INTRODUCTION:

A critical contributor to losses in crop yield across the globe is abiotic stress. In particular, drought is the most common form of abiotic stress that results in agricultural yield losses, since it occurs in almost all climate zones¹. Sustainable agriculture around the world can be negatively impacted even by short-term water shortage, resulting in significant losses in annual crop yield². Given the context of climate change and global warming, it is incredibly likely that the duration and frequency of instances of drought will increase, thereby making drought stress an extremely relevant field of research³.

Drought is defined as a condition of limited productivity of plants in an agricultural or natural setting, caused by a period of below-average precipitation levels^4,5. This condition may either be persistent, in regions characterized by generally low water availability or intermittent, as a result of unpredictable water supply⁶.

Abiotic stress may also be induced by a multitude of other factors such as high salinity and abnormal ambient temperature⁴. Such conditions adversely affect all important physiological processes, such as respiration and photosynthesis⁷.

Regardless of the cause for this drought condition or decrease in water potential, certain effects are commonly seen in plants as a result. Some of these effects include the inhibited assimilation of carbon dioxide, closure of stomata⁸ and the overproduction of reactive oxygen species (ROS) such as superoxide anion radicals, singlet oxygen, nitric oxide, hydroxyl radicals, and hydrogen peroxide, amongst others⁹. The increased production of reactive oxygen species also results in a consequent increase in the oxidation of lipids, nucleic acids and proteins, thereby causing significant changes in essential plant biochemical properties such as enzyme activity, cellular integrity, transport efficiency, and gene expression¹⁰. In addition to this, the deficiency of water also negatively affects cell division, leaf surface expansion, stem growth, and root proliferation¹¹. Dehydration of plant tissue to a larger degree alters intermolecular and intramolecular interactions between cells, and eventually results in diminished plant growth and ultimately, death^10,12.

For the development of new methods to increase plant drought resistance, it is necessary to develop drought stress models. This will enable researchers to understand the changes in the plant metabolome and proteome. For this purpose, media infused with PEG-6000 is used (Fig 1), as a means to simulate the drought stress conditions seen in naturally arid environments¹³.

Drought Stress Conditions:

Quantitatively, environmental water deficit can be defined as a decrease in water potential (Ψ_W) of the plant’s growth medium¹⁴. The Van’t Hoff equation indicates that a decrease in free energy of substrate water as a result of drought conditions causes water uptake by plant tissues to be thermodynamically unfavourable. Water potential values between 0 and -0.3 MPa are generally representative of well-watered plants, whereas values between -0.4 MPa and -1.4 MPa represent moderate water stress and values below -1.4 MPa correspond to severe water stress and permanent turgor loss¹⁵.

These values differ based on the plant species being studied and the drought model being examined, but a range of -0.3 to -0.8 MPa is generally accepted as the standard for moderate drought stress in plant growth environments. Plant tissue is generally found to be affected not only by the degree of water potential decrease but also the duration of treatment under these conditions¹⁶.

Fig 1: Model of the PEG induced drought stress

Plant Responses:

Plants employ one of three main strategies to survive the deficit in water, namely, drought escape, drought tolerance, and drought avoidance¹⁷. Plants implement all three of these strategies to resist drought conditions and maintain favourable levels of turgidity and water balance. As part of the drought tolerance strategy adopted by plants, there are adjustments made to osmotic conditions, and plant antioxidant activity is enhanced. In addition to this, a tolerance to desiccation is also developed, along with increased water uptake and reduced water loss¹⁷. Commonly seen drought stress response in plants are reflected at the biochemical, physiological, photosynthetic and metabolic level (Table 1).

During the early stages of drought stress response in plants, closure of stomata is observed. In addition to this, enhanced root growth and expansion is observed, to provide the plant with as much water as possible in a water-deficient environment¹⁴. Due to these mechanisms, crop plants can usually maintain their productivity under short term drought conditions, but this is accompanied by a drastically slower photosynthesis rate, reduced CO₂ uptake, and redirection of plant transport systems to facilitate enhanced root growth¹⁸.

