International Journal of

Molecular Sciences

Review
Methodology of Drought Stress Research:
Experimental Setup and
Physiological Characterization
Natalia Osmolovskaya 1,† , Julia Shumilina 2,3,† , Ahyoung Kim 3 , Anna Didio 2,3 ,
Tatiana Grishina 2 , Tatiana Bilova 1,3 , Olga A. Keltsieva 4 , Vladimir Zhukov 5 ,
Igor Tikhonovich 5,6 , Elena Tarakhovskaya 1,7 , Andrej Frolov 2,3, * and
Ludger A. Wessjohann 3, *
1 Department of Plant Physiology and Biochemistry, St. Petersburg State University, St. Petersburg 199034,
Russia; natalia_osm@mail.ru (N.O.); bilova.tatiana@gmail.com (T.B.); elena.tarakhovskaya@gmail.com (E.T.)
2 Department of Biochemistry, St. Petersburg State University, St. Petersburg 199904, Russia;
schumilina.u@yandex.ru (J.S.); didio1992@yandex.ru (A.D.); tgrishina@mail.ru (T.G.)
3 Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle (Saale) 06120, Germany;
ariyong1002@gmail.com
4 Institute of Analytical Instrumentation, Russian Academy of Science, St. Petersburg 190103, Russia;
keltcieva@gmail.com
5 All-Russia Research Institute for Agricultural Microbiology, St. Petersburg 196608, Russia;
vladimir.zhukoff@gmail.com (V.Z.); arriam2008@yandex.ru (I.T.)
6 Department of Genetics and Biotechnology, St. Petersburg State University, St. Petersburg 199034, Russia
7 Department of Scientific Information, Russian Academy of Sciences Library, St. Petersburg 199034, Russia
* Correspondence: afrolov@ipb-halle.de (A.F.); wessjohann@ipb-halle.de (L.A.W.);
Tel.: +49-(0)-34555821350 (A.F.); +49-(0)-34555821301 (L.A.W.)
† These authors contributed equally on the manuscript.




Received: 23 November 2018; Accepted: 14 December 2018; Published: 17 December 2018


Abstract: Drought is one of the major stress factors affecting the growth and development of plants.
In this context, drought-related losses of crop plant productivity impede sustainable agriculture all
over the world. In general, plants respond to water deficits by multiple physiological and metabolic
adaptations at the molecular, cellular, and organism levels. To understand the underlying mechanisms
of drought tolerance, adequate stress models and arrays of reliable stress markers are required.
Therefore, in this review we comprehensively address currently available models of drought stress,
based on culturing plants in soil, hydroponically, or in agar culture, and critically discuss advantages
and limitations of each design. We also address the methodology of drought stress characterization
and discuss it in the context of real experimental approaches. Further, we highlight the trends of
methodological developments in drought stress research, i.e., complementing conventional tests with
quantification of phytohormones and reactive oxygen species (ROS), measuring antioxidant enzyme
activities, and comprehensively profiling transcriptome, proteome, and metabolome.

Keywords: drought stress; drought models; drought tolerance; oxidative stress; phytohormones;
polyethylene glycol (PEG); stress markers

1. Introduction
Being a natural climatic feature, drought occurs in almost all climate zones with varying frequency,
severity, and duration, and is one of the most deleterious factors of environmental stress [1,2]. Indeed,
even a short-term water deficit results in essential annual losses of crop yields [3,4], impeding

Int. J. Mol. Sci. 2018, 19, 4089; doi:10.3390/ijms19124089 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018, 19, 4089 2 of 25

sustainable agriculture all over the world [5–7]. Due to oncoming climate changes, the frequency and
duration of drought periods will increase, making this factor one of the most important threats of the
current century [8,9].
In the context of agriculture, drought is defined as a period of below-average precipitation [10],
when the amounts of available water in the plant rhizosphere drop below the limits required for
efficient growth and biomass production [11]. Such a soil water deficit can be persistent in climate
zones characterized by low water availability, or by intermittent and unpredictable water supply during
the vegetative period [12]. Because of this, drought is the major environmental stressor, affecting the
plant’s growth and development by disrupting its water status [13]. This dramatically affects all key
physiological processes, such as photosynthesis, respiration, and uptake of mineral nutrients [14,15].
First, drought compromises stomata function, impairs gas exchange, and leads to overproduction
of reactive oxygen species (ROS) and development of oxidative stress [16]. Second, water deficit
inhibits cell division, expansion of leaf surface, growth of stem, and proliferation of root cells [7].
In concert, all these factors dramatically reduce plant productivity and might lead to the death of
drought-sensitive plants upon prolonged exposure to drought [17].
At the quantitative level, water deficit in the environment can be characterized by a decrease of soil
water potential (Ψw ) [18]. According to the van’t Hoff equation, it indicates a decrease in free energy of
substrate water that makes water uptake from the medium under these conditions thermodynamically
unfavorable and loss of water by the plant more probable. Values of Ψw from 0 to −0.3 MPa are
characteristic for well-watered plants, whereas values below −0.4 MPa correspond to moderate water
stress, and potentials of −1.5 to −2.0 MPa represent severe stress and permanent loss of turgor [19].
However, these values vary among species and drought models. They are based on experience with
seeds and seedlings, which are commonly more drought tolerant. Thus, in our experience, Ψw values
of −0.3 to −0.8 MPaare more typical for experimentally useful, i.e., recoverable, moderate drought
stress in plants beyond the seedling stage (v.i.). In general, leaf Ψw can be determined by several
approaches. In the easiest but most reliable way, Ψw can be addressed by the gravimetric method [20].
It can also be accomplished with a Scholander pressure chamber and thermocouple psychrometer [21]
or tensiometer [22]. Thermocouple psychrometry is one of the most popular methods, and is usually
accomplished with press saps or freeze-thawed leaf disks [23]. Recently, a new method was proposed
for determination of Ψw in leaf cell apoplast, relying on the measurement of photosynthetic CO2 /H2 O
gas exchange [24].
It is important to mention that not only the degree of Ψw decrease, but also its duration, can
affect the plant organism [25]. Therefore, water stress often develops upon minimal reduction of
soil Ψw . To avoid this scenario, plants adopt various strategies to prevent water loss, to preserve
water supply even under reduced Ψw , and to sustain periods of unfavorable water regimen
accompanied by low water content in tissues [10]. These drought-induced alterations can affect plant
morphology, physiology, and biochemistry in degree -depending on plant species, developmental
stage, and duration and severity of drought [4,6,7,26,27].
The main strategies employed by plants to sustain water deficit are (i) drought escape, (ii) drought
avoidance, and (iii) drought tolerance [28]. Generally, all three strategies impact the development of
the state known as drought resistance, which can be defined as the ability to maintain favorable
water balance and turgidity under drought conditions. In the escape strategy, plants complete
their life or growth cycle before the impact of drought causes harm, i.e., they use a seasonal
response [4]. The strategy of drought avoidance relies on enhanced water uptake and reduced water
loss, whereas drought tolerance is mediated by osmotic adjustment, extension of antioxidant capacity,
and development of desiccation tolerance [28]. On one hand, these strategies represent different steps
of drought response (Figure 1). On the other hand, they might indicate different climatic and ecological
specializations of plant species [29]. This concept of stress avoidance and stress tolerance proposed by
Levitt [30] provides insight into plant responses to a relevant decrease of Ψw at the cell and organism
levels [10].
 of reduced CO2 uptake, a dramatic drop in photosynthesis rate, and redirection of assimilate
transport for enhancement of root growth [33,34]. When drought persists for a long time and adaptive
capacities of the avoidance strategy are not sufficient to sustain plant growth and productivity, other
mechanisms might be involved. At this step, mechanisms such as accumulation of compatible solutes
and protective proteins (so-called metabolic adjustment), cell wall hardening, ROS detoxification, and
Int. J. Mol. Sci. 2018, 19, 4089 3 of 25
metabolic changes are involved in establishing drought tolerance [10].

Figure 1. The
Figure 1. The main
main drought
drought resistance
resistance strategies
strategies employed
employed byby plants
plants to
to counter
counter water
water deficit
deficit periods
periods
(drought
(drought escape, drought avoidance, and drought tolerance) and the main steps of the plant response
escape, drought avoidance, and drought tolerance) and the main steps of the plant response
to
to dehydration.
dehydration.

As can be seen from Figure 1, the first response of the plant organism to drought as a
Thus, plant drought resistance is a complex process that requires a global view to understand
drought-resistance strategy relies on avoiding water deficit [31] by maintaining tissue Ψw by increasing
its underlying mechanisms. Obviously, the majority of molecular events triggered by a decrease of
water uptake or restricting water loss [32]. At the early steps of drought response, it is mainly achieved
tissue Ψw cannot be unambiguously attributed solely to avoidance or tolerance strategy. Therefore, a
by stomata closure, triggered by abscisic acid (ABA). However, according to Muller et al. [33], the rapid
complex multilevel regulatory network controlling plant adaptive responses to drought stress is
expansion of roots and young leaves (as a major C sink) is affected earlier and more intensely than
required. Studies of responses to water deficit such as stomata closure, expression of stress-specific
photosynthesis (C source); accordingly, root growth is enhanced to provide sufficient water uptake
genes, accumulation of osmolytes, and up regulation of antioxidant systems recently made
under drought conditions. These avoidance mechanisms can secure the maintenance of crop plant
considerable progress [17,35–38]. It was shown that the mechanisms underlying stress resistance are
productivity during short-term periods of water deficiency [18]. However, this is achieved at the price
crucial for plant survival and are associated with significant changes in the patterns of metabolites
of reduced CO2 uptake, a dramatic drop in photosynthesis rate, and redirection of assimilate transport
and proteins [10,15,35]. Hence, analyzing the changes in plant metabolome and proteome associated
for enhancement of root growth [33,34]. When drought persists for a long time and adaptive capacities
with the onset of drought might be an important step in breeding and engineering plants with
of the avoidance strategy are not sufficient to sustain plant growth and productivity, other mechanisms
increased drought resistance [15,35] or developing plant protectants against drought stress [39].
might be involved. At this step, mechanisms such as accumulation of compatible solutes and protective
proteins (so-called metabolic adjustment), cell wall hardening, ROS detoxification, and metabolic
changes are involved in establishing drought tolerance [10].
Thus, plant drought resistance is a complex process that requires a global view to understand
its underlying mechanisms. Obviously, the majority of molecular events triggered by a decrease of
tissue Ψw cannot be unambiguously attributed solely to avoidance or tolerance strategy. Therefore,
a complex multilevel regulatory network controlling plant adaptive responses to drought stress is
required. Studies of responses to water deficit such as stomata closure, expression of stress-specific
genes, accumulation of osmolytes, and up regulation of antioxidant systems recently made considerable
progress [17,35–38]. It was shown that the mechanisms underlying stress resistance are crucial
for plant survival and are associated with significant changes in the patterns of metabolites and
proteins [10,15,35]. Hence, analyzing the changes in plant metabolome and proteome associated with
Int. J. Mol. Sci. 2018, 19, 4089 4 of 25

the onset of drought might be an important step in breeding and engineering plants with increased
drought resistance [15,35] or developing plant protectants against drought stress [39].
Recently, Wang et al. [15] comprehensively reviewed drought-related effects on the plant
proteome, including changes in signal reception and transduction, ROS scavenging, osmotic regulation,
protein synthesis/turnover, modulation of cell structure, and carbohydrate and energy metabolism.
These functional patterns of plant response to drought gave access to understanding of fine mechanisms
underlying the process of stress tolerance. Apparently, for successful study of plant responses to
drought stress under experimental conditions, reliable and adequate stress models are required.
Accordingly, various drought models have been established (Table 1). However, the available
information is often complex, incomplete, and inconsistent. A comprehensive literature search for
drought tolerance research shows great variability and inconsistency in the experimental designs
and methods for stress characterization [15]. Therefore, here we systematically address different
experimental setups for establishing drought stress models and consider physiological and biochemical
methods for their characterization.

2. Experimental Models of Drought Stress

Despite a large variety of available drought models, according to their basic setup, all of these
techniques can be classified as soil-based, aqueous culture–based, or agar-based. The common feature
of all drought stress models is reduction of the water potential in the substrate or medium surrounding
plant roots. However, individual methods have different applicability limitations and vary in terms of
the scientific questions they can address. Therefore, the advantages and disadvantages of each model
need to be carefully considered prior to experiment planning.

2.1. Soil-Based Drought Models

The obvious advantage of this model strategy is the close similarity of experimental conditions
to actual drought in nature and agriculture. In this case, the decrease of soil Ψw is established by
gradual decline or immediate interruption of plant watering [40]. Such models adequately simulate
short-term drought, which represents the most frequent case in the European agricultural practice
due to varying weather conditions [41,42]. However, difficulty controlling the substrate Ψw represents
an essential limitation of this approach. Indeed, in this experimental setup, the severity of drought
stress is determined by the rates of water evaporation from the soil surface and consumption by the
plant [43]. As these cannot be defined by the researcher and depend on multiple factors, reproducibility
and predictability of such experiments are always questionable. Moreover, as the rates of water
consumption and evaporation are relatively high, this model does not allow probing long-term
drought responses, such as accumulation of osmoprotective metabolites or proteins and cell wall
modifications [10]. Therefore, many important aspects of plant drought tolerance and adaptation to
low Ψw , such as, for example, accumulation of osmoprotective proteins and hardening of cell walls,
can be overlooked in this experimental setup, although using large and deep pots might improve this
situation [10].
Despite the above-mentioned problems, several improvements can be made to increase the
reproducibility and reliability of soil models. First of all, in this type of experiment, the size and
structure of soil particles, as well as their water capacity, should be taken into account. Thus, to achieve
moderate (i.e., less severe) drought conditions, in an optimized variant of this model, plants are grown
in foil-sealed vessels to prevent water evaporation from the soil surface [5]. Thereby, each pot can be
equipped with a piece of tubing inserted into the soil to facilitate rewatering of plants. Due to the
water supply, in this model, water deficit can be increased gradually, making it possible to address
long-term plant responses to drought [44]. Moreover, stability of the water regimen can be improved
by an automated irrigation system.
Recently, Todaka et al. [40] introduced an automatic irrigation system to relay monitoring of
actual water content in soil. Using this approach, the authors proposed a drought model able to ensure
Int. J. Mol. Sci. 2018, 19, 4089 5 of 25

the desired values of Ψw (−9.8, −31.0, and −309.9 KPa). However, this system failed to reproduce
the conditions of severe dehydration. Although the optimized method described above is reliable
and reproducible enough, repeated measurements of leaf and soil Ψw are laborious and require large
amounts of plant material, which are hardly available in long-term experiments under reproducible
laboratory conditions. For example, such a restriction can be critical when mutants or transgenic plants
are dealt with, in particular those with reduced stomata density or small leaf area [45].
An elegant way to avoid this complication is to culture mutant or transgenic plants in the same pot
with the reference plants, e.g., the wild-type (wt) counterparts [10]. In this case, leaf Ψw determination
can be limited to the reference (or wt) plants, which are commonly more suitable for assessing stress
markers. The obtained results can be extrapolated to the mutants. In this case, both reference or wt and
experimental or mutant plants would grow in the same medium and therefore be exposed to the same
soil Ψw if they are planted in a suitable scheme and position. The best way to provide a quantitative
characteristic of drought stress by this approach is to complement it with a measurement of soil Ψw at
the end of the dehydration period. Analogously, this method can be applied to untreated and treated
plants in assays for chemical drought tolerance enhancers or other phytoeffectors (v.i.) to be tested.
It is important to mention the setups that rely on inert substrate, such as vermiculite or perlite,
as soil substitutes. The advantage of this approach is that the roots of experimental plants can be
pulled out easily and without damage to investigate drought-related changes in water potential [46]
or oxidative and metabolic responses [47] at the root level. Inert substrates are suitable for studying
the effects of drought in legume–rhizobial nodule symbiosis [48]. On the other hand, the certain
disadvantage is that watering, unlike soil culture, is carried out not with water, but with a nutrient
solution, so the impact of drought by cessation of watering is accompanied by the appearance of
another stress factor, i.e., a deficiency of mineral elements.

2.2. Drought Models Based on Hydroponic Aqueous Culture

Despite the high relevance of soil-based drought models because of their similarity to natural
conditions, they all have a common intrinsic limitation: the difficulty of adequately controlling Ψw
in the root microenvironment. However, this is critical when a precise definition of substrate Ψw is
required, as in multiple or long-term experiments comparable over months (and seasons). Therefore,
the models, based on aqueous hydroponic culture with predictably decreased Ψw of nutritional
solution, might be advantageous for such applications. The easiest way to reduce the Ψw of growth
medium is to decrease its level in pots and partially exposure roots to air, as was shown for lettuce by
Koyama et al. [49]. To simulate severe dehydration, plant roots can be left under air for up to eight
hours [50], allowing the severity of simulated drought to be defined by the duration and repetitions of
the dehydration process. This approach is based on the fact that Ψw of leaves, at least to some extent,
corresponds to the index of water availability for plants, which in turn depends on water potentials of
soil and plant roots [51]. Thus, experimentally affecting the Ψw of roots influences the Ψw of leaves as
well. When using this approach, however, one needs to keep in mind that the degree of dehydration
and kinetics would strongly depend on air humidity. Further, it is important to remember that in this
case the plant response is dependent on root distribution (e.g., long vs. short roots).
Despite the ease of the above approach, most often desired Ψw values of plant rhizosphere are
obtained by supplementing nutrient solutions with osmotically active substances (osmolytes, which
reduce available water), taken in calculated concentrations. This approach is based on the simulation
of drought by application of osmotic stress, i.e., increasing the medium osmotic pressure compared
to that of plant tissues [52]. Similar events occur in soil when the water content decreases (due to
evaporation and absorption by the plant) and the concentrations of solutes grow, resulting in an
increased osmotic component of the water potential [53]. Thus, the described setup corresponds
well with natural drought. This strategy allows precise adjustment of Ψw and efficient monitoring
of its magnitude, resulting in high accuracy, reproducibility, and interexperimental comparability of
acquired data [54]. However, when working with this kind of drought model, selecting an appropriate
Int. J. Mol. Sci. 2018, 19, 4089 6 of 25

osmolyte requires special attention. Thus, low-molecular-weight osmolytes (e.g., sugar alcohols and
sodium chloride) routinely used in early studies [55] demonstrate strong negative side effects when
applied in experimental drought. Indeed, these compounds easily penetrate cell walls and plasma
membranes, increasing intracellular osmotic pressure and leading to plasmolysis [56]. Any salts
also change ion titers and distribution in plants, affect ionic strength, and trigger the process of ion
transport. On the other hand, nonionic carbohydrate-related osmolytes (e.g., sorbitol and mannitol)
are readily involved in cellular metabolism themselves, and thus might directly affect the results of
the experiment [56], as they are often toxic to plants [57]. They can also increase mold growth under
commonly nonsterile conditions. Because of this, the use of biologically inert polymeric osmolytes
is preferable and advantageous [58]. Therefore, currently, drought stress models rely on presumably
nonpermeable high-molecular-weight osmolyte polyethylene glycol (PEG) with an average molecular
weight of 6000 Da or more [55,59].
It is well documented that PEG effectively decreases medium Ψw , thereby disrupting absorption
of water by plant roots [60]. In terms of this approach, 5–20% (w/v) [61] or even 40% (w/v) [62] PEG in
growth medium enables a stabile decrease of Ψw during any desired period of time [63]. Importantly,
PEG-based aqueous models allow the setup of recovery experiments by transfer of stressed plants
to PEG-free nutrient solution or exchange of the PEG solution [10]. Therefore, PEG-based models of
drought stress represent the method of choice in molecular biology and plant protectant studies and
screening experiments [64]. One issue yet underexplored in the PEG model is the complexing ability of
PEGs on metal ion species and thus the altered availability of the various ions for the plant. However,
also under drought conditions, ion availability eventually changes and decreases.
One of the most promising applications of aqueous PEG-based models of osmotic stress is
screening for potential drought-protective compounds. Substances that influence plant performance
(without being plant protectants against biotic stress, e.g., from pathogens) in agrochemistry are
defined as phytoeffectors and include drought stress tolerance enhancers. Phytoeffectors are able to
prime crop plants against short-term drought and ensure that their productivity is sustained under
drought conditions with spatiotemporal control, largely independent of the crop species or variety
used. Such effects were described for salicylic acid and its derivatives [65], as well as for various
fungicides of the triazole [66] and imidacloprid [67,68] families. The drought-protective effects of
small molecules on a plant organism are usually mediated by inhibition of enzymes, involved in plant
response to stress, as was described for poly(ADP-ribose) polymerase (PARP)in the beginning of this
decade [68], although later at least direct involvement of PARP appeared doubtful [69]. If a molecular
target for drought stress effects is known, and ideally the active site too, methods of computational
chemistry like virtual screening and molecular docking approaches [70] allow virtual screening of
thousands of structures with millions of conformers. The most promising candidates for wet lab testing
can thus be identified.
For rapid screening of such compounds, a reliable model based on a Lemna minor culture was
recently developed in our group [68]. This technique (Figure 2A) relies on a microtiter plate format
and assumes treatment of plants with PEG6000 or PEG8000 supplementing the growth medium in the
presence and absence of potential phytoeffectors. After a 24 h stress period, plants are transferred to
a PEG-free medium, and stress recovery is monitored for further 48 h, before the protective effect is
assessed by attenuation of growth inhibition via measurement of leaf peak area increase by means of a
2D-photodocumentation visualization system.
The Lemna system has several advantages over classical spraying systems: Plants are all clones,
reproducing by budding, and they are small and can be grown in microtiter plates (6-, 12-, or 24-well
format) under sterile conditions. The small scale allows medium-throughput screening with small
amounts of compounds. Most importantly, these can be applied in a concentration-dependent manner
to the multiwell plate well (while spraying or dumping delivers only uncertain amounts to plants),
and both root and leaf uptake is ensured. The leaves are flat and 2D phenotyping is easily done with
Int. J. Mol. Sci. 2018, 19, 4089 7 of 25

the respective software [68]. For better reproducibility, initial root length should be unified, and until
Int. J. Mol. Sci. 2018, 19, x 7 of 26
termination of the experiments, plant growth should not be limited by well size.

Figure 2. Experimental drought models based on osmotic stress and established by supplementation
of growth medium with polyethylene glycol (PEG): (A) Lemna minor model, established with aqueous
growth
Figure medium supplemented
2. Experimental droughtwith PEG6000
models based([68]); (B) Brassica
on osmotic stressnapus model, established
and established with aerated
by supplementation
aqueous
of growthculture supplemented
medium with PEG8000;
with polyethylene and (C)
glycol (PEG): (A) agar-based
Lemna minorPEG infusion
model, Arabidopsis
established thaliana
with aqueous
model,
growthestablished by overlaying solidified
medium supplemented agar medium
with PEG6000 with
([68]); (B) PEG8000
Brassica solution
napus for established
model, five days. with
aerated aqueous culture supplemented with PEG8000; and (C) agar-based PEG infusion Arabidopsis
Despite their established
thaliana model, wide use, by PEG-based models have
overlaying solidified some intrinsic
agar medium limitations
with PEG8000 solution that need
for five days.to be
taken into account when planning experiments [63]. First, PEG-containing nutrient solutions are
characterized by high
Despite their wideviscosity, which models
use, PEG-based compromises diffusion
have some of oxygen
intrinsic to the
limitations thatroots,
need to especially
be taken
in deeper
into vessels,
account whenandplanning
can cause experiments
hypoxia [10]. [63].
To prevent
First, the development nutrient
PEG-containing of hypoxia, additional
solutions are
aeration needs to be provided for plants grown in PEG-containing medium. For this,
characterized by high viscosity, which compromises diffusion of oxygen to the roots, especially in air is continuously
supplied by pumps
deeper vessels, andthrough silicone
can cause tubes connected
hypoxia to the culture
[10]. To prevent vessels [71]. Although
the development of hypoxia, thisadditional
approach
can be easily established for larger plants (as it was done in our lab
aeration needs to be provided for plants grown in PEG-containing medium. For this, with B. napus; Figure 2B air
[72]),
is
small model plants like Arabidopsis, typically grown in small vessels on large scale
continuously supplied by pumps through silicone tubes connected to the culture vessels [71]. for highly replicated
biological
Althoughexperiments,
this approach cannot
can bebeeasily
supplied with air individually
established and are
for larger plants (astypically grown
it was done under
in our labhypoxic
with B.
conditions [73]. Small and flat vessels like the wells used in the Lemna system [68]
napus; Figure 2B [72]), small model plants like Arabidopsis, typically grown in small vessels on large are usually not
prone to such
scale for problems.
highly replicated biological experiments, cannot be supplied with air individually and are
Another
typically grownpossible issue isconditions
under hypoxic absorption and
[73]. accumulation
Small of like
and flat vessels PEGthe with
wellsmolecular
used in theweight
Lemna
4000–8000 Da in plant roots, which might
system [68] are usually not prone to such problems. result in damage [74]. The accompanying partial root
Another possible issue is absorption and accumulation of PEG with molecular weight 4000–8000
Da in plant roots, which might result in damage [74]. The accompanying partial root dysfunction
might impact leaf dehydration in an unpredictable way. Thus, stress responses observed in plant
shoots are only partly related to osmotic stress applied by PEG solution. The impact of PEG-related
Int. J. Mol. Sci. 2018, 19, 4089 8 of 25

dysfunction might impact leaf dehydration in an unpredictable way. Thus, stress responses observed
in plant shoots are only partly related to osmotic stress applied by PEG solution. The impact of
PEG-related root damage on these responses is difficult to estimate, but is obviously increased when
plant transfer on PEG-containing medium is accompanied with wounding of roots, which should be
avoided [75].

2.3. Agar-Based Drought Models

In general, growing plants in agar allows avoiding or reducing development of the hypoxic state.
As this is especially relevant for Arabidopsis, agar-based models are widely used in plant biology, and
specifically in drought stress experiments with Arabidopsis thaliana seedlings [76]. Thus, van der Weele
et al. proposed an agar-based PEG infusion model relying on saturation of solidified agar (filled in
Petri plates) with Murashige and Skoog medium supplemented with PEG8000 during two days [77].
Unfortunately, PEG affects the solidification of agar, therefore adding it directly to the agar medium
under preparation is not advisable [73]. Because of this, generating a desired Ψw of agar medium
is achieved by diffusing PEG from a concentrated overlay solution into preformed, solidified agar.
Adjusting the concentration of the overlay solution, the equilibrium in Ψw between aqueous overlay
medium and agar is achieved after 24 h of diffusion [76]. After decantating the PEG solution, seedlings
can be transferred to the now PEG-containing agar medium (stress application), and eventually plants
can be replanted to a PEG-free one after a defined treatment period (recovery).
Due to a constant character of Ψw , the agar-based PEG infusion model is advantageous compared
to those based on soil or (nonaerated) aqueous culture. Thus, the Ψw of seedlings can achieve
equilibrium with the agar medium during treatment time. Under soil drying conditions, this is
impossible, as soil Ψw changes continuously along with water evaporation and consumption by the
plant. On the other hand, due to PEG interfering with root integrity [78], this equilibrium is also hardly
achievable in aqueous PEG solutions (especially when PEG concentration is high). Thus, the agar-based
model system currently is an ideal choice to address dehydration avoidance and mechanisms of
dehydration tolerance [10]. Most commonly, PEG concentrations in the agar medium do not exceed
the values needed for medium to medium-high drought stress, i.e., Ψw < −1.2 MPa [57]. However,
based on the solubility of PEG8000 in water, the agar-based infusion model can be established in a
broad range of overlay medium Ψw values from −0.47 MPa to −3.02 MPa [79].
The agar-based PEG infusion model was successfully applied to different plants and fungi [75].
Further, a similar setup (10% w/v PEG6000 in the overlay medium) was used to probe the effect of water
stress on rape oilseed (Brassica napus) germination and seedling development [80,81]. An essential
limitation of the setup, originally proposed by Verslues and co-workers [10], was its applicability
to only the early steps of plant ontogenesis, seed germination and seedling development. Thus,
this method was inapplicable to mature plants, and corresponding stress responses characteristic of
later stages of ontogenesis could not be addressed.
Therefore, to extend the agar-based approach to mature organisms, we modified the method of
Verslues and co-workers to 5-to-7-week-old A. thaliana plants [35]. This setup combined germination
on agar in truncated polypropylene tubes, growth for 4–5 weeks in aqueous culture, and transfer to
agar medium preinfused with PEG8000 solution with a polymer concentration corresponding to the
targeted substrate Ψw (Figure 2C).
In general, our observations confirmed earlier data indicating higher sensitivity of mature plants
to drought compared to seedlings [10], although stress tolerance varies essentially between species.
Thus, in contrast to seedlings, application of Ψw below −0.6 MPa led to reduced survival of plants over
a period of 7 days, whereas a drop to −0.4 MPa was accompanied by significant alterations in plant
metabolome and proteome, indicating metabolic adjustment and changes in redox metabolism [35].
Soybean turned to be more resistant to osmotic stress applied in an agar-based model and successfully
survived osmotic stress applied by 8 and 16 (w/v) PEG for two weeks for pre- and post-flowering
treatments [82].
Int. J. Mol. Sci. 2018, 19, 4089 9 of 25

To summarize, compared to other setups, the agar-based PEG infusion model has two
fundamental advantages. First, it provides a stable and reproducible decrease of substrate Ψw that
cannot be achieved with the soil-based model. On the other hand, compared to models based on
aqueous culture, it has higher relevance to the conditions of a real field, as it relies on a solid substrate.
Second, the agar-based model allows precise Ψw setting in plant rhizosphere without accompanying
hypoxia and PEG-related root toxicity. It is important to keep in mind, however, that this setup
does not allow a direct extrapolation of drought effects to the field or ecosystem due to the model’s
simplicity, which doesnot consider water gradient in soil and heterogeneity in terms of water holding
capacity. For fast (pre-)screening of phytoeffectors, especially if only small amounts of test compounds
are available, the Lemna minor aqueous system has advantages [68] but must be complemented later by
the solid medium method for validation [35].

3. Physiological and Biochemical Characterization of Drought Stress

Adequate and correct application of an experimental drought stress model requires
comprehensive characterization at the levels of physiology, biochemistry, and molecular biology.
These experiments deliver objective information on the actual functional state of the plant organism
and its metabolic response to stress. This block of data is necessary to confirm the stressed state of
experimental plants (i.e., development of stress response) and to estimate the severity of stress-related
alterations. Accordingly, a panel of physiological and biochemical markers of drought stress ideally
accompanies any study relying on modeling setups. Importantly, these markers can be used
for dynamic characterization of plant adaptive responses throughout the whole experiment, i.e.,
acquisition of stress kinetics (Table 2). Thus, ideally, selection of the markers needs to consider all steps
of drought response, starting from drought perception. It is assumed that drought is recognized by
roots, which send a chemical message to the shoot [83]. Abscisic acid (ABA) plays a key role in this
signaling [84]. This effector is synthesized in response to hydraulic signals in vascular tissues and
further transported to leaf epidermis cells. Resulting stomata closure results in suppression of xylem
transport, decreased turgor, and root growth arrest [37].

3.1. Water Status and Photosynthetic Parameters as Markers of Drought Stress

One of the first detectable symptoms of drought is dehydration of plant tissues, which is
characterized by a decrease of Ψw and loss of leaf turgor [6]. Due to its simplicity, low time expense,
and robustness, the measurement of leaf water potential prior to sunrise is one of the most commonly
used tests for this marker [85]. Although critical values of tissue water potentials are species-specific,
Ψw of less than −0.8 MPa is commonly recognized as a sign of drought stress [86]. On the other hand,
the degree of water loss can be reliably assessed by a decrease of leaf relative water content (LRWC) [87].
In the easiest and most straightforward way, this parameter can be addressed by the gravimetric
method and calculating dry weight/fresh weight ratios [88]. Despite its simplicity, this approach yields
highly reproducible data. An obvious disadvantage of this method is its destructive character, i.e.,
consumption of plant material for each determination [85]. In this context, a nondestructive technology
based on automatic assessment of short-wave infrared irradiation reflected from the leaf surface might
be a good alternative [85]. Another nondestructive approach relies on long-term phytomonitoring,
i.e., continuous measurement of leaf transpiration, turgor, and xylem flow by means of nondamaging
sensors attached to the plant [89].
One of the primary plant responses to dehydration is stomata closure, which is intended toprevent
transpiration-related water loss, and is essential for the success of the drought avoidance strategy [90].
Similarly to dehydration itself, this parameter can be quantitatively characterized [91]. Experimentally,
it can be done by measuring the rate of gas flow through a leaf surface or the electrical conductivity
of the water film (of constant ionic strength) on the leaf surface [92]. Therefore, stomata conductance
is usually expressed in mmol/m2 /s [92]. Technically, such experiments are based on porometric
Int. J. Mol. Sci. 2018, 19, 4089 10 of 25

measurements, i.e., determining times required for increased air humidity in an isolated chamber with
a leaf inside [93].
Since stomata closure disrupts the supply of parenchyma cells with carbon dioxide, drought
ultimately negatively affects the efficiency of photosynthesis by inhibiting carbon assimilation and
light reactions [5]. In the simplest way, photosynthetic activity can be addressed by quantitative
determination of pigments: chlorophylls (at least chlorophyll a) and carotenoids [94]. Thereby,
decreased chlorophyll level is considered a symptom of oxidative stress and may be the result
of pigment photo-oxidation and chlorophyll degradation [95]. Accordingly, as was shown in a
comparative screening of barley genotypes, higher chlorophyll content was generally associated with
higher drought tolerance [94,96]. This allows us to consider this indicator as an important marker of
plant functional state under drought conditions.
Besides degradation of photosynthetic pigments, dehydration negatively affects the whole
photosynthetic apparatus [97]. One of the most reliable markers of this process is decreased
photosystem II (PS II) activity [98]. Both relative chlorophyll content and PS II efficiency can
be easily quantified with pulse amplitude modulation (PAM) fluorometry [99,100]. Thereby, the
ratio of minimum (background) and potentially maximum chlorophyll fluorescence (Fv/Fm) is
interpreted as the maximum of PS II photochemical activity and might be considered as a reliable
marker of PS II photoinhibition and one of the most important indicators of drought stress [101].
Importantly, the chlorophyll fluorescence is registered in vivo, thus it does not require sampling of
plant material [102]. Interestingly, in some cases, drought does not cause any alterations of PS II
activity. This result, observed with potato leaves, can be explained by photochemical quenching of
excess light energy by increased photorespiration [103]. It needs to be taken into account that besides
drought stress, the onset of senescence can underlie a decrease in Fv/Fm ratio [104].
In agreement with the described mechanisms, the features protecting the chloroplast
photosynthetic machinery from oxidative damage might increase stress tolerance. This was illustrated
in a comparative study of two B. napus cultivars grown for 3 weeks in aerated aqueous nutrient
medium with Ψw of −0.6 MPa (18% w/v PEG8000) [105]. The developing stress could be recognized
in both cultivars by a pronounced decrease in growth and photosynthetic parameters, including PS
II activity and chlorophyll a content. However, the cultivar with higher leaf contents of chlorophyll
a and carotenoids, as well as higher Fv/Fm ratios, demonstrated a clearly higher drought tolerance.
Thus, it could be concluded that the quantum yield of photosynthesis and the chlorophyll a content
could be effective selection criteria in screening for cultivars of crop plants with drought tolerance [105].

3.2. Changes in Phytohormone Patterns as Markers of Drought Stress

Plant response to environmental stress is a complex process, precisely tuned by multiple
regulatory systems [12,106]. In particular, dehydration triggers activation of signal transduction
cascades, including long-distance transport steps mediated by phytohormones [107]. Specifically,
drought-induced stomata closure is regulated by abscisic acid (ABA) and relies on ABA-dependent
signaling pathways [108]. Upon dehydration, ABA tissue content in Arabidopsis leaves can be
increased up to 30-fold [107]. In a time-course study of the drought-avoidance response performed with
Arabidopsis, early accumulation of ABA and induction of associated signaling genes coincided with a
decrease in stomata conductance, as revealed by a panel of physiological, biochemical, and molecular
biology methods [12]. Therefore, increased ABA in leaf cells represents a reliable marker of drought
stress in model experiments [35].
Besides ABA, several other hormones and their interaction networks have an impact on the control
of stomata conductance during water deficit. Thus, auxins, cytokinins, and ethylene are prone to inhibit
the ABA-mediated stomata closure mechanism, whereas brassinosteroids, isoleucinyl jasmonates,
and jasmonic and salicylic acids support the effects of ABA [109]. Jasmonic acid and its derivatives play
a significant role in plant response to drought in terms of opening and closing of stomata [110], acting in
an interplay with ABA and starting ABA signaling transduction [111]. In contrast to jasmonates and
Int. J. Mol. Sci. 2018, 19, 4089 11 of 25

ABA, ethylene is involved in the stimulation of stomata opening via inhibition of nicotinamide adenine
dinucleotide phosphate, reduced form (NADPH) oxidase in the leaves of plants, responsible for the
launch of ROS-dependent stomata closure pathways [112], but ethylene also induces senescence. Thus,
despite their impact on drought response, the mentioned phytohormones have complex patterns of
effects [107]. Therefore, their use as drought stress markers is hardly possible. Similarly, their potential
to be applied as phytoeffectors in the field is limited. Apart from cost, bioavailability, and stability
issues, it would require an extremely balanced mixture of suitable hormones, adapted in each case to
the plant species, developmental stage, and status.

3.3. Metabolites as Markers of Drought Stress

Various abiotic stressors are known to affect the profiles of plant metabolites [113]. Indeed,
the process of metabolic adjustment, i.e., accumulation of osmotically active and metabolically
neutral solutes, such as different sugars, amino acids (predominantly proline and glycine), betaine,
polyamines, and organic acids, under drought conditions is well documented [20]. Metabolic
adjustment is the second step in plant adaptation to drought (after stomata closure) and is critical
in maintaining the water status and physiological activity of plant cells, especially during relatively
short-term drought [5,114]. To address the tissue contents of drought-protective metabolites, different
methodological approaches can be employed. On the one hand, each group of metabolites
can be analyzed individually (for example, analysis of betaine [115] and inositol [116] levels).
On the other hand, entire profiles of primary metabolites can be addressed by comprehensive
gas chromatography–mass spectrometry (GC-MS)-based hyphenated techniques, giving access to
relative [117,118] and absolute [119] amounts of individual analytes. For a complete understanding
of plant response to drought, an analysis of plant hormones and secondary metabolites can be
equally essential. In this regard, Ahmed et al. reported upregulation of phenolic metabolites in
the leaves of Gossypium barbadense L. under water deficit conditions [120], and Ma et al. demonstrated a
drought-related increase of the expression levels of flavonoid genes and upregulation of leaf flavonoids
in Triticum aestivum [121].
It is important to mention that accumulation of sugars in the background of reactive oxygen
species (ROS) overproduction (usually accompanying plant response to drought) might result in
enhanced formation of reactive carbonyl compounds (RCCs) and glycation of plant proteins [72,122],
similar to the mechanism recently reported to occur under plant aging [123]. Additional in vitro
experiments with peptide and protein models showed formation of various glycoxidative modifications
of lysyl and arginyl residues [124–127] prospectively, with an impact on pro-inflammatory properties of
glycated proteins [128]. Hence, these modifications might affect nutritional properties of plant-derived
foods. Moreover, the processes of DNA damage and reparation (associated also with the PARP/PARG
system [69]) can impact protein glycation as well [129,130].
Remarkably, metabolic adjustment in different plants has both common and species-specific
features. Thus, some osmoprotective metabolites, like glycine betaine, are specific for certain plant
species, e.g., sugar beet (Beta vulgaris), spinach (Spinacia oleracea), and barley (Hordeum vulgare) [131],
while increased proline content, which is apparently a crucial and the most conserved response to
drought, is characteristic for a wide range of plants [132]. Obviously, such metabolites can be used as
nonspecific and species-specific markers of drought stress. It is important to remember that metabolic
adjustment is efficient only on a relatively short time scale, whereas when drought persists for longer
times, increased accumulation of compatible solutes can be energy- and resource-intensive for the
plant. In cases of severe stress, when soil water content is largely depleted, metabolic adjustment may
have only a small effect on water uptake, or even be detrimental by taking too many resources from
the plants [18,133].
Int. J. Mol. Sci. 2018, 19, 4089 12 of 25

3.4. Protective Proteins as Markers of Drought Stress

Underlying the long-term adaptation of plant organisms to drought is a pronounced increase in the
expression of drought-specific genes, such as Solanum tuberosum DS2 (StDS2) [134], late embryogenesis
abundant (LEA) [135]. Accordingly, biosynthesis of a broad array of drought-protective proteins,
predominantly chaperones, LEA proteins, and enzymes of antioxidant defense (referred to below
in detail), is upregulated. Chaperones form the group of proteins involved in the formation and
maintenance of the native protein structure [136], mostly represented by so-called heat shock proteins:
the ubiquitous polypeptides, originally described with respect to a heat shock response, but actually
involved in an array of stress adaptation responses [137]. Currently, special attention is being paid
to the role of heat shock proteins in drought tolerance [138]. Xiang et al. found that overexpression
of the heat shock protein Osnsp50.2 in rice leaf reduced water loss and increased resistance of
plants to drought-related osmotic stress [139]. It was also shown that increased expression of
chaperone-like proteins ERD10 and ERD14 in A. thaliana cells impacted the prevention of luciferase,
alcohol dehydrogenase, and citrate synthase inactivation in firefly [140].
LEA proteins represent another class of polypeptides involved in adaptation to water deficiency.
These proteins were discovered more than 35 years ago in a study of embryogenesis and germination of
cotton seeds [141]. The key feature of the LEA proteins underlying their drought-protective properties
is their high hydrophilicity [142]. These molecules are known to prevent mechanical damage of
mitochondria, chloroplasts, and other cellular structures by forming a membrane-protecting shield,
thereby preventing peroxidation of membrane lipids [111,143]. The constitutive expression level of
LEA proteins can be considered as a marker of drought resistance. Thus, it was shown that more LEA
genes are overexpressed in drought-resistant Gossypium tomentosum cultivars than drought-sensitive
ones [144].

3.5. Oxidative Stress Associated with Drought

Water deficiency results in a disturbance of the balance between ROS (and RNS) generation
and detoxification, triggering oxidative stress, and upregulation of ROS production under drought
conditions is well documented (comprehensively reviewed by de Carvalho et al. [145]). Due to their
high reactivity, ROS are extremely toxic and can damage proteins, lipids, and nucleic acids [146].
Under persistent oxidative stress, this damage can become irreversible and might lead to cell death.
Indeed, excessive ROS production is a central process in response to infection (killing the intruder cells
or tissues surrounding it).
Although ROS as singlet oxygen can be produced by the energy transfer from triplet chlorophyll
to molecular oxygen [147] (Figure 3A), the main reason underlying overproduction of cellular
ROS in response to plant dehydration is the overload of electron transport chains in chloroplasts
and mitochondria due to overproduction of reduced forms of nucleotides [145,148]. Indeed,
even under normal conditions, light reactions of photosynthesis are associated with continuous ROS
production [149], and PS II is the main contributor to chloroplast photosynthesis [150]. The superoxide
anion radical (O2 − ) is formed on the electron acceptor side of PS II by electron leakage into molecular
oxygen (Figure 3A). Due to the drought-related stomata closure and overload of electron-transport
chains, the rate of this process is essentially increased [151]. The formed O2 − can be dismutated to
hydrogen peroxide (H2 O2 ), which can further yield highly toxic hydroxide radical (OH· ), for example,
by the Fenton reaction in the presence of certain transition metal ions [152]. On the PS II donor side,
incomplete water oxidation also leads to H2 O2 production. Dehydration affects the function of PS II,
resulting in higher production of H2 O2 and faster transformation into OH radical [145,150].
Another important source of ROS in chloroplasts is the Mehler reaction (Figure 3A), i.e., partial
reduction of O2 to O2 − (with subsequent formation of H2 O2 ) by components of the PS I–Fe-S centers
and reduced ferredoxin and thioredoxin. Under stress conditions, reactions of the Calvin cycle are
inhibited by lack of CO2 due to the stomata closure. This situation provokes an overreduction of the
chloroplastic electron transport chain, which results in a higher leakage of electrons to O2 in the Mehler
 monodehydroascorbate is rapidly reduced to ascorbic acid by dehydroascorbate reductase, parallel
to the oxidation of glutathione to glutathione disulfide (GSSG). The subsequent regeneration of
glutathione (GSH) is catalyzed by glutathione reductase, which plays a key role in maintaining the
pool of reduced glutathione required for survival under stress conditions [161,162].
The ratio of reduced to oxidized forms of ascorbate and glutathione is crucial for maintaining a
Int. J. Mol. Sci. 2018, 19, 4089 13 of 25
favorable redox status of living cells, being an informative indicator of plant stress adaptation
capacity [162]. Therefore, addressing the expression or activity of antioxidant enzymes may be
important in screening different plant species and cultivars for drought tolerance. The enzymes of
reaction [145]. Importantly, the deficit of CO2 might result in enhancement of H2 O2 production in
the ascorbate–glutathione cycle were recently considered as targets for the engineering of transgenic
peroxisomes, with photorespiration contributing over 70% of the total H2 O2 production in C3 plants
stress-resistant plants [163].
subjected to drought stress [153].

Figure 3. (A) The main pathways of reactive oxygen species (ROS) generation in plants and (B) the
major pathways of plant enzymatic antioxidant defense. SOD, superoxide dismutase; CAT, catalase;
APx, Figure 3. (A)peroxidase;
ascorbate The main pathways
MDHA, of reactive oxygen species (ROS)
monodehydroascorbate; generationmonodehydroascorbate
MDHAR, in plants and (B) the
major pathways of plant enzymatic antioxidant defense. SOD, superoxide dismutase; CAT, catalase;
reductase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; GSH, reduced glutathione;
APx, ascorbate peroxidase; MDHA, monodehydroascorbate; MDHAR, monodehydroascorbate
GSSG, oxidized glutathione; GR, glutathione reductase.
reductase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; GSH, reduced glutathione;
GSSG, oxidized glutathione; GR, glutathione reductase.
Mitochondria also can represent an important source of stress-related excess ROS. Normally,
approximately 1–2% of the oxygen consumed by plant mitochondria is converted to O2− and H2 O2 .
Underlying this increase in ROS production are complexes I and III of the mitochondrial electron
transport chain, which can act as electron donors for molecular oxygen and enhance the generation
of O2 − and H2 O2 [148]. It is assumed that the excess NADH produced during glycine oxidation
in the photorespiratory pathway results in an overload of the mitochondrial electron transport
chain [154]. Interestingly, the activity of alternative oxidase, and probably rotenone-insensitive
NAD(P)H-dehydrogenase, is involved in detoxification of ROS under these conditions and contribute
to plant drought tolerance [148].
In general, ROS production correlates well with the severity of drought stress [145]. This allows
the use of some compounds associated with oxidative stress as biochemical markers of drought.
ROS readily attack double bonds in polyunsaturated fatty acids, resulting in the formation of
lipid hydroperoxides [155]. Consequently, shorter and reactive carbonyl products result from
their breakdown, such as, e.g., malondialdehyde, known as a reliable marker of lipid oxidative
damage [156,157]. The contents of these compounds increase in plant leaves under stress conditions
and can be used as drought stress markers. Similarly, H2 O2 tissue contents are often used for
estimations of drought stress severity in plants, as this molecule represents the most stable and
easily measurable form of ROS [158].
The mechanisms of plant drought tolerance necessarily include pathways that reduce ROS
content in stressed cells. The most efficient antioxidant defense relies on the activities of specific
antioxidant enzymes (Figure 3B). The enzymes of the ascorbate–glutathione cycle play a central
role in detoxification of H2 O2 under drought stress conditions [5,159]. Ascorbate peroxidase, the key
antioxidant enzyme neutralizing H2 O2 in plant cells, relies on ascorbic acid as a donor of electrons [160].
The resulting dehydroascorbate can be regenerated (i.e., reduced to monodehydroascorbate) by
Int. J. Mol. Sci. 2018, 19, 4089 14 of 25

the reaction with NADPH catalyzed by monodehydroascorbate reductase [161]. The formed toxic
monodehydroascorbate is rapidly reduced to ascorbic acid by dehydroascorbate reductase, parallel
to the oxidation of glutathione to glutathione disulfide (GSSG). The subsequent regeneration of
glutathione (GSH) is catalyzed by glutathione reductase, which plays a key role in maintaining the
pool of reduced glutathione required for survival under stress conditions [161,162].
The ratio of reduced to oxidized forms of ascorbate and glutathione is crucial for maintaining
a favorable redox status of living cells, being an informative indicator of plant stress adaptation
capacity [162]. Therefore, addressing the expression or activity of antioxidant enzymes may be
important in screening different plant species and cultivars for drought tolerance. The enzymes of
the ascorbate–glutathione cycle were recently considered as targets for the engineering of transgenic
stress-resistant plants [163].

4. Conclusions
The comprehensive literature survey clearly demonstrates the importance of an appropriate
experimental design of reversible stress induction under reproducible and long-term stable laboratory
conditions. Currently, PEG-induced drought stress models in particular are state of the art. Stress
characterization methods include a set of standards but also species-specific small-molecule metabolites
and enzymes indicative of the elucidation of drought tolerance mechanisms in plants. In this context,
multiple modifications of the drought model experimental setups allow monitoring different aspects of
plant functional states, in agreement with specific objectives. Currently, the progress of studies focused
on improving plant drought resistance is associated with molecular biology and omics techniques,
in an effort to eventually understand and genetically or chemically influence plant responses to
drought periods.

Table 1. Overview of drought stress model setups.

Species Drought Stress Model Osmotically Active Agent Age of Plant Duration of Stress Reference
Arabidopsis thaliana L. Agar system 50, 300 mmol/L mannitol 7 days 2 weeks [164]
Arabidopsis thaliana L. Agar system 100, 200, 300 mmol/L mannitol 8 days 1 day [165]
Arabidopsis thaliana L. Agar system 17% PEG8000 2 weeks 3 days [100]
Hydroponic system
PEG6000 or 8000
Lemna minor L. (Microtiter plate Adult 24 h [68]
Variable conc.
formate possible)
Every 15 days until
Hordeum vulgare L. Soil system No Adult [166]
physiological maturity
Every 15 days until
Zea mays L. Soil system No Adult [166]
physiological maturity
Zea mays L. Hydroponic system 15% PEG6000 5 weeks 24 h [167]
Populuseuphratica Soil system No 2 months 0, 4, 8 24, 48, 96 h [168]
Sorbitol (0.1, 0.2, 0.3, and 0.4 m) and
Solanum tuberosum L. Agar system 2 weeks 3 weeks [169]
PEG8000 (0%, 4.8%, and 9.6%)
Lolium perenne L. Hydroponic system 10, 20% PEG6000 1 week 4 weeks [170]
Solanum lycopersicum L. Hydroponic system 15% PEG8000 25 days 0, 3, 6, 24, 48 h [171]
Medicago sativa Hydroponic system 15% PEG6000 28 days 24 h [172]
Pistacia lentiscus Soil system 5, 10, 15, 20, 25% PEG6000 1,5 months 20, 23 days [173]
Brachypodiumdistachyon Soil system No Vegetativestage 4, 8, 12 days [174]
Transgenicplum
Soil system No 8 weeks 7, 15 days [175]
“Claudia verde”
Trefoilstage
Stipapurpurea Soil system No (about 3 weeks’ 7, 15 days [176]
growth)
Saccharum spp. Soil system No 2 months 17 days [177]
Hordeumvulgare L. Hydroponic system 20% PEG6000 31 days 9 days [178]
Brassica campestris ssp. Hydroponic system 60, 120%PEG6000 34 days 7 days [179]
Reproductive
Oryzasativa L. Soil system No – [180]
stage
Cucumissativus L. Hydroponic system 2% PEG6000 2 weeks 7 days [181]
Int. J. Mol. Sci. 2018, 19, 4089 15 of 25

Table 2. Markers of drought stress in plants.

Parameter Growth Model Plant Object Method Reference
Physiological Markers
Leaf water Cotton (Gossypium hirsutum
Soil Pressure chamber technique [182]
potential (MPa) L.)
RWC (%) = [(FW − DW)/(SW − DW)] × 100, where FW,
Relative water content Potato (Solanum tuberosum
Soil DW, and SW are fresh, dry, and saturated [183]
(RWC; %) L.)
(turgid)weights of leaf tissues, respectively
Tomato (Lycopersicon Abaxial stomatal conductance measurement with
Stomatal conductance Soil [90]
esculentum Mill.) diffusion porometer (AP4, Delta-T, Cambridge, UK)
Determination of leaf chlorophyll using chlorophyll
meter (SPAD-502, Minolta, Japan); measurement of
Photosynthetic chlorophyll fluorescence with portable fluorescence
parameters (chlorophyll Soil Barley (Hordeum vulgare L.) spectrometer (Handy PEA, Hansatech Instruments, [96]
content and PSII activity) Norfolk, UK)
Fluorescence value Fv/Fm represents maximum
quantum yield of PSII: Fv = Fm−Fo
Biochemical Markers
Clover (Trifolium
Soil ABA analysis in xylem sap by ELISA [184]
Phytohormones subterraneum L.)
Soil Wheat (Triticum aestivum L.) ABA analysis by HPLC [185]
LMW drought stress–responsive metabolites in root and
leaf samples of 7 wild and domesticated wheatrelatives
Metabolites Soil Triticum spp. [186]
revealed by GC-MS based comparative
metabolomicsapproach
Expression pattern analysis of OsHSP50.2, an HSP90
Soil Rice (Oryza sativa L.) [139]
family gene
Protective proteins Cotton (Gossypium
Soil tomentosum, Gossypium LEA gene expression analysis and profiling [144]
hirsutum)
ROS and antioxidant Water culture +
Wheat genotypes – [187]
enzymes PEG6000

PS, photosystem II; ABA, abscisic acid; LMW, low molecular weight; LEA, late embryogenesis abundant.

Author Contributions: N.O., J.S., A.D., and E.T. wrote the draft; O.A.K. and A.K. prepared tables and figures and
contributed to the manuscript; T.G. and T.B. supervised the work of A.D., O.A.K., and J.S.; V.Z. and I.T. contributed
to writing the manuscript and critical reading; A.F. and L.A.W. initiated and supervised the whole work and
wrote the final version of the manuscript.
Funding: This research was funded by the Russian Science Foundation (project number 17-16-01042).
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
2D two-dimensional
ABA abscisic acid
APx ascorbate peroxidase
CAT catalase
DHA dehydroascorbate
DHAR dehydroascorbate reductase
GC-MS gas chromatography–mass spectrometry
GR glutathione reductase
GSH reduced glutathione
GSSG oxidized glutathione
LEA late embryogenesis abundant
LRWC leaf relative water content
MDHA monodehydroascorbate
MDHAR monodehydroascorbate reductase
NADPH nicotinamide adenine dinucleotide phosphate
PAM pulse amplitude modulation
PARP poly (ADP-ribose) polymerase
PEG polyethylene glycol
PS II photosystem II
RCC reactive carbonyl compound
Int. J. Mol. Sci. 2018, 19, 4089 16 of 25

ROS reactive oxygen species

SOD superoxide dismutase
St Solanum tuberosum

References
1. Drought Assessment, Management, and Planning: Theory and Case Studies; Wilhite, D.A. (Ed.) Springer: Boston,
MA, USA, 1993; ISBN 978-1-4613-6416-0.
2. Zhang, X.; Lu, G.; Long, W.; Zou, X.; Li, F.; Nishio, T. Recent progress in drought and salt tolerance studies in
Brassica crops. Breed. Sci. 2014, 64, 60–73. [CrossRef] [PubMed]
3. Shao, H.B.; Chu, L.-Y.; Jaleel, C.A.; Manivannan, P.; Panneerselvam, R.; Shao, M.-A. Understanding water
deficit stress-induced changes in the basic metabolism of higher plants—Biotechnologically and sustainably
improving agriculture and the ecoenvironment in arid regions of the globe. Crit. Rev. Biotechnol. 2009, 29,
131–151. [CrossRef] [PubMed]
4. Basu, S.; Ramegowda, V.; Kumar, A.; Pereira, A. Plant adaptation to drought stress. F1000Research 2016, 5.
[CrossRef] [PubMed]
5. Farooq, M.; Wahid, A.; Kobayashi, N.; Fujita, D.; Basra, S.M.A. Plant drought stress: Effects, mechanisms
and management. Agron. Sustain. Dev. 2009, 29, 185–212. [CrossRef]
6. Jaleel, C.A.; Manivannan, P.; Wahid, A.; Farooq, M.; Al-Juburi, J.; Somasundaram, R.; Panneerselvam, R.
Drought Stress in Plants: A Review on Morphological Characteristics and Pigments Composition. Int. J.
Agric. Biol. 2009, 11, 7.
7. Anjum, S.A.; Xie, X.; Wang, L.; Saleem, M.F.; Man, C.; Lei, W. Morphological, physiological and biochemical
responses of plants to drought stress. Acta Physiol. Plant. 2015, 37. [CrossRef]
8. Trenberth, K.E.; Dai, A.; van der Schrier, G.; Jones, P.D.; Barichivich, J.; Briffa, K.R.; Sheffield, J. Global
warming and changes in drought. Nat. Clim. Chang. 2014, 4, 17–22. [CrossRef]
9. Zhao, T.; Dai, A. The Magnitude and Causes of Global Drought Changes in the Twenty-First Century under
a Low–Moderate Emissions Scenario. J. Clim. 2015, 28, 4490–4512. [CrossRef]
10. Verslues, P.E.; Agarwal, M.; Katiyar-Agarwal, S.; Zhu, J.; Zhu, J.-K. Methods and concepts in quantifying
resistance to drought, salt and freezing, abiotic stresses that affect plant water status. Plant J. 2006, 45,
523–539. [CrossRef] [PubMed]
11. Deikman, J.; Petracek, M.; Heard, J.E. Drought tolerance through biotechnology: Improving translation from
the laboratory to farmers’ fields. Curr. Opin. Biotechnol. 2012, 23, 243–250. [CrossRef] [PubMed]
12. Harb, A.; Krishnan, A.; Ambavaram, M.M.R.; Pereira, A. Molecular and Physiological Analysis of Drought
Stress in Arabidopsis Reveals Early Responses Leading to Acclimation in Plant Growth. Plant Physiol. 2010,
154, 1254–1271. [CrossRef] [PubMed]
13. Sun, W.; Zhao, X.; Ling, Q.; Li, H.; Gao, X. Exotic shrub species (Caragana korshinskii) is more resistant
to extreme natural drought than native species (Artemisia gmelinii) in a semiarid revegetated ecosystem.
Agric. For. Meteorol. 2018, 263, 207–216. [CrossRef]
14. Chirkova, T.V. Physiologicheskiie Osnovy Ustojchivosti Rastenii; Izdatelstvo Sankt-Peterburgskogo Universiteta:
Saint Petersburg, Russia, 2002.
15. Wang, X.; Cai, X.; Xu, C.; Wang, Q.; Dai, S. Drought-Responsive Mechanisms in Plant Leaves Revealed by
Proteomics. Int. J. Mol. Sci. 2016, 17, 1706. [CrossRef] [PubMed]
16. Kar, R.K. Plant responses to water stress: Role of reactive oxygen species. Plant Signal. Behav. 2011, 6,
1741–1745. [CrossRef] [PubMed]
17. Gill, S.S.; Tuteja, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop
plants. Plant Physiol. Biochem. 2010, 48, 909–930. [CrossRef] [PubMed]
18. Boyer, J.S.; Kramer, P.J. Water Relations of Plants and Soils; Academic Press Inc.: Waltham, MA, USA, 1995;
ISBN 978-0-12-425060-4.
19. Haswell, E.S.; Verslues, P.E. The ongoing search for the molecular basis of plant osmosensing.
J. General Physiol. 2015, 145, 389–394. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2018, 19, 4089 17 of 25

20. Paudel, G.; Bilova, T.; Schmidt, R.; Greifenhagen, U.; Berger, R.; Tarakhovskaya, E.; Stöckhardt, S.;
Balcke, G.U.; Humbeck, K.; Brandt, W.; et al. Changes in Arabidopsis thaliana advanced glycated proteome
induced by the polyethylene glycol-related osmotic stress. J. Exp. Bot. 2016, 67, 6283–6295. [CrossRef]
[PubMed]
21. Dos Santos, C.L.; de Borja Reis, A.F.; Mazzafera, P.; Favarin, J.L. Determination of the Water Potential
Threshold at Which Rice Growth Is Impacted. Plants 2018, 7, 48. [CrossRef] [PubMed]
22. Btittelli, M. Measuring Soil Water Potential for Water Management in Agriculture: A Review. Sustainability
2010, 2, 1226–1251. [CrossRef]
23. Kikuta, S.B.; Richter, H. Leaf discs or press saps? A comparison of techniques for the determination of
osmotic potentials in freeze-thawed leaf material. J. Exp. Bot. 1992, 43, 1039–1044. [CrossRef]
24. Voronin, P.Y.; Rakhmankulova, Z.F.; Shuyskaya, E.V.; Maevskaya, S.N.; Nikolaeva, M.K.; Maksimov, A.P.;
Maximov, T.C.; Myasoedov, N.A.; Balnokin, Y.V.; Rymar, V.P.; et al. New method for quantitative
determination of water potential of mesophyll cells’ apoplast in substomatal cavity of the leaf. Russ.
J. Plant Physiol. 2017, 64, 452–456. [CrossRef]
25. Razmkhah, H. Comparing Threshold Level Methods in Development of Stream Flow Drought
Severity-Duration-Frequency Curves. Water Resour. Manag. 2017, 31, 4045–4061. [CrossRef]
26. Grover, A.; Kapoor, A.; Lakshmi, O.S.; Agarwal, S.; Sahi, C.; Katiyar-Agarwal, S.; Agarwal, M.; Dubey, H.
Understanding molecular alphabets of the plant abiotic stress responses. Plant Mol. Biol. 2001, 80, 206–216.
27. Duque, A.S.; de Almeida, A.M.; da Silva, A.B.; da Silva, J.M.; Farinha, A.P.; Santos, D.; Fevereiro, P.;
Araujo, S.S. Abiotic Stress Responses in Plants: Unraveling the Complexity of Genes and Networks to Survive.
In Abiotic Stress—Plant Responses and Applications in Agriculture; InTech: London, UK, 2013; pp. 49–101.
[CrossRef]
28. Zhang, Q. Strategies for developing Green Super Rice. Proc. Natl. Acad. Sci. USA 2007, 104, 16402–16409.
[CrossRef] [PubMed]
29. Carboni, M.; Zeleny, D.; Acosta, A.T.R. Measuring ecological specialization along a natural stress gradient
using a set of complementary niche breadth indices. J. Veg. Sci. 2016, 27, 892–903. [CrossRef]
30. Levitt, J. Responses of Plants to Environmental Stresses; Academic Press: New York, NY, USA, 1972. [CrossRef]
31. Joao, Z.; Ziany, B.; Silva, I.; Josiane, R.; Valdinei, S. Cotton response to water deficits at different growth
stages. Rev. Caatinga 2017, 30, 980–990. [CrossRef]
32. Antunes, C.; Pereira, A.J.; Fernandes, P.; Ramos, M.; Ascensão, L.; Correia, O.; Maguas, C. Understanding
plant drought resistance in a Mediterranean coastal sand dune ecosystem: Differences between native and
exotic invasive species. J. Plant Ecol. 2018, 11, 26–38. [CrossRef]
33. Muller, B.; Pantin, F.; Génard, M.; Turc, O.; Freixes, S.; Piques, M.; Gibon, Y. Water deficits uncouple growth
from photosynthesis, increase C content, and modify the relationships between C and growth in sink organs.
J. Exp. Bot. 2011, 62, 1715–1729. [CrossRef] [PubMed]
34. Li, L.J.; Gu, W.R.; Meng, Y.; Wang, Y.L.; Mu, J.Y.; Li, J.; Wei, S. Physiological and biochemical mechanism
of spermidine improving drought resistance in maize seedlings under drought stress. Ying Yong Sheng Tai
Xue Bao 2018, 29, 554–564. [CrossRef] [PubMed]
35. Frolov, A.; Bilova, T.; Paudel, G.; Berger, R.; Balcke, G.U.; Birkemeyer, C.; Wessjohann, L.A. Early responses
of mature Arabidopsis thaliana plants to reduced water potential in the agar-based polyethylene glycol
infusion drought model. J. Plant Physiol. 2017, 208, 70–83. [CrossRef] [PubMed]
36. Lipiec, J.; Doussan, C.; Nosalewicz, A.; Kondracka, K. Effect of drought and heat stresses on plant growth
and yield: A review. Int. Agrophys. 2013, 27, 463–477. [CrossRef]
37. Bhargava, S.; Sawant, K. Drought stress adaptation: Metabolic adjustment and regulation of gene expression.
Plant Breed. 2013, 132, 21–32. [CrossRef]
38. Tatrai, Z.A.; Sanoubar, R.; Pluhar, Z.; Mancarella, S.; Orsini, F.; Gianquinto, G. Morphological and
Physiological Plant Responses to Drought Stress in Thymus citriodorus. Int. J. Agron. 2016, 10, 1–8.
[CrossRef]
39. Lamaoui, M.; Jemo, M.; Datla, R.; Bekkaoui, F. Heat and drought stresses in crops and approaches for their
mitigation. Front. Chem. 2018, 6, 26. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2018, 19, 4089 18 of 25

40. Todaka, D.; Zhao, Y.; Yoshida, T.; Kudo, M.; Kidokoro, S.; Mizoi, J.; Kodaira, K.-S.; Takebayashi, Y.;
Kojima, M.; Sakakibara, H. Temporal and spatial changes in gene expression, metabolite accumulation and
phytohormone content in rice seedlings grown under drought stress conditions. Plant J. 2017, 90, 61–78.
[CrossRef] [PubMed]
41. Vinocur, B.; Altman, A. Recent advances in engineering plant tolerance to abiotic stress: Achievements and
limitations. Curr. Opin. Biotechnol. 2005, 16, 123–132. [CrossRef] [PubMed]
42. Ford, K.L.; Cassin, A.; Bacic, A. Quantitative proteomic analysis of wheat cultivars with differing drought
stress tolerance. Front. Plant Sci. 2011, 2, 44. [CrossRef] [PubMed]
43. Gernot, B.; Alireza, A.; Kaul, H.-P. Management of crop water under drought: A review. Agron. Sustain. Dev.
2015, 35. [CrossRef]
44. Thompson, A.J.; Thorne, E.T.; Burbidge, A.; Jackson, A.C.; Sharp, R.E.; Taylor, I.B. Complementation of
notabilis, an abscisic acid-deficient mutant of tomato: Importance of sequence context and utility of partial
complementation. Plant Cell Environ. 2004, 27, 459–471. [CrossRef]
45. Vrablova, M.; Vrabl, D.; Hronkova, M.; Kubasek, J.; Santrucek, J. Stomatal function, density and pattern, and
CO2 assimilation in Arabidopsis thaliana tmm1 and sdd1-1 mutants. Plant Biol. 2017, 19, 689–701. [CrossRef]
[PubMed]
46. Seminario, A.; Song, L.; Zulet, A.; Nguyen, H.T.; González, E.M.; Larrainzar, E. Drought stress causes a
reduction in the biosynthesis of ascorbic acid in soybean plants. Front. Plant Sci. 2017, 8, 1042. [CrossRef]
[PubMed]
47. Ahmad, N.; Malagoli, M.; Wirtz, M.; Hell, R. Drought stress in maize causes differential acclimation responses
of glutathione and sulfur metabolism in leaves and roots. BMC Plant Biol. 2016, 16, 247. [CrossRef] [PubMed]
48. Staudinger, C.; Mehmeti-Tershani, V.; Gil-Quintana, E.; Gonzalez, E.M.; Hofhansl, F.; Bachmann, G.;
Wienkoop, S. Evidence for a rhizobia-induced drought stress response strategy in Medicago truncatula.
J. Proteom. 2016, 136, 202–213. [CrossRef] [PubMed]
49. Koyama, R.; Itoh, H.; Kimura, S.; Morioka, A.; Uno, Y. Augmentation of Antioxidant Constituents by Drought
Stress to Roots in Leafy Vegetables. HortTechnology 2012, 22, 121–125. [CrossRef]
50. Ito, Y.; Katsura, K.; Maruyama, K.; Taji, T.; Kobayashi, M.; Seki, M.; Shinozaki, K.; Yamaguchi-Shinozaki, K.
Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene
expression in transgenic rice. Plant Cell Physiol. 2006, 47, 141–153. [CrossRef] [PubMed]
51. Zhou, S.; Duursma, R.A.; Medlyn, B.E.; Kelly, J.W.G.; Prentice, I.C. How should we model plant responses to
drought? An analysis of stomatal and non-stomatal responses to water stress. Agric. Forest Meteorol. 2013,
182, 204–214. [CrossRef]
52. Ji, H.; Liu, L.; Li, K.; Xie, Q.; Wang, Z.; Zhao, X.; Li, X. PEG-mediated osmotic stress induces premature
differentiation of the root apical meristem and outgrowth of lateral roots in wheat. J. Exp. Bot. 2014, 65,
4863–4872. [CrossRef] [PubMed]
53. Hellal, F.A.; El-Shabrawi, H.M.; Abd El-Hady, M.; Khatab, I.A.; El-Sayed, S.A.A.; Abdelly, C. Influence of
PEG induced drought stress on molecular and biochemical constituents and seedling growth of Egyptian
barley cultivars. J. Genet. Eng. Biotechnol. 2018, 16, 203–212. [CrossRef]
54. Amist, N.; Singh, N.B. PEG imposed water deficit and physiological alterations in hydroponic cabbage.
Iran. J. Plant Physiol. 2016, 8, 1653–1658.
55. Hohl, M.; Schopfer, P. Water Relations of Growing Maize Coleoptiles: Comparison between Mannitol and
Polyethylene Glycol 6000 as External Osmotica for Adjusting Turgor Pressure. Plant Physiol. 1991, 95,
716–722. [CrossRef] [PubMed]
56. Munns, R. Comparative physiology of salt and water stress. Plant Cell Environ. 2002, 25, 239–250. [CrossRef]
[PubMed]
57. Verslues, P.E.; Ober, E.S.; Sharp, R.E. Root growth and oxygen relations at low water potentials. Impact
of oxygen availability in polyethylene glycol solutions. Plant Physiol. 1998, 116, 1403–1412. [CrossRef]
[PubMed]
58. Hassan, N.S.; Shaaban, L.D.; Hashem, E.S.A.; Seleem, E.E. In vitro selection for water stress tolerant callus
line of Helianthus annus L. cv. Myak. Int. J. Agric. Biol. 2004, 6, 13–18.
59. Zhong, Y.-P.; Li, Z.; Bai, D.-F.; Qi, X.-J.; Chen, J.-Y.; Wei, C.-G.; Lin, M.-M.; Fang, J.-B. In Vitro Variation of
Drought Tolerance in Five Actinidia Species. J. Am. Soc. Hortic. Sci. 2018, 143, 226–234. [CrossRef]
Int. J. Mol. Sci. 2018, 19, 4089 19 of 25

60. Chutia, J.; Borah, S.P. Water Stress Effects on Leaf Growth and Chlorophyll Content but Not the Grain Yield
in Traditional Rice (Oryza sativa Linn.) Genotypes of Assam, India II. Protein and Proline Status in Seedlings
under PEG Induced Water Stress. Am. J. Plant Sci. 2012, 3, 971. [CrossRef]
61. Meher; Shivakrishna, P.; Reddy, K.A.; Rao, D.M. Effect of PEG-6000 imposed drought sress on RNA content,
relative water content (RWC), and chlorophyll content in peanut leaves and roots. Saudi J. Biol. Sci. 2018, 25,
285–289. [CrossRef] [PubMed]
62. Liu, J.; Yang, H.; Gosling, S.N.; Kummu, M.; Flörke, M.; Pfister, S.; Hanasaki, N.; Wada, Y.; Zhang, X.;
Zheng, C.; et al. Water scarcity assessments in the past, present, and future: REVIEW ON WATER SCARCITY
ASSESSMENT. Earth’s Future 2017, 5, 545–559. [CrossRef] [PubMed]
63. Bressan, R.A.; Hasegawa, P.M.; Handa, A.K. Resistance of cultured higher plant cells to polyethylene
glycol-induced water stress. Plant Sci. Lett. 1981, 21, 23–30. [CrossRef]
64. Rao, S.; Ftz, J. In vitro selection and characterization of polyethylene glycol (PEG) tolerant callus lines and
regeneration of plantlets from the selected callus lines in sugarcane (Saccharum officinarum L.). Physiol. Mol.
Biol. Plants 2013, 19, 261–268. [CrossRef] [PubMed]
65. Senaratna, T.; Touchell, D.; Bunn, E.; Dixon, K. Acetyl salicylic acid (Aspirin) and salicylic acid induce
multiple stress tolerance in bean and tomato plants. Plant Growth Regul. 2000, 30, 157–161. [CrossRef]
66. Vanden Bossche, H.; Marichal, P.; Gorrens, J.; Geerts, H.; Janssen, P.A.J. Mode of action studies: basis for the
search of new antifungal drugs. Ann. N Y Acad. Sci. 1988, 544, 191–207. [CrossRef] [PubMed]
67. Thielert, W. A unique product: The story of the imidacloprid stress shield. Pflanzenschutz Nachrichten-Bayer
2007, 59, 73.
68. Geissler, T.; Wessjohann, L.A. Whole-Plant Microtiter Plate Assay for Drought Stress Tolerance-Inducing
Effects. J. Plant Growth Regul. 2011, 30, 504–511. [CrossRef]
69. Rissel, D.; Heym, P.P.; Thor, K.; Brandt, W.; Wessjohann, L.A.; Peiter, E. No silver bullet-Canonical Poly
(ADP-Ribose) Polymerases (PARPs) are no universal factors of abiotic and biotic stress resistance of
Arabidopