Sustainability in agriculture depends on growing suitable crops for a particular climate in the defined areas. Prolonged exposure to high temperatures drastically affects crop productivity. Elevated temperatures also result in osmotic stress from the water evaporation within the soil causing excessive salt accumulation. Heat, drought, and salt are the major abiotic stresses affecting crop yield. These stresses in combination are becoming quite common in heat and drought-hit areas. Among cereals, wheat is domesticated first and considered a major staple food crop globally (Tack et al., 2015; Abhinandan et al., 2018). Heat negatively affects the wheat grain yield. It is estimated that every 1°C rise in temperature results in 6% yield losses in wheat crops; however, it depends on the specific growth stage of the crop, time, duration, and intensity. An increase in temperature above the optimum value before and during anthesis results in embryo abortion in developing seeds, reducing the grain number/ear without affecting the grain weight, whereas, after anthesis, the onset of high temperature does not affect the number of grains per ear but reduce the grain size and weight by hampering grain filling ultimately affecting the crop yield (Foulkes et al., 2002; Weldearegay et al., 2012; Schmidt et al., 2020).

Plants are sessile eukaryotes and are very sensitive to even slight changes in their environment. There are some receptors present on the plant cell membrane that perceive stress (abiotic/biotic) signals and transduce this information downstream for the activation of certain stress-responsive genes. The ultimate product of coordinated action of these genes results in signal transcription/proteins synthesis, protein modification like ubiquitination, glycosylation, methylation, adaptors attachment, and subsequently scaffolding of the plants to adapt/survive under harsh environmental conditions (Trewavas and Malho, 1997; McCarty and Chory, 2000; Gilroy and Trewavas, 2001; Mahajan and Tuteja, 2005; Tuteja, 2007; Tuteja and Sopory, 2008).

Under extreme temperatures, plants tend to maintain their membrane integrity and fluidity, acting as a permeable barrier. According to a rough estimate, the membrane surface of a plant cell is recycled every 90–120 min (Munnik et al., 2021). These lipids have amphipathic properties and can be differentiated as sphingolipids, glycerolipids, and sterols based on their unique chemical structure and biophysical properties (Enrique Gomez et al., 2017). Among glycerolipids, phospholipids are predominantly present in the mitochondrial envelope and plasma membrane (PM), which play a vital role in the development of the plant, regulating their responses against particular environmental stimuli (Dubots et al., 2012; Niu and Xiang, 2018; Wang X. et al., 2020). Plant phospholipases are involved in the hydrolysis of phospholipids and can be divided into four categories, that is, PLA 1 (phospholipase A1), PLA 2 (phospholipase A2), PLC (phospholipase C), and PLD (phospholipase D). Within each category, there are subfamilies with different structures, substrates, and binding sites (Wang X. et al., 2020). Three types of PLCs are reported based on their cellular function and substrates specificity: (1) PI-PLC (Phosphatidylinositol-specific PLCs) hydrolyzes phosphoinositide (PPI); (2) PC-PLC/NPC (phosphatidylcholine-specific PLC/Non-specific phospholipase C) hydrolyzes the commonly present phospholipids like PC and PE; and (3) GPI-PLC (Glycosyl phosphatidylinositol PLC) hydrolyzes the proteins attached to the glycosylphosphatidylinositol (GPI) (Hong et al., 2016).

Extracellular signals activate the PLCs responsible for the production of inositol 1,4,5 trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 travels to the cytoplasm to bind and activate the ligand-gated calcium channel also known as the InsP3 receptor to release Ca⁺² from intracellular channels, whereas, DAG deals with the protein kinase C (PKC) family which has a C1 conserved domain. Massive intracellular processes due to increase or decrease in calcium and phosphorylation levels result in the activation and deactivation of various target proteins to respond against extracellular changes (Shiva et al., 2020; Hayes et al., 2021).

The signaling pathway of plant PI-PLC is somewhat different from mammals, for example, in plants, inositol 1, 4, and 5 trisphosphates (InsP3) could phosphorylate further to inositol hexakisphosphate (InsP6), which is responsible for the release of calcium ions from intracellular calcium reserves and similarly, phosphatidic acid (PA) which is a product of diacylglycerol (DAG) might act as a second messenger in this pathway (Munnik, 2014). PIP₂ is presumably a substrate of PLC, hardly found in the plasma membrane of flowering plants (Simon et al., 2014; Zhang et al., 2018c). PLC hydrolyze PIP (Phosphatidylinositol 4 monophosphate), that is, also known as the precursor of PLC and can be found in abundance in the plasma membrane, but to date, the typical precursor of PLC in plants is unknown in in vivo analysis (Munnik, 2014). Likewise, DGPP (Diacylglycerol pyrophosphate) can function as an attenuator of PA signaling and as a generator of new signals, but it needs to be investigated further (van Schooten et al., 2006).

Plants tend to upregulate many PLC genes upon the onset of various biotic and abiotic stresses. In Arabidopsis thaliana, 9 PI-PLCs and 6 NPCs genes (Munnik, 2014), 4 PI-PLC and 5 NPCs in Oryza sativa (Rice) (Singh et al., 2013), 12 PI-PLCs and 9 NPCs genes in Gossypium spp. (Cotton) (Zhang et al., 2018a), 5 PI-PLC and 4 NPCs genes in Zea mays L. (Maize), while 12 PLC genes in Glycine max (Soybean) (Wang F. et al., 2015) are reported. An increase in PIP₂ and PA had been observed in response to heat, salt, cold, drought, and ABA stresses (Alcázar-Román and Wente, 2008; Darwish et al., 2009; Mishkind et al., 2009; Arisz et al., 2013; Balogh et al., 2013; Simon et al., 2014; Zhang et al., 2018c). Earlier it is reported that PLC is involved in plant growth and development, for example, PLC1 is known to contribute to pollen tube growth in tobacco and petunia (Dowd et al., 2006; Helling et al., 2006), over-expression of the PLC2 gene can increase drought tolerance and regulate phytochrome level in Brassica napus (Das et al., 2005; Nokhrina et al., 2014), PLC3 and PLC9 contributing in generating thermotolerance in Arabidopsis thaliana (Zheng et al., 2012; Gao et al., 2014), upregulation of AtPLC5 in response to drought stress could lead to subsequent novel phenotype including stunted root hair growth, reduced lateral root development, stomatal closure, and inhibition/reduction of seed germination (Zhang et al., 2018b,c). These findings are inconsistent with previous studies as reported on maize, tomato, and potato (Apone et al., 2003; Apostolakos et al., 2008; Wang et al., 2008; Vossen et al., 2010).

PI-PLC was initially reported in wheat in root plasma membrane vesicles (Melin et al., 1992). Based on their subfamily, genomic homology, and chromosomal position, a total of 26 TaPLC genes including 7 NPC genes have been reported in Triticum aestivum (wheat) which are located unevenly on 14 chromosomes (Wang X. et al., 2020), but to date, TaPLC1 (Zhang et al., 2014; Wang X. et al., 2021) and TaPI-PLC1-2B have been cloned and investigated for salt, drought, heat, and cold stress (Khalil et al., 2011; Wang X. et al., 2020). However, TaPLC5 has yet to be reported in the already identified wheat PLCs. There is growing evidence that phosphoinositide signaling is a major element of stress responses. It proposes that changes in the lipid signal levels are one of the early consequences of abiotic stresses. Therefore, this study focuses on investigating signaling phospholipids levels in response to high temperature and osmotic stresses. We observed that AtPLC5 over-expression causes a dramatic increase in PIP₂ and PA levels at tillering and anthesis stages. These are the crucial stages for wheat grain development at various duration in varying intensity levels of heat and osmotic stresses.

Abiotic stresses can elicit a series of plant responses. Membrane plays an important role in vesicle transport and cell signaling not only through host-specific proteins but also provides a substrate for the production of lipid (as a second messenger). In addition to the role of lipids as components of membrane structure, they also work as a signal transducer, component of coordinated regulatory activator, and stimulate the expression of specialized proteins and trigger cellular responses to environmental cues (Hou et al., 2016; Kosová et al., 2018; Munnik et al., 2021). Phospholipases on the plasma membrane are the first receptors to receive environmental signals and respond accordingly. PLCs due to their regulatory roles in stress management have been extensively investigated in different plant species. It has been established that stress causes a synergistic increase in PIP₂ levels and free calcium, which enhances IP₃ synthesis and further releases cytosolic calcium through PI-PLC activity (Hunt et al., 2004; Gao et al., 2014; Zhang et al., 2014). Heat shock induces a rapid increase of Ca⁺² in the cytoplasm, probably from intracellular reserves and extracellular sources. It is reported that Ca⁺²/calmodulin pathway is involved in thermotolerance. It is logical to claim that Ca⁺² channels could be used as a thermosensor (Gao et al., 2012; Hayes et al., 2021). However, it is still a challenging task to identify the primary heat-activated Ca⁺² channel.

