Myoinositol and its metabolites in abiotic stress tolerance in plants

Plants tend to develop tolerance mechanism under adverse environmental conditions (Ashraf and Harris, 2004). Over-production of different kinds of soluble substances is one of the most common stress responses in plants (Serraj and Sinclair, 2002; Ashraf et al., 2012). Usually, plants tide over the stress condition through different mechanisms such as cellular osmotic regulation, detoxification of reactive oxygen species, protection of membrane integrity, and stabilization of proteins (Bohnert and Jensen, 1996; Mechri et al., 2015; Per et al., 2017).

Many studies demonstrate the importance of metabolite accumulation attributed to osmotic adjustment leading to water retention and protection of biochemical pathways. Such accumulations are called osmoprotectans which protect cellular components from osmotic damages. Thus, when single enzyme based characters were scrutinized in transgenic plants, the accumulation of mannitol, proline, fructans, trehalose, glycine betaine, or ononitol (Tarczynski et al., 1993; Kishor et al., 1995; Holmström et al., 1996; Hayashi et al., 1997; Sheveleva et al., 1997) were found to provide higher salinity and cold or drought tolerance. In plants, cold, drought, and salt stresses, all stimulate the accumulation of compatible osmolytes and antioxidants (Hasegawa et al., 2000). Cyclitols, such as quercitol, pinitol or quebrachitol, are well stable and common among the candidate classes of compatible solutes (Merchant et al., 2006).

Inositol is a cyclohexanehexol, a cyclic carbohydrate with six hydroxyl groups one on each of the ring carbons. Myo -inositol (MI) is the oldest known inositol and was first isolated from muscle extracts by Scherer in 1850 (Posternak, 1965). Among the nine possible geometrical isomers of inositol. The myo -isomer is the most abundant form in nature (Fig. 1). It occupies a unique place in inositol metabolism because this is the only isomer synthesized de novo from D-glucose-6-phosphate; all other isomers are derived from myoinositol (Loewus, 1990; Loewus and Murthy, 2000). MI can be divided into two mirror image halves (Fig. 2) – a perpendicular plane passes through C-2 and C-5

and splits the molecule into non-superimposable mirror images. Therefore, any modification that will disturb the symmetry of the molecule will render the molecule chiral, thus MI is prochiral.

MI and its stereo-forms, play a plethora of functional role in plants, in signal transduction, cell wall formation, regulation of tissue growth, osmotic adjustment, membrane transport (Stevenson-Paulik et al., 2005; Zhai et al., 2016) etc. The loss of viability called inositolless death was found in case of MI requiring mutants of Saccharomyces cerevisiae (Culbertson and Henry, 1975) and Neurospora crassa (Strauss, 1958). Increased activity of 1L-myo-Inositol-1-phosphate synthase (MIPS) and higher rate of inositol production in plants have been directed to stress resistance. They have been known to act as osmoprotectants. In this review we discuss the role of myo-inositol and its metabolites that plays critical role in abiotic stress tolerance in plants on having been produced as a response to such stressful conditions.

Inositols and ROS accumulation

One of the most commonly known effect of stress in plants is the increase of reactive oxygen species (ROS) level. ROS damage the photosynthetic pigments, cellular lipids, proteins and nucleic acids by oxidation and also regulates apoptosis (Gadjev et al. 2008). A systematic up-regulation of ROS-scavenging genes was observed in sweet potato plants overexpressing IbMIPS1- under salt and drought stresses (Zhai et al., 2016). Methylated derivatives of inositol have been found to be effective in checking ROS level and thereby protecting photosynthetic machinery (Patra et al., 2010). Various reports show that inositol caused decline in the negative effects of osmotic stress thereby increasing the tolerance to drought stress of plants (Cevik et al. 2014; Sambe et al., 2015). SOD (superoxide dismutase) is in the first line of defence against the toxic effects of elevated levels of ROS (Gill and Tuteja, 2010). Total SOD activities in pepper plants were affected by myoinositol treatment and drought stress. Cu,Zn-SOD II isoform is enhanced by myo-inositol treatment in droughted plants (Aytunç, et. al., 2018). Mechri et al. (2015) reported that sugar alcohols may function as scavengers of activated oxygen species, by preventing peroxidation of lipids and resulting cell damage. Perhaps, MI is effective as H2O2 scavenger in response to stress conditions. Accumulation of ROS also impacts the photosynthetic machinery and the total chlorophyll content of a plant (Sarkar et al., 2014). Chlorophyll reduction under abiotic stress denotes osmotic stress, which is an outcome of pigment photo-oxidation and chlorophyll degradation (Rai et al., 2013, Farooq et. al, 2009). Transgenic 7PcI O1-plants (Porteresia coarctata) expressing inositol, retained the chlorophyll content thus maintaining normal photosynthetic potential even under high salt concentration, while untransformed line showed maximum chlorophyll depletion (Mukherjee et al., 2019).