In the case of persistent drought conditions, adaptive measures taken by plants are no longer sufficient to sustain productivity and growth in plants. Under these circumstances, plants execute measures such as cell wall hardening, ROS detoxification and other metabolic changes specific to drought tolerance⁴.

Plant drought resistance is, therefore, a complex process involving various molecular events and adaptive responses in plants. It requires a comprehensive understanding of the multi-level regulatory network employed by plants to survive during drought conditions. Careful analysis of these mechanisms can aid research aimed at engineering and breeding plants that have an increased resistance to drought conditions, and therefore, higher practical value⁷. Information currently available regarding plant drought stress mechanisms and their underlying processes, however, is often inconsistent, complex, or incomplete. It, therefore, becomes important to conduct further research in a controlled laboratory environment and gain an enhanced understanding of the mechanisms that plants employ to survive despite a water shortage.

Properties of PEG 6000:

PEG is chemically a polymer with ethylene oxide as monomer units (Fig 2). PEG is also a widely used compound with a multitude of applications in this regard is polyethylene glycol. Polyethylene glycol can be used to change the osmotic potential of a nutrient solution culture, thereby inducing drought stress in a controlled manner conducive for experimental procedures¹⁹. PEG is an ideal substrate material to recreate soil-like conditions in vitro wherein osmotic potential can be readily manipulated.

Fig 2: PEG Chemical Structure

Specifically, variants with higher molecular weights (greater than 6000) are preferred due to the reason that the relatively larger molecular size is effective in preventing diffusion through cell walls²⁰. Owing to this large size preventing entry into plant cells, PEG 6000 has been shown to cause water loss by cytorrhysis rather than plasmolysis²¹. Thus, as a result of its large molecular size, PEG 6000 is an ideal solution for the simulation of osmotic stress.

Moreover, PEG 6000 is preferred over its commonly used alternative for the purpose of water potential reduction – mannitol – for the reason that the latter has been shown to be taken up by plant cells, thereby resulting in negative effects on growth due to toxicity²². PEG is a hydrophilic compound and can readily be dissolved in water, as well various other compounds such as methanol, ethanol, and benzene. It is also biologically inert and is considered a safe chemical in terms of toxicity. This also makes it suitable as a media component in experiments concerning plant cultures.

Use of PEG has also been proved to induce enhanced accumulation of phenolic compounds in medicinal plants²³. Phenolic compounds contribute to the medicinal properties of plants^24,25. Many of these plants are used in ethnobotanical and folklore practices^26-28. Medicinal properties of plants^29-31and antioxidant properties^32,33are due to the diverse presence of secondary metabolites. Therefore the use of PEG in inducing stress on medicinal plants important and may be utilize for elicitation of secondary metabolites.

Agar-based Drought Stress Model:

There are multiple methods and models available to simulate drought conditions for experimental purposes. These various models can all be classified into one of three categories, namely, soil-based, agar-based, or aqueous culture-based models. All of these categories of drought stress models share the common feature of water potential reduction in the medium or substrate being used for culture growth. Each method has its own set of unique applications and must be employed based on the research objective at hand.

Soil-based drought stress models have the advantage of being able to simulate actual drought conditions seen in agricultural and natural environments by gradually reducing or immediately interrupting plant watering in a soil medium to reduce substrate water potential³⁴. However, a significant disadvantage to this approach is the difficulty in controlling changes to the substrate water potential. Factors such as soil surface evaporation rate and plant water consumption rate are difficult to measure and reproduce in an experimental setting. In addition to this, such a drought stress model involves relatively high levels of water consumption and evaporation, due to which long term drought responses such as cell wall modifications and osmoprotective metabolite accumulation cannot be comprehensively analyzed^4,35.

Similarly, drought stress models involving plant cultivation in PEG-containing hydroponic aqueous media have their own set of limitations³⁶. The high viscosity caused by the presence of PEG results in reduced oxygen diffusion to roots, thereby leading to hypoxia in some cases⁴. Additional measures for aeration need to be provided, involving the continuous supply of air pumped via silicone tubes connected to the culture vessel³⁷. Additionally, the accumulation of some of the heavier variants of PEG (4000-8000 Da) in plant roots can also lead to damage, partial root dysfunction, and eventually, dehydration³⁸.

An alternative method to conduct drought stress experiments and tackle some of the issues faced in the case of soil-based or aqueous culture models is with the use of agar-based models. In this case, substrate water potential can be controlled more easily for experimental procedures involving the analysis of drought stress activity in plants⁴.

Agar-based drought stress models usually employ PEG as a means to reduce the water potential in the substrate, in a controlled manner that can both be measured as well as be reproduced with relative ease. Generally, 5-20% (w/v)³⁹. PEG content in the growth medium of choice has been shown to decrease water potential of the substrate, but this has been shown to occur even up to 40% (w/v)⁴⁰. PEG content in some cases. The use of agar-based models helps to reduce or completely avoid the state of hypoxia which may result in the case of some of the other models mentioned above.

Since PEG affects the process of solidification in agar, it is recommended not to add it directly to agar containing media prior to solidification⁴¹. Instead, the desired reduction in substrate water potential can be achieved by diffusing PEG in a concentrated overlay solution onto pre-solidified agar media¹³.

In summary, this method is especially advantageous over soil-based or aqueous culture-based models since the substrate water potential remains constant following treatment with PEG. The seedlings or plantlets growing on such media, therefore, exhibit results that are consistent with a constant value of drought stress throughout, unlike in the case of soil-based models wherein water potential values constantly change. Moreover, the issue of PEG interfering with root integrity as in the case of aqueous cultures is also solved by employing an agar-based model.

Biochemical and Physiological Effects:

An important part of experiments aimed at understanding the effects of drought conditions under various models is the characterization of biochemical and physiological changes that accompany drought conditions in plants. This knowledge allows for obtaining critical information regarding the functional changes and the metabolic response displayed by plants as a result of drought stress. To better understand these changes comprehensively, it is of paramount importance to develop a list of biochemical and physiological markers for a more real-time characterization. The selection of these markers needs to be done by taking all the major steps of plant drought response into account, starting from drought perception itself.

Drought is assumed to be recognized first by the roots, which subsequently chemically signal the same to the shoot⁴². A key role is played by abscisic acid (ABA) in this signalling process⁴³. Following the reception of hydraulic signals in vascular tissues, ABA is synthesized and transported to leaf epidermis, resulting in stomatal closure, decrease in turgor, xylem transport suppression⁴⁴.

Photosynthetic activity:

The first noticeable effect that results from drought stress conditions is plant tissue dehydration, typically characterized by loss of leaf turgor⁴⁵. A quantitative method to assess the extent of water loss in plant tissue is by the measurement of leaf relative water content⁴⁶. This straightforward measurement involves the calculation of dry weight to fresh weight ratio of plant matter via a gravimetric approach⁴⁷. This method is not only simple but also results in the collection of highly reliable and reproducible data.

One of the first responses displayed by plants when subjected to dehydration is the closure of stomata. This is intended to prevent the loss of water by transpiration⁴⁸. The extent of stomatal closure can be quantitatively measured and characterized by experimental procedures involving the measurement of gas flow rate or the measurement of electrical conductivity of a water film of constant ionic strength on the surface of the leaf^49,50.

The closure of stomata also negatively affects several photosynthetic processes, since parenchymal cells are not supplied with normal levels of carbon dioxide⁸. The magnitude of reduction in photosynthetic activity can be assessed quantitatively by measuring the amount of chlorophylls and carotenoids present⁵¹. Oxidative stress caused by drought conditions usually results in reduced chlorophyll content, possibly due to degradation or pigment photo-oxidation⁵². Apart from pigment degradation, various photosynthetic processes such as photosystem II activity are also negatively impacted by the presence of drought conditions⁵³.