Previously, PIP₂ and PA abundance had been observed in Arabidopsis within 2 min of onset of heat (40°C) stress, and it was mediated by PLD and PIPK (Mishkind et al., 2009). In the current study, we investigated the stimulation of heat-induced PIP₂ and PA accumulation in Triticum aestivum L. and observed that their induction proceeded in a time-dependent manner. The rapid rise in PIP₂ level was evident with the onset of heat (40°C) that reached 2.2-folds in just 15 min and continue to increase with the increase in the duration of heat stress. However, after 60 min, the PIP₂ level started to decline, which might indicate the stress-induced damage caused to the plasma membrane. In the current study, it was observed that the PA accumulation started just after 7.5 min of the onset of heat stress, and kept on increasing continuously with the increase in the duration of heat stress. The quick abundance of PIP₂ and PA indicates the synthesis of these signaling lipids associated with thermosensing. Although it is still unclear how the elevated temperature activates these lipid-modifying enzymes, this increase in PIP₂ and PA is either caused by PIP5K, PLC, or PLD activity, which is yet to be determined. It is reported previously that PA induction is closely associated with the activation of PLD under heat stress (Shiva et al., 2020; Hayes et al., 2021); however, it has been observed that in wheat at 40°C, PBut level seems to decrease while the total PBut content remains in lower limit. In contrast, PA level seems to increase in a time-dependent manner. It is still unknown which other factors are involved in the generation of PA through PLD or PLC. Similarly, it is yet to be explored what circumstances help in the activation/inhibition of PLD or PLC.

Plant leaves serve as a sensor for biotic and abiotic stresses. A slight change in the surrounding temperature is usually sensed by the plant through their leaves. The present study investigated PIP₂ and PA responses in younger to older leaves against heat stress. We also observed PIP (Phosphatidylinositol monophosphate) response. Upon onset of heat stress (40°C), the young leaves depicted minor elevation in PIP₂, PIP, and PA and contributed accordingly to stress responses as compared to mature leaves which showed a gradual increase up to 3.4-folds in PIP₂ and PA accumulation, while illustrated 1-fold decrease in PIP level. Therefore, it could be suggested that although the younger leaves have actively dividing cells, they are quite sensitive to heat stress Zhang et al. (2014) reported a 16-fold increase in TaPLC1 expression level in older leaves upon salt and drought stress. This could be implied that an increase in expression in response to environmental changes might be considered an adaptive mechanism to manage abiotic stresses.

In the current study, PIP₂ response was observed to be similar in wild-type (Faisalabad-2008) and AtPLC5 over-expressing lines of wheat under normal conditions (20°C). However, heat stress (40°C) at the anthesis stage caused a stronger and significant rise in PIP₂ level in AtPLC5 over-expression lines (Figure 3B) as compared to wild-type that ultimately helped the plant to adapt/tolerate fluctuations in temperature and grain formation sustaining the crop yield. We also compared the strength of two constitutive promoters (CaMV35S and UBQ10). UBQ10 promoter indicated relatively higher expression of AtPLC5 in OE3 and OE4 lines with a consequent significant increase of ∼4.5-folds in PIP₂ accumulation as compared to AtPLC5 expression driven under CaMV35S promoter in OE1 and OE2 transgenic wheat. Zhang et al. (2018c) reported a 12-fold increase in PIP₂ level at the onset of osmotic stress in PLC5OE lines containing UBQ10 promoter in 6-day-old seedlings of Arabidopsis thaliana (Zhang et al., 2018c), which is in agreement with our findings and increase in PLC activity.

PI-PLC as a stress mediator had been reported along with their isoforms in many plants including maize (Apostolakos et al., 2008), rice (Darwish et al., 2009; Singh et al., 2013), tobacco (Helling et al., 2006), tomato (Vossen et al., 2010), cotton (Zhang et al., 2018a), soybean (Wang F. et al., 2015), brassica (Das et al., 2005), Arabidopsis (Gao et al., 2014), and wheat (Wang X. et al., 2021). Recent findings illustrated the over-expression of TaPLC1 aided in improved salt, drought, heat, and cold stress tolerance in wheat (Khalil et al., 2011; Wang Y. et al., 2020; Wang X. et al., 2021). PIP₂, as a PLC substrate is hardly detected in plants’ plasma membrane under normal conditions, while its level significantly increased under osmotic stress, for example, cold, salinity, or heat stress (Darwish et al., 2009; Mishkind et al., 2009; Arisz et al., 2013; Munnik, 2014; Zhang et al., 2014). In the present study, it was observed that the lines that showed more PIP₂ accumulation also revealed more transcript levels through real-time quantitative PCR. In addition, we also observed that these lines retained their stay green character relatively for a longer period of time when exposed continuously for 14 days to heat stress.

The structural lipids like PC (Phosphatidylcholine), PG (Phosphatidylglycerol), and PA (Phosphatidic acid) at the anthesis stage of wheat were reported to drop under high temperatures (Narayanan et al., 2016; Djanaguiraman et al., 2020). Likewise, we also observed a slight decrease in PA in our AtPLC5 over-expression lines of wheat during anthesis at 40°C. However, ∼2.2-fold increase in PA accumulation was observed in the wild when subjected to heat stress (40°C for 30 min). This increase might reflect the activity of PLD as previously reported by Hayes et al. (2021).

Upon rising environmental temperature, plants with sufficient water resources transpire more rapidly to keep their leaves cool, while on water scarcity in hot conditions, leaves close their stomata to prevent water loss through evaporation and to maintain their cells membrane integrity. Lee et al. (2007) reported PIP₂ to be an important precursor for stomatal opening, as detected previously in the closed stomata phenotype of the PLC5OE line in Arabidopsis. In this study, sorbitol was used to mimic drought/osmotic stress in wheat and to observe its effect on the PIP₂ level. Interestingly, a significant increase in PIP₂ level was observed upon osmotic stress in AtPLC5 overexpression lines of wheat, this might result in the enhanced hydrolytic activity of PLC5 which might lead to an increase in PIP₂ hydrolysis resulting in a subsequent increase in IP₃ that might further be metabolized into IP₆ which facilitate the stomatal closure by activating the release of Ca⁺² from intracellular channels (Zhang et al., 2018b,c). In addition, in the current study, we observed that wheat transgenic lines containing CaMV35S promoter induced significantly higher PIP₂ that matched with findings previously reported by Zhai et al. (2013). It is explained that ZmPI-PLC1 expressed under CaMV35S promoter induced drought tolerance in transgenic tobacco (Ruelland et al., 2015).

However, a detailed phosphoimager-based densitometry study demonstrated a meager decrease in PI and PE levels and a slight increase in PC and PG levels, when exposed to osmotic stresses. An increase in the level of cardiolipin (CL) was observed in OE lines of transgenic wheat. In plants, PG (phosphatidylglycerol) was found to be mainly present in the thylakoid membrane of chloroplast and supposed to be involved in the photosynthetic electron transport chain (Hagio et al., 2002; Kobayashi et al., 2017). Previous reports have suggested the prerequisite presence of PG for chloroplast biogenesis, as its deficiency yielded a pale-yellow green phenotype, indicating the failure of establishing thylakoid membrane networks inside leaf chloroplast (Haselier et al., 2010; Kobayashi et al., 2015). Interestingly, an increase in PG level of overexpressor lines of wheat was observed, which means they remained photosynthetically active when exposed to abiotic stress and could accumulate more synthates, more synthates mean more nutrients available to be assimilated during grain filling leading to enhanced crop productivity, which might ultimately yield higher grain and biomass.

Phosphatidylinositol 4,5-bisphosphate is claimed to be a PLC substrate in animals, its concentration is relatively hard to detect in the plasma membrane of plants where PLC activity mostly resides (Van Leeuwen et al., 2007; Munnik, 2014). In contrast to PIP₂, PI4P is 20–30 times more abundant in plasma membrane under normal conditions. Under stress conditions such as abscisic acid (ABA), salinity, heat, or hyperosmotic stress, the level of PIP₂ increased (Darwish et al., 2009; Mishkind et al., 2009; Zhang et al., 2018b), while the level of PI4P has been reported to drop in response to these stresses (Arisz et al., 2013). But does it go down due to conversion into PIP₂ or PIP is an assumed substrate of PLC in the plant? Also, it remained debatable, whether this reflected the hydrolysis by phosphatase or a PLC or is a result of PIP5K activation. Further research is needed to decipher the exact role of PLC in wheat and the downstream process of PA, PPIs, and IPPs production and accumulation.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

NA, MA, and NS conceived and designed the research. NA conducted the research experiments. NA and KI evaluated the data. MA, NS, and MT provided the research material. NA and MA wrote the manuscript. SM, MT, and NS critically reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.