Malondialdehyde (MDA) is a widely used marker of lipid injury caused by environmental stress. MDA content was observed to be higher in drought stressed plants than well-watered plants. Fan et al. (2017) reported an increase in MDA content in cucumber during drought stress. Low MI treatment significantly increased the level of MDA in both droughted and well watered plants. In transgenic sweet potato plants, overexpression of IbMIPS1 significantly decreased their MDA content compared to watered plants, indicating a marked improvement of their salt tolerance (Wang et al., 2016) under stress, as has been postulated for other polyols (Loewus and Loewus, 1983; Bohnert et al., 1995). Inositols have also been reported to mimic the structure of water and maintain an artificial sphere of hydration around macromolecules (Mechri et al., 2015). MI is known to accumulate in response to stresses in many plants through an induced expression of MIPS ( elson et al., 1998, Majee et al. 2004, Tan et al., 2013). Therefore, an attempt to understand and determine the role and mechanisms of MI in providing abiotic stress tolerance in higher plants is significant.

Osmolyte accumulation as stress response

Several studies indicated that osmo-protective compounds play a role in mutants or transgenic plants with different capabilities, accumulating these metabolites for stress tolerance (Szabados et al., 2011). MI may function as a compatible solute for protection against abiotic stresses and can be converted to other compatible solutes as well (Taji et al., 2006). Omolytes help in osmotic adjustment of the cells and also protects the cells and macromolecules by maintaining membrane integrity, preventing protein degradation and protecting against oxidative damage by scavenging free radicals and lowering Tm value of nucleic acids (Crowe et al., 1987; omura et. al., 1995). Osmolytes not only help in osmotic adjustment in the cellular milieu but also act as scavengers of ROS. D-ononitol and myo-inositol are the potential protectants of enzymes and membranes from damage by ROS (Sheveleva et. al., 1997). Isomerization and methylation of MI leads to the formation of O-methyl inositol, which also directly participates in stress-related responses of plants (Loewus and Murthy, 2000). The plant species which are constantly exposed to saline conditions, accumulate cyclic sugar alcohols, pinitol and ononitol (Paul and Cockburn, 1989). Thus, Mesenbryanthemum crystallinum has been seen to accumulate these compounds when subjected to such stress (Bartels and elson, 1994). The upregulation of inositol biosynthesis by subjecting plants to salinity stress may be exploited for the enhanced production of myo-inositol from glucose-6phosphate (RayChaudhuri and Majumder, 1996).

Physiological, biochemical, metabolic and molecular changes occur in plants under various environmental stresses. Increased activity of the rate limiting enzyme, myo-Inositol synthase (MIPS) in addition to higher rate of inositol production is one such mechanism in plants to resist stress. The genes encoding MIPS have been isolated from several plant species, such as Arabidopsis (Johnson, 1994), rice (Yoshida et al., 1999), maize (Larson and Raboy, 1999), soya bean (Hegeman et al., 2001), smooth cordgrass (Joshi et al., 2013) and Medicago falcata (Tan et al., 2013). The upregulation of these genes confer stress tolerant character to plants. In higher plants, the inositol that accumulates in response to salt stress may function as an osmolyte, in addition to its critical roles concerning the cellular machinery (Bohnert et. al., 1995). The extent to which osmolyte levels rise is dependent on changes in the external osmotic potential (Hasegawa et at., 2000; Chen and Murata, 2002). Compounds such as gums, cell wall- located carbohydrates, glycoproteins and mucilages, which are involved in protective functions during stress, are also produced from inositol and inositol-1-phosphate (Shen et. al., 1997, Crowe et. al., 1992).

Myo-inositol in salinity stress tolerance

Polyols and sugar alcohols such as pinitol, mannitol, ononitol and inositol is known to play vital role as osmoprotectants against salt stress in various plants (Ghosh et. al., 2006). Tomato ( Lycopersicon esculentum Mill. cv. ew Yorker) plant subjected to aCl stress showed an increased MI content which remained elevated throughout (Sacher and Staples, 1985). Plants alternated daily between salt and control solutions accumulated less MI and exhibited less growth than the continuously salt stressed plants. The facultative halophyte, Mesenbryanthemum crystallinum has been seen to accumulate pinitol and ononitol when the plant is stressed (Bartels and elson, 1994). The content of free MI in Ulva lactuca was found to increase proportionately with the increase of surface salinity of the Chilika Lagoon, Odisha, India (Basu, 2019). It was proposed that myo -inositol could serve not only as a substitute for the production of compatible solutes, but also as a leaf-to-root signal that promotes sodium uptake ( elson et al ., 1999). When subjected to salinity stress by aCl treatment, the salt tolerant varieties of rice ( Oryza sativa ), exhibited enhanced activity in the chloroplast form of MIPS. The enhanced MIPS activity in the highly salt tolerant varieties of rice due to salinity stress was found to be comparable to that of Porteresia coarctata , a halophytic wild rice species. This ultimately suggests a role of inositol pathway in osmoregulation (RayChaudhury and Majumder, 1996).

MIPS gene isolated from the wild halophytic rice Porteresia coarctata, and introgressed into IR64 indica rice showed its role in conferring salt-tolerance. The PcI O1 transformed transgenic rice lines exhibited significantly higher salt tolerance with negligible compromises in other physiological parameters compared to the untransformed system grown without stress. Further introgression of PcI O1 in Brassica juncea also imparted a substantial tolerance to salt and oxidative stress and such plants could be a potential salt tolerant genetically modified crop.

Also, an attempt was made to generate salt-tolerant indica rice plants through transgenic functional cointrogression of both PcI O1 and PcIMT1 (Inositol-o-methyl transferase) which showed comparatively higher accumulation of inositol in stress conditions (Mukherjee et al., 2019). Overexpression of MIPS in B. juncea, Arabidopsis, tobacco and rice enhanced their tolerance to salt, dehydration and chilling respectively due to the increased production of inositol (Kaur et al., 2013; Majee et al., 2004; Tan et al., 2013).

MI has been implicated in salt tolerance of many plants as a facilitator of uptake and long-distance transport of sodium ( elson et al., 1999). Increased tolerance to salinity (150 mM aCl) were seen in transgenic Arabidopsis plants expressing SaI O1 ( Spartina alterniflora myo-inositol 1-phosphate synthase), at both germination level and during plant growth and development. ormal growth was observed after a week exposure to salt stress in transgenic plants whereas the wild type (WT) plants struggled to survive and eventually perished. Results indicated that under salinity stress, Arabidopsis transgenic plants were less sensitive to photoinhibition as compared to WT plants. Increased MI due to expression of SaI O1, served as the substrate for accumulating metabolic end products and that could facilitate sodium sequestration and protect photosynthesis ( elson et al., 1999, Bohnert et al., 1995). There was also indication of a substantial protection of photosystems especially PSII in SaI O1- Arabidopsis plants and the possible role of MI in protecting the chloroplast.

Does myo-inositol help in drought tolerance?

In plants, the limitations in available water imposes both hyperionic and hyperosmotic stress that can upset homeostasis both at cellular level and whole-plant level. Altered osmotic potential results in molecular damage that ultimately hinders growth and can even lead to death (Hasegawa et al., 2000). Water deficit or osmotic effects are probably the major physiological mechanisms for growth reduction as both stresses lower the soil water potential (Hu and Schmidhalter, 2005).

Plants respond to drought stress by synthesis of different metabolites including polyols in ripe olive fruit (Martinelli, 2013), grape berry (Conde et al. , 2014) etc. Six different polyols (mannitol, sorbitol, galactitol, myoinositol, glycerol and dulcitol) were significantly accumulated in the pulp of grape berries in response to water deficit. MI was the most abundant of the quantified polyols in mature leaves and tissues which helps the plant with water deficit, either directly as an osmolyte or indirectly as a precursor of galactinol and raffinose family oligosaccharides (Conde et al., 2014). MI is closely related to the accumulation of RFOs conferring further stress tolerance (Elsayed et al., 2014). There is a close relationship between the metabolism of MI and RFO and the yield performance of maize under drought stress. Galactinol synthase (Go1S EC 2.4.1.123) is the key enzyme that catalyzes the production of galactinol from MI which is the key regulatory factor in RFO biosynthesis (Taji et al., 2002; Kerner et al., 2004; Sengupta et al., 2015).

Expression of a stress responsive gene (SaI O1) for osmotic stress tolerance mentioned earlier was induced through accumulation of MI (Joshi et al., 2013). The findings of Wei et al. (2010) suggested that upregulation levels of the rcMIPS gene and their increased rcMIPS activities might be involved in tolerating drought stress in Ricinus communis. In the leaves, the activities increased by 88.5% under 30% PEG stress conditions for 72 h compared to the control (Wei et. al., 2010). There are seven Go1S-related genes in the Arabidopsis thaliana and named as AtGo1S 1, 2, 3, 4, 5 6 and 7 respectively. Among these, AtGo1S1 is the drought responsive gene, which mainly functions in drought stress tolerance (Taji et al., 2002). The model plant Ajuga reptans expresses two distinct Go1S, ArGo1S1 and ArGo1S2 which regulates raffinose family oligosaccharide (RFO) metabolism (Sprenger and Keller, 2000). RFOs have long been suggested to act as anti-stress agent in both generative and vegetative tissues (Taji et al., 2002). XvGo1S gene encoding galactinol synthase was also identified in the leaves of Xerophyta viscosa. This gene shows negative correlation between RFO accumulation and MI depletion which was reversed after rehydration. This suggests that myo- inositol is channelled into RFO synthesis during water deficit and channelled back to metabolic pathway during rehydration to repair desiccation-induced damages (Peter et al., 2007). In addition to Go1S, MIPS also control the levels of galactinol and raffinose because it controls the production of MI, the galactinol precursor. Therefore, both Go1S and MIPS are proved to play important roles in drought stress tolerance (Taji et al., 2002; Evers et al., 2010). Different genes associated with MIPS and stress tolerance in plants have been enumerated in table 1.

Myo-inositol in cold stress

Low and high temperature stress is very crucial for determining the health of plants. Plants exhibit a maximum rate of growth and development at an optimum temperature or over a diurnal range of temperatures (Fitter and Hay, 1981). Stress tolerance can be induced by exposure to reduced temperature and is known as chilling tolerance or cold acclimation. Chilling tolerance is the ability of a plant to tolerate low temperatures (0–15 ºC) without injury or damage (Somerville, 1995), while cold acclimation is an enhanced tolerance to the physical acclimation. Chilling tolerance involve an array of biochemical, molecular and metabolic processes (Thomashow, 1999; Larkindale et al., 2005; Kotak et al., 2007; Zhu et al., 2007).

Plants experience cold or chilling stress at temperatures from 0–15 °C. Under such conditions, plants try to uphold homeostasis to acquire freezing tolerance and this involves extensive reprograming of gene expression and metabolism (Thomashow, 1999; Cook et al., 2004). Accumulation of sugars and altered gene expression are two major processes that has been shown to confer freezing tolerance in plants. Among cold-responsive genes, cD A encoding MIPS had the highest abundance in the library, entailing that MIPS might play an important role in the cold tolerance of M. falcata (Tan et al., 2013). Expression analysis of MfMIPS1 ( Medicago falcata MIPS1) in tobacco exhibited an enhanced tolerance to chilling stress by increased levels of inositol, galactinol and raffinose sugars (Tan et al., 2013).

MI is known to induce galactinol synthase gene

(GolS) expression and participates in cold-induced MfGolS1 (galactinol synthase gene from Medicago falcata ) expression (Zhuo et al., 2013). A full length cD A encoding a MI transporter-like protein, MfI T-like, was cloned from Medicago sativa subsp. falcata a species with better cold tolerance than M. sativa subsp. sativa. Higher levels of MI was observed in leaves of transgenic tobacco plants overexpressing MfI T-like than the wild-type suggesting that transgenic plants had higher MI transport activity than the wild-type. Transgenic plants had a better endurance to freezing temperature, implied to have a higher maximal photochemical efficiency of photosystem II (Fv/Fm) after chilling treatment.

Under freezing conditions, the presence of cyclitol function as cryoprotective solutes. Accumulation of cryoprotectans prevents freeze induced shrinkage of by balancing the concentration of cryotoxic substance during ice formation. Cyclitols like pinitol, quebrachitol, quercitol, O-methyl-muco-inositol have all been found to be accumulated in low temperature (Diamantoglou, 1974; Ericsson, 1979; Popp et al., 1997). In mistletoe ( Viscum album ) more than 25% of its dry matter is occupied by the cyclitols during winter (Richter, 1989). Similarly enhanced storage of cyclitols in the living bark tissue and buds has been found in a number of tree species during the onset of cold season (Popp and Smirnoff, 1995; Popp et al., 1997). The transcription of the enzyme, myo -inositol-o-methyl-transferase, a key enzyme for ononitol and pinitol biosynthesis has been induced in the Mediterranean species M. crystallium when the plant was exposed to 4^oC for 78hr. Similarly, accumulation of pinitol in chickpea in the thylakoid membrane functions as cryoprotective solutes (Orthen, 2000). Raffinose also plays a crucial role as compatible solutes in Arabidopsis in freezing tolerance (Hannah, et al., 2006).

Myo-inositol in high temperature stress

In plants, high temperature stress (HS) causes photosynthetic acclimation and alters physiological processes directly and changes the pattern ofdevelopment indirectly (Wahid et al., 2007).

It consequently influences the reproductive growth by increasing flower abortion and reducing seed size (Talwar et al., 1999). In response to HS, plants are endowed with different mechanisms and regulatory networks, viz. regulating vital genes, managing numerous physiological and biochemical adaptations and so forth. One such mechanism to counter the effect of HS is by increasing the level of inositol in plants.

Increasing the amount of cytoplasmic free Ca²⁺ under temperature stress could reduce the effect of stress and decrease the lipid peroxidation (Wu et al. 1997). Overexpression of Cicer arietinum MIPS (CaMIPS2) is known to enhance the tolerance of Saccharomyces pombe cells under salinity and high temperature (Kaur et al., 2008). Also Ca²⁺ levels were seen to contribute to the cell water use efficiency by exogenous MI treatments. TaMIPS2, identified from a heat subtractive cD A library from wheat for its tolerance to HS was found to be expressed during various developing seed stages upon infliction with HS. The transcript levels increased in unfertilized ovaries and significant amount of the same was found during the recovery period, indicative of the pivotal role of MIPS in heat stress recovery and flower development. Any abiotic stress condition, whether drought, cold, heat or salinity essentially deprives the plant from water. Therefore, osmotic stress is rather universal in all of these forms of stress. The plant cell utilizes different mechanisms to tide over the various stress conditions mentioned. They act by activating various genes, and synthesizing a plethora of chemicals. Some of these genes and biochemical molecules derived from MI is presented in fig. 3.

Khurana et al., 2017, studied the overexpression of TaMIPS2 in Arabidopsis under different abiotic stress conditions and revealed that TaMIPS2 transgenic plants have reduced sensitivity to HS, divulging a role of MI during this stress. Increased levels of inositol upon TaMIPS2 overexpression in Arabidopsis transgenics and increasd MI levels were correlated with enhanced tolerance towards HS and other abiotic stresses. TaMIPS2 transgenics when quantitatively analyzed showed better survival during heat as well as cold and salt stress. However, in salt stress, the length of transgenics were shorter compared to wild type plants

Increased membrane stability index, FV/FM ratio along with total chlorophyll content in the transgenics were found when compared to the wild type under stress conditions. On evaluation, TaMIPS2 overexpressing Arabidopsis transgenic during stress were found to have increased level of oligosaccharides. Significant increase in MI level was observed upon heat and ABA treatment that associated with decrease in stress sensitivity of transgenics. This might also be responsible for increased stachyose levels resulting in enhanced stress tolerance.

Table 1: Different genes coding for myo -inositol and its associated derivatives potentially responsible for abiotic stress tolerance in plants.

SL. NO.	Type of Stress	Chemical/Molecular Response	Gene Responsible	Reference
1.	Osmotic	Myo-inositol o- methyl transferase	IMT 1	Rammesmayer et al., 1995; Vernon & Bohnert, 1992
2.	Salinity	D-pinitol	Imt 1, Inps 1 transcript	Ishitani et al. 1996
3.	Cold	RFO	AtGolS3	Taji et al., 2002
4.	Drought & salinity	RFO	AtGolS1 andAtGolS2	Taji et al., 2002
5.	Drought, salinity, cold	Raffinose	DREB1A/CBF3 and DREB1B/CBF1	Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Liu et al., 1998
6.	High temperature	Raffinose	AtGolS1	Panikulangara et al., 2004)
7.	Salinity	myo-inositol-O-methyl transferase	Imt	elson et al. 1998
8.	Salinity, high temperature	inositol	MIP2	Kaur et al., 2008; Khurana et al. 2012
9.	Drought	up-regulation of many stress-responsive genes	AKI 11	Umezawa et al., 2004
10.	Drought and heat	expression of downstream stressinducible genes	DREB2A	Mizoi et al., 2018
11	Heat Stress	Heat shock factor (HSF)-	Os02g0496100	Kikuchi et al., 2003
12	Cold stress	Expression of the cold-Regulated genes	WCOR410	Danyluk et al., 1994
13	Cold stress	Expression of the cold-Regulated genes	WCS120	Ganeshan et al., 2008
14	Salinity stress	PM ADPH oxidase-dependent H(2)O(2) generation	LOC543151	Yang et al., 2007
15	Salt stress	Regulates the proline synthesis	P5CS	Karthikeyan et al., 2011
16	Salt stress	Proline accumulation	P5CSF129A	Kumar et al., 2010
17	Salt stress	Increases the level of spermidine and spermine	SAMDC	Roy and Wu, 2002
18	Salt stress	Increased glycine betaine content	betA	He et al., 2010
19	Salt stress	Biosynthesis of mannitol	mtlD	Abebe et al., 2003
20	Salt stress	Increases the ion flux, germination rates, chlorophyll content and antioxidant enzyme activities	SmCP	Zheng et al., 2018
21	Salt stress	Increases the level of proline biosynthesis enzyme (P5CS)	DH -5	Saibi et al., 2015
22	Salt stress and oxidative stress	Expression of stress-related genes were up-regulated, and antioxidant enzyme activity were increased	PnF3H	Li et al., 2017
23	Salt stress	Increased proline synthesis	ADC	Espasandin et al., 2018

24	Salt stress	Increased Glycine betaine content	AhCMO	Zhang et al., 2009
25	Salt stress and drought	Increases proline and chlorophyll content	BADH	Rezaei et al., 2020
26	salt stress	Phosphatidylinositol 5-phosphate	PtdIns5P	Pical et al., 1999
27	Salt stress	Myo -inositol oxygenase	OsMIOX	Duan et al., 2012

Figure 1: Structure of nine stereo isomers of inositol (*source– Murthy, 2006)

Figure 2: Mirror image of inositol (*source – Murthy, 2006)

Figure 3: Some genes that regulates the expression of MI derivatives responsible for different types of abiotic stress tolerance in plants

ACKNOWLEDGMENTS

We acknowledge the Department of Botany, Sikkim University, Gangtok 737102, India for providing essential infrastructure and ambience for writing the paper.

CONFLICTS OF INTEREST

The authors declare that they have no potential conflicts of interest.