**2. The use of Ins and phosphorylated Ins-derivatives in plant cells under stress**

Although there are several articles addressing the involvement of Ins and its derivatives in plant stress responses, so far no report has described the overall transcriptional orchestration of these components covering the metabolic pathways related to them. Information is available only in particular assays covering few genes and their expression modulations, some individ‐ ual gene knockout analyses or a specific GMO (genetically modified organism) assay. In most cases, Ins and its phosphorylated derivatives have positive impact in plant tolerance/resistance to several stresses, promoting a biotechnological interest in these compounds.

molecular mechanisms for physiological adjustments to inadequate conditions for develop‐ ment, resulting from adverse conditions. In this way, plants have a diverse and active cellular machinery at different stratified levels, covering perception, signaling, transcriptional control

Among the molecules functioning in more than one of the aforementioned levels, inositol (Ins; C6H12O6) is a biomolecule of great interest. It is a cyclic carbohydrate (polyalcohol) that anchors in each of the six carbons forming the ring, a hydroxyl group. Along with their derivatives, Ins has multiple effects on plant metabolism. They act from the production of secondary messengers to the synthesis of osmolytes and antioxidants (more details in the reviews of [2, 3]). Phosphorylated Ins-derivatives [(poly)phosphoinositides and inositol (poly)phosphates] are versatile and dynamic signaling molecules in all eukaryotes, particularly in plants [4]. These two classes of compounds [highlighted in red and orange respectively, in Figure 1] are interdependent. While (poly)phosphoinositides are used in the synthesis of inositol (poly)phosphates through the action of phospholipases; the breaking of inositol (poly)phos‐ phates produces inositol, which is a substrate for the synthesis of (poly)phosphoinositides. Moreover, according to Ins metabolism, shown in Figure 1, another branch realizes the synthesis of methylated derivatives (highlighted in green). These compounds act as important osmoregulators during periods of unfavorable conditions [5]. Additionally, oxygenated Insderivatives are observed (highlighted in yellow in Figure 1), which are involved in increasing plant tolerance to stresses by decreasing oxidative damage [6]. Still associated with Ins is the metabolism of the Raffinose Family Oligosaccharides (RFOs) [7]. In this biosynthetic pathway, the galactinol synthase (GolS; EC 2.4.1.123) uses myo-inositol and UDP-galactose to produce galactinol, which serve as galactose donors for subsequent synthesis of RFO members ([8, 9]; highlighted in red in Figure 2). Recent reports indicate that RFOs may assist in the reactive oxygen species (ROS) cleaning process. In periods of stress, ROS accumulation favors the

Due to the impacts related to the Ins and its derivatives in a plant cell, assays have been conducted to understand how these biomolecules affect the physiology of plants. Thus, overexpression or knockout of genes present in these pathways has been shown as interesting strategy for generating more efficient plants capable of growing under abiotic stress condi‐ tions. In this chapter, studies using molecular genetic tools will be presented, which affect the

**2. The use of Ins and phosphorylated Ins-derivatives in plant cells under**

Although there are several articles addressing the involvement of Ins and its derivatives in plant stress responses, so far no report has described the overall transcriptional orchestration of these components covering the metabolic pathways related to them. Information is available only in particular assays covering few genes and their expression modulations, some individ‐ ual gene knockout analyses or a specific GMO (genetically modified organism) assay. In most

above-mentioned metabolic pathways and the studied organisms.

of key metabolic pathways and synthesis of molecules responsive to stresses [1].

82 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

physiological imbalance of plants [10].

**stress**

Among the Ins-derivatives, those that are phosphorylated [(poly)phosphoinositides (high‐ lighted in red in Figure 1) and inositol (poly)phosphates (highlighted in orange)] are the most discussed in the literature. This fact reflects the importance of these compounds in plant physiology in signaling activities. An example is a work developed by Hunt et al. [11] with transgenic tobacco plants (*Nicotiana tabacum* cv. Wisconsin 38) expressing reduced levels of a gene encoding phospholipase C (PI-PLC; EC 3.1.4.11). This enzyme catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to InsP3 [inositol (1,4,5)–trisphosphate; (Figure 1)], affecting from this point on the rest of the pathway. The obtained transgenic plants showed a partial inhibition of stomatal opening due to the action of ABA (abscisic acid phytohormone). A possible role of PI-PLC enzyme and InsP3 in ABA-dependent signaling pathway was also reported, thus suggesting that a complete response to ABA phytohormone in guard cells requires PI-PLC. However, other calcium-mobilizing pathways could also help in ABA guard cell signaling. The stomatal movement is a critical process for the plant under drought conditions by enabling better use of its water supply.

Mills et al. [12] provide more details on this ABA-mediated stomatal regulation in transgenic plants obtained by Hunt et al. [11]. A three-day assay under drought and in dark-adapted conditions, to reopen the stomata in response to light, was carried out. The results showed that transgenic plants with reduced PI-PLC as compared to control plants (with the empty vector without the transgene insert) have a greater increase in stomatal conductance. Thus, there is a strengthening of the role of inhibition due to PI-PLC in ABA-mediated stomatal opening. Further analysis indicated tobacco PI-PLC acting on the inhibition of stomatal opening by ABA, but not in promoting ABA-induced stomatal closure.

There are also reports of the involvement of Ins-derivatives in ABA-independent mechanisms during periods of drought. Perera et al. [13] obtained *A. thaliana* plants transformed with human type I gene for inositol polyphosphate 5-phosphatase (InsP5-ptase; EC 3.1.3.56; Figure 1). This enzyme hydrolysis InsP3, which is an essential element of the signal transduction pathway in general response to stresses. Looking at the impact on plant response to drought, the authors observed that transgenic plants showed increased stress tolerance after 12 days of watering suspension. After this period of stress, wild and controls plants (with the empty vector without the transgene insert) became brown and dry, while InsP5-ptase transgenic lines remained green and turgid. Furthermore, transgenic plants under drought conditions showed reduced levels of ABA compared with wild plants in the same condition as well as no induction of several genes regulated by the phytohormone. The analyses of stomatal responses in transgenic plants observed that guard cells are less responsive to the inhibition of opening stomata promoted by ABA. Nevertheless, there is an increase in sensitivity to the closing of the stomata, induced by the phytohormone. The transgenic plants showed irregular behavior in coordinated processes via ABA. Despite this, these plants showed a compensatory overex‐ pression of an ABA-independent pathway involving the transcription factor (TF) DREB2A (dehydration-responsive element-binding protein 2A) and a subset of genes regulated by this TF. In this way, the drought tolerance of Ins5-ptase plants was mediated in part via DREB2A-

**Figure 1.** Inositol phosphate metabolism from the KEGG Pathway database. The stereoisomers (highlighted in blue); the phosphorylated derivatives [inositol (poly)phosphates (highlighted in orange) and (poly)phosphoinositides (high‐ lighted in red)]; the methylated derivatives (highlighted in green); the oxygenated derivatives (highlighted in green); and others (highlighted in purple).

dependent and that constitutive dampening of InsP3 signal revealed unforeseen interconnec‐ tions between signaling pathways.

In a similar assay, Khodakovskaya et al. [14] reported physiological consequences in transgenic lines of *Solanum lycopersicum* (cv. Micro-Tom) overexpressing a human type I InsP5-ptase gene. The transgenic lines presented a content of 15–30% of InsP3 observed in the wild-type plants. This reduction led to increases in: (1) the total vegetative biomass (two- to fourfold) with an increased ratio of root:shoot dry weight; (2) lycopene levels (in fruit); and (3) the hexose concentration (on fruits and leaves). After 13 days of water stress, the leaf water potential in transgenic plants was about -0.4 MPa higher than in control plants. Drought tolerance in transgenic lines was associated with increased hexoses in the leaves. This would contribute to maintaining a greater potential for water in transgenic leaves under drought. Furthermore, increases in the number of root biomass may have contributed to this improved performance.

The involvement of InsP3 in other stress tolerance processes, beyond drought, has also been demonstrated. Alimohammadi et al. [15] obtained transgenic tomato plants (*Lycopersicon esculentum* cv. MicroTom,) overexpressing a human type I InsP5-ptase gene. These plants presented a decreased level of InsP3 and supported a continuous exposure to light longer than wild plants. Prolonged exposure to light causes oxidative stress in plant cells and can result in irreversible damage. However, the molecular mechanism involved in this tolerance process was not reported, but these transgenic lines were characterized in more detail by Alimoham‐

The Transcriptional Modulation of Inositols and Raffinose Family Oligosaccharides Pathways in Plants — An... http://dx.doi.org/10.5772/61341 85

**Figure 2.** The Galactose metabolism from the KEGG Pathway database. Highlighted in red are the enzymatic reactions associated with the metabolism of Raffinose Family Oligosaccharides.

dependent and that constitutive dampening of InsP3 signal revealed unforeseen interconnec‐

**Figure 1.** Inositol phosphate metabolism from the KEGG Pathway database. The stereoisomers (highlighted in blue); the phosphorylated derivatives [inositol (poly)phosphates (highlighted in orange) and (poly)phosphoinositides (high‐ lighted in red)]; the methylated derivatives (highlighted in green); the oxygenated derivatives (highlighted in green);

In a similar assay, Khodakovskaya et al. [14] reported physiological consequences in transgenic lines of *Solanum lycopersicum* (cv. Micro-Tom) overexpressing a human type I InsP5-ptase gene. The transgenic lines presented a content of 15–30% of InsP3 observed in the wild-type plants. This reduction led to increases in: (1) the total vegetative biomass (two- to fourfold) with an increased ratio of root:shoot dry weight; (2) lycopene levels (in fruit); and (3) the hexose concentration (on fruits and leaves). After 13 days of water stress, the leaf water potential in transgenic plants was about -0.4 MPa higher than in control plants. Drought tolerance in transgenic lines was associated with increased hexoses in the leaves. This would contribute to maintaining a greater potential for water in transgenic leaves under drought. Furthermore, increases in the number of root biomass may have contributed to this improved performance.

The involvement of InsP3 in other stress tolerance processes, beyond drought, has also been demonstrated. Alimohammadi et al. [15] obtained transgenic tomato plants (*Lycopersicon esculentum* cv. MicroTom,) overexpressing a human type I InsP5-ptase gene. These plants presented a decreased level of InsP3 and supported a continuous exposure to light longer than wild plants. Prolonged exposure to light causes oxidative stress in plant cells and can result in irreversible damage. However, the molecular mechanism involved in this tolerance process was not reported, but these transgenic lines were characterized in more detail by Alimoham‐

tions between signaling pathways.

84 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

and others (highlighted in purple).

madi et al. [16]. These authors observed that under stress conditions, the transgenic plants maintained high chlorophyll content and accumulated low levels of hydrogen peroxide. This fact was attributed to the induction of genes related to multiple antioxidants [LeAPX1 (*L. esculentum* ascorbate peroxidase 1), SICAT2, LeSOD (*L. esculentum* superoxide dismutase)] during continuous exposure to light. Other effects included overexpression of the LePHYB photoreceptor (*L. esculentum* phytochrome B) and a key enzyme [LeCHS1 (*L. esculentum* chalcone synthase)] in the biosynthesis pathway of flavonoids, which are plant nonenzymatic antioxidants. There was also an overexpression of the SIMYB12 transcription factor, leading to an increase in flavonoids in tomato plants by up-regulation of the LeCHS1 expression. A relationship was established between change in phosphoinositol signaling pathway and increases tolerance to continuous exposure to light, through the activation of ROS-scavenging enzymes, and up-regulation of molecular activators of non-enzymatic antioxidants.

The biotechnological potential through the manipulation of compounds shown in Ins-related metabolic pathways may also be seen in the work of Ahmad et al. [17]. These authors per‐ formed a comprehensive analysis of *A. thaliana* genome, using the activation tagging technique in dedifferentiated calli, to identify salt-tolerant mutants (NaCl 150 mM). To this end, plants were modified with pRi35ADEn4 binary vector. Such vector contains four copies of the 339 bp long cauliflower mosaic virus (CaMV) 35S enhancer in the construct that induces the expres‐ sion of adjacent genes after proper insertion. Of the 18 potential tolerant mutants (150 mM NaCl), a line (stc1; salt-tolerant callus 1) presented the gene for myo-inositol-1-P-synthase-1 [At4g39800; MIPS1; EC 5.5.1.4; Figure 1] with induced expression in callus, with or without salt. This gene expressed 45 and 15 times higher compared to wild-type under the control condition. MIPS catalyzes the first step in the biosynthesis of inositol from glucose-6-P (Figure 1). The referred induction was greater in the mutant line than in the wild type (approximately, 260 times higher), both under stress. The *mips* gene transcription in the wild type reduced dramatically under stress condition. The tolerance analysis revealed that the mutant plants regenerated from calli showed salt tolerance in germination and growth. However, the mechanism involved was not disclosed, but the authors suggested that MIPS protect the calli and the plants from salt stress as osmolytes or by providing a precursor in the regulation of signal transduction pathways.

Kusuda et al. [18] went beyond the study of transformed lines overexpressing enzymes from the Ins-related pathways. They analyzed the differences among wild type and transformants lines in regard to salt tolerance in 3.5 days in medium with concentrations up to 250 mM NaCl. They also sought for differences by mining the metabolomes (the fourth leaf tissue harvested at 0, 6, and 12 h after NaCl stress induction) of the studied plants. To this end, a rice cultivar (*Oryza sativa* cv. Kitaake) was transformed with the construction Act::RINO1. The RINO1 gene encodes an MIPS (EC 5.5.1.4, Figure 1). It has been demonstrated that the constitutive overex‐ pression of rice MIPS when compared to a wild type, results in greater tolerance to salt stress. Furthermore, it leads to a range of metabolic changes, with increased production of various metabolites (such as inositol, raffinose, ascorbate, amino acids). These handle the protection of plants from abiotic stresses. Additionally, activation of basal metabolisms such as glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle has been observed during induction of the Ins metabolism in those plants overexpressing MIPS.

Ins metabolism and phosphorylated Ins-derivatives are also associated with response to biotic stresses in plants. This fact shows the plurality of actions of these compounds. Murphy et al. [19] report evidence in this direction. They obtained transgenic potato [*S. tuberosum* L. (cv. Desiree)] and *A. thaliana* lines, synthesizing low levels of phytic acid (1-D-myo-inositol-P6 or InsP6, Figure 1). The transgenic potato lines were obtained by: (1) constitutive expression of an antisense sequence of the myo-inositol 3-phosphate synthase gene. This enzyme (IPS, EC 5.5.1.4, Figure 1) catalyzes the first step in the InsP6 biosynthesis; (2) plants expressing the *Escherichia coli* polyphosphate kinase (PPK, EC 2.7.4.1; Figure 1). PPK inserts inorganic phosphate into chains of phosphate residues linked by phospho-anhydride bonds, and this decreases the phosphate available to InsP6 biosynthesis. Genetically modified potatoes presenting both (1) and of (2) showed increased susceptibility to avirulent pathogen potato virus Y and the virulent pathogen tobacco mosaic virus (TMV). In relation to *A. thalianai*, the authors obtained three loss-of-function mutants [two (atips1 and atips2) involving the gene for IPS (EC 5.5.4.1, Figure 1); and one (atipk1) involving the gene encoding inositol polyphos‐ phate kinase (IPK1; EC 2.7.1.158, Figure 1)]. IPK1 catalyzes the final step in InsP6 and without this enzyme, very little InsP6 is done. The disruption of InsP6 biosynthesis in *A. thaliana* resulted in an increased susceptibility to viruses (tobacco mosaic virus), bacteria [*Pseudomonas syringae* pv tomato (Pst) DC3000 and Pst DC3000 AvrB], and fungus (*Botrytis cinerea*). The increased susceptibility only occurred for atips2 and atipk1 mutants. For atips1 mutants showing also InsP6 depletion, the resistance to pathogens was not compromised. This suggests either that a particular pool of InsP6 regulates defense against pathogens in *A. thaliana*, or IPS1 and IPs2 are differentially regulated, and one cannot compensate for the other. It was observed with regard to the typical responses of both species that levels of salicylic acid (SA), a key molecular signal for establishing local and systemic acquired resistance, were not diminished. Therefore, SA was not responsible for increased susceptibility to pathogens analyzed. Fur‐ thermore, a relationship between the inositol phosphate metabolism and basal resistance to pathogens (fungi, bacteria, and viruses) has been established. This indicates that InsP6 is also required as an essential operation signal for regulation and defense in plants, together with several other well-known defensive signals (NO, cADPR, SA and Ca2+ ).

sion of adjacent genes after proper insertion. Of the 18 potential tolerant mutants (150 mM NaCl), a line (stc1; salt-tolerant callus 1) presented the gene for myo-inositol-1-P-synthase-1 [At4g39800; MIPS1; EC 5.5.1.4; Figure 1] with induced expression in callus, with or without salt. This gene expressed 45 and 15 times higher compared to wild-type under the control condition. MIPS catalyzes the first step in the biosynthesis of inositol from glucose-6-P (Figure 1). The referred induction was greater in the mutant line than in the wild type (approximately, 260 times higher), both under stress. The *mips* gene transcription in the wild type reduced dramatically under stress condition. The tolerance analysis revealed that the mutant plants regenerated from calli showed salt tolerance in germination and growth. However, the mechanism involved was not disclosed, but the authors suggested that MIPS protect the calli and the plants from salt stress as osmolytes or by providing a precursor in the regulation of

86 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

Kusuda et al. [18] went beyond the study of transformed lines overexpressing enzymes from the Ins-related pathways. They analyzed the differences among wild type and transformants lines in regard to salt tolerance in 3.5 days in medium with concentrations up to 250 mM NaCl. They also sought for differences by mining the metabolomes (the fourth leaf tissue harvested at 0, 6, and 12 h after NaCl stress induction) of the studied plants. To this end, a rice cultivar (*Oryza sativa* cv. Kitaake) was transformed with the construction Act::RINO1. The RINO1 gene encodes an MIPS (EC 5.5.1.4, Figure 1). It has been demonstrated that the constitutive overex‐ pression of rice MIPS when compared to a wild type, results in greater tolerance to salt stress. Furthermore, it leads to a range of metabolic changes, with increased production of various metabolites (such as inositol, raffinose, ascorbate, amino acids). These handle the protection of plants from abiotic stresses. Additionally, activation of basal metabolisms such as glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle has been observed during

Ins metabolism and phosphorylated Ins-derivatives are also associated with response to biotic stresses in plants. This fact shows the plurality of actions of these compounds. Murphy et al. [19] report evidence in this direction. They obtained transgenic potato [*S. tuberosum* L. (cv. Desiree)] and *A. thaliana* lines, synthesizing low levels of phytic acid (1-D-myo-inositol-P6 or InsP6, Figure 1). The transgenic potato lines were obtained by: (1) constitutive expression of an antisense sequence of the myo-inositol 3-phosphate synthase gene. This enzyme (IPS, EC 5.5.1.4, Figure 1) catalyzes the first step in the InsP6 biosynthesis; (2) plants expressing the *Escherichia coli* polyphosphate kinase (PPK, EC 2.7.4.1; Figure 1). PPK inserts inorganic phosphate into chains of phosphate residues linked by phospho-anhydride bonds, and this decreases the phosphate available to InsP6 biosynthesis. Genetically modified potatoes presenting both (1) and of (2) showed increased susceptibility to avirulent pathogen potato virus Y and the virulent pathogen tobacco mosaic virus (TMV). In relation to *A. thalianai*, the authors obtained three loss-of-function mutants [two (atips1 and atips2) involving the gene for IPS (EC 5.5.4.1, Figure 1); and one (atipk1) involving the gene encoding inositol polyphos‐ phate kinase (IPK1; EC 2.7.1.158, Figure 1)]. IPK1 catalyzes the final step in InsP6 and without this enzyme, very little InsP6 is done. The disruption of InsP6 biosynthesis in *A. thaliana* resulted in an increased susceptibility to viruses (tobacco mosaic virus), bacteria [*Pseudomonas syringae* pv tomato (Pst) DC3000 and Pst DC3000 AvrB], and fungus (*Botrytis cinerea*). The

induction of the Ins metabolism in those plants overexpressing MIPS.

signal transduction pathways.

Recently, Meng et al. [20] found that *A. thaliana* ips1 loss-of-function mutant (atips1; IPS; EC 5.5.4.1, Figure 1) exhibits spontaneous cellular death and increased resistance to oomycete *Hyaloperonospora arabidopsis* pathogen. This result, together with the above, supports the premise that a particular InsP6 pool may regulate defense pathways, since atips1 mutants did not show increased susceptibility to the variety of testes pathogens (Tobacco mosaic virus; *Pseudomonas syringae* pv. tomato (Pst) DC3000 and Pst. DC3000 AvrB; and *Botrytis cinerea*). Additionally, it was observed that spraying the atips1 mutant plants with Ins could suppress the formation of spontaneous lesions, indicating that Ins production and not AtIPS1 protein is required to prevent the lesion formation. The authors also found evidence for the role of Ins (or Ins-derivatives) in the regulation of programmed cell death.

Reports presented in Table 1 also show the broad roles of Ins and its phosphorylated deriva‐ tives in plant cells. From the wide spectrum of analyzed genes, a range of effects on plants at different levels was observed. These effects have shown associations with hormone signaling pathways, such as ABA [12], influences in photosystems [21, 22], with reactive oxygen species (ROS; [23]), with relative water content, with osmotic adjustment [24], among others (Table 1).
