000-fold lower potassium supply (1 μmol/L) [90].

root surface. Thus, diffusion of K+

plant species (1 mmol/L K+

decrease due to prolonged K deficiency [81]. Hence, cytosolic K<sup>+</sup>

pH, ion homeostasis and electrical membrane potential [77, 78, 83, 85].

Thus, it can be concluded that activation and induction of nitrate transport and mobilization of nitrate from its reserve pools likely are an important component of

Potassium is the second (after nitrogen) most abundant mineral element in plants

was shown to be essential in the activation of 50–60 key enzymes, involved in critical

As mentioned above, the lack of potassium might suppress various enzymatic

metabolic processes [81, 82], including photosynthesis, oxidative metabolism and protein synthesis [2]. Thus, this cation might be involved in the regulation of metabolite

activities [81, 82], but this can occur only when potassium cytosolic contents

crucial for the central cell metabolism [84], plant growth and adaptation to the environment and must, therefore, be finely controlled [78]. In addition, K+

crucial role in the establishment of cell turgor and osmoregulation, neutralization/ scavenging of anions (e.g. those of organic and inorganic acids), control of cytosolic

Potassium availability in soil is the main factor, affecting supply of terrestrial plants with this element. Indeed, although potassium is the seventh most abundant element in the Earth crust [86], only a small part of the whole soil potassium pool is present in a form readily available for plants, whereas most of the soil potassium constitutes hardly soluble minerals [87]. Thus, due to the low rates of their solu-

low and varies between 0.1 and 1.0 mmol/L [82, 88]. Moreover, due to a rapid local depletion at the root surface, in reality these values can be even lower, and supply of the plant with potassium is highly dependent on the rates of its liberation from minerals and transport in soil solution [89]. Both rates are typically relatively slow: growing plants can deplete soil solution to yield potassium concentrations between 1 and 2 μmol/L. On the other hand, this might result in enhancement of potassium release and mobilization in soil [89]. Obviously, the amounts of soil water (i.e. soil water contents) essentially affect the gradients of potassium concentrations on the

According to the available literature data, under sufficient nutritional supply, the average potassium contents in the most of the plant species vary between 4 and 8% (w/w) in dry matter [84]. However, for multiple other species, the optimal potassium contents are essentially lower and lie in the range of 0.5–2.0% (w/w) [80]. Thus, a comparative study of potassium contents in 14 hydroponically grown

in the range of 153–274 mmol/kg fresh weight with a drop to 15–53 mmol/kg under a

adaptation to the environment [78]. In contrast to nitrogen, potassium is not involved in metabolism and remains in ionic form to execute its specific functions in plant cells [79]. Potassium is associated with or directly involved in several physiological processes supporting plant growth and development—photosynthesis, protein and starch biosynthesis; transport of sugars and nutrients; and stomatal closure [77, 80]. Moreover,

) in plant cells [77] and is essential for plant growth and

homeostasis is

in soil solution is rather

is essentially restricted in dry soil but is signifi-

in the nutritional solution) revealed potassium contents

plays a

**3. The role of ion homeostasis in the mechanisms of plant responses** 

*DOI: http://dx.doi.org/10.5772/intechopen.89398*

the cell response to nitrate deficiency.

**to potassium deficiency**

[2]. It is the main cation (K+

K+

*Cell Growth*

[66], low concentrations of NO3

so far. The tonoplast efflux NO3

osmotic balance [33, 50, 70].

required turgor in the vacuoles.

playing a role in xylem-to-phloem transfer [76].

gradient [52, 67]. It was proposed that the NO3

to the vacuole is mainly mediated by the vacuole H<sup>+</sup>

Later, it was demonstrated that AtCLCa operates as a NO3

the NR activity [66].

<sup>−</sup> in the cytosol are sufficient to ensure NR activity,

<sup>−</sup> transport from the cytoplasm

<sup>−</sup>/H<sup>+</sup>

<sup>−</sup> antiport system [68].

exchanger with a 2:1

/ NO3

<sup>−</sup> transporters were not identified as well [31].

<sup>−</sup> between

whereas relatively high amounts of nitrate in the vacuole only minimally influence

Based on the ability to sustain nitrate deficiency, tolerant and sensitive genotypes can be distinguished [51]. At least to some extent, this difference might depend on the rates of nitrate transport across the tonoplast from vacuole into cytoplasm during nitrate starvation and on the ability to restore the vacuolar pool under normal nitrate supply afterwards [52]. Thereby, the tonoplast proton

vacuole and cytoplasm [59] by building an electrochemical transmembrane proton

nitrate-proton stoichiometry, building an up to 1:50 nitrate gradient between the two compartments [57]. In this context, the nitrate/proton symport system might be responsible for the remobilization of the vacuolar nitrate [69]. However, the mechanisms behind nitrate efflux from vacuoles to cytoplasm are mostly unknown

However, three members of the NRT1/NPF family, localized in the tonoplast— NPF5.11, NPF5.12 and NPF5.16—have been proposed to be the vacuolar nitrate efflux transporters in Arabidopsis [67]. They are predominantly expressed in the cells of root pericycle and xylem parenchyma, thus acting as important players in modulation of nitrate allocation between roots and shoots and being crucial for plant adaptation to the changing environment [67]. Generally, the vacuole is not only a storage location for nitrate but also impacts on turgor maintenance and

Usually, the highest concentrations of nitrate are found in stems and petioles, whereas the contents of nitrate are lower in leaf blades [50]. According to our own observations, in lettuce and Chinese cabbage, the corresponding differences in nitrate concentrations can reach two- to eightfold under high nitrate supply [71]. In response to a 7-day long nitrate exclusion from the nutrient solution, the nitrate contents decreased primarily in vascular tissues. Essentially this reduction in nitrate contents was accompanied with a pronounced increase in the levels of chloride and anions of organic acids, obviously acting to compensate for nitrate to maintain the

It is logical to assume that the ability of different plants to generate, maintain and mobilize the reserve nitrate pools in vascular tissues is one of the strategies for their survival under adverse conditions of nitrogen deficiency. In particular, nitrate storage pools enable survival of winter cultivars during the periods of nitrate depletion in soils due to intensive rains [32]. This strategy is in agreement with the plant inherited nitrate mobilization and transport from older (source) to younger (sink) leaves during plant ontogenesis or when the external nitrate availability is decreasing [50, 72]. This was confirmed by the results of gene expression analysis. Thus, it was shown that the expression of the AtNRT1.7 gene is induced by nitrogen starvation in the sieve elements/companion cells of the leaf minor veins and facilitates phloem loading in older leaves [73]. Analogously, AtNRT2.4 and AtNRT2.5 are induced by nitrogen starvation and repressed by nitrogen resupply [74]. NRT2.4 is expressed in the phloem of leaf major veins and might retrieve nitrate under nitrogen starvation towards minor veins [75]. In turn, NRT2.5 is expressed in minor veins and, together with NRT2.4, may affect leaf remobilization and phloem transport of nitrate [51]. Moreover, NRT1.11 and NRT1.12, localized in the companion cells of the leaf major veins, were shown to impact on phloem loading, besides

pumps (V-ATPase and V-PPase) play a key role in the distribution of NO3

**66**

Thus, it can be concluded that activation and induction of nitrate transport and mobilization of nitrate from its reserve pools likely are an important component of the cell response to nitrate deficiency.