Metabolic activity:

Plants are the source of secondary metabolites and natural products^54,55. There are several successful examples of plants, when cultivated in vitro under stress conditions, overproducing useful secondary metabolites^55-57. Plants also have unique trichomes for successful delivery of secondary metabolites^58,59. When plants are subjected to drought conditions, they undergo a process of metabolic adjustment, involving the accumulation of metabolically neutral, osmotically active solutes such as amino acids, sugars and organic acids⁶⁰. These measures adopted by plants are part of the second step in the strategy adopted by plants to adjust to drought conditions, following the closure of stomata^8,61. The amounts of these various accumulated metabolites can be assessed and quantitatively analyzed by various methods such as gas chromatography-mass spectrometry, or by simpler methods involving the analysis of relative and absolute levels of individual metabolites^62-64.

An understanding of secondary metabolite production and plant hormone levels is also paramount while trying to gain a comprehensive understanding of the effect of drought on plant activity. In particular, it has been observed that phenolic metabolites show a marked increase in plants that have been subjected to drought conditions for a significant period of time⁶⁵. There have also been cases wherein an upregulation of leaf flavonoids has been observed under conditions of water shortage⁶⁶.

The presence of drought conditions leads to the upregulation of ROS production, thereby disturbing the balance between ROS production and detoxification⁶⁷. Since ROS are extremely reactive, their presence in higher than normal amounts has a toxic effect and leads to damage of nucleic acids, proteins and lipids[68]. If the conditions causing ROS upregulation persist for an extended period, the damage caused can become irreversible, eventually resulting in cell death.

The overproduction of reactive oxygen species paired with the accumulation of sugars leads to an increase in the production of reactive carbonyl compounds (RCCs) along with plant protein glycation^69,70. These effects have been reported to negatively impact the nutritional properties of plant-derived foods, and have been reported to be associated with the activity seen in plants that are aging⁷⁰.

Metabolic adjustment so seen in plants is only a practical option in the case of short-term drought stress, and the accumulation of solutes can be expensive for the plant both in terms of energy and resources over a longer timeframe. Furthermore, if the soil water content is shallow and the stress subjected is especially severe, the adjustment of plant metabolic activity will have a negligible effect on water uptake, if any^14,71.

CONCLUSION:

This literature review consolidates many of the physiological and metabolic changes exhibited in plants as a result of drought stress and explores the PEG-based drought stress model employed for laboratory research. The drought stress exhibited in plants as a result of water shortage can be quantitatively characterized by measuring several plant metabolites, enzymes and pigments, and comparing these quantities with the levels at normal conditions.

The improvement of plant resistance to drought is an objective that is being explored in the domains of molecular biology and multiple ‘omics’ disciplines. Various approaches involving chemical or genetic alterations to plants to allow drought resistance are being explored and will, hopefully, provide a long term, robust solution to the problem of drought conditions affecting crop productivity around the world.

Table 1: Commonly seen drought responses in plants

Sl. No.	Response Category	Plant Response
1.	Physiological changes	Decrease in turgor⁴⁴
		Increased tolerance to desiccation¹⁷
		Increased water uptake¹⁷
		Enhanced root growth¹³
2.	Photosynthetic changes	Stomatal closure⁴⁴
		Slower photosynthetic rate¹⁸
		Reduced CO₂uptake¹⁸
		Pigment degradation⁵²
3.	Metabolic changes	Increased antioxidant activity¹⁰
		Accumulation of osmotically active solutes⁶⁰
		Increase in phenolic metabolites⁶⁵
		Upregulation of leaf flavonoids⁶⁶
		Increase in ROS, RCC production^9,69

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Received on 06.05.2020 Modified on 29.09.2020

Research J. Pharm. and Tech 2021; 14(11):6173-6178.

DOI: 10.52711/0974-360X.2021.01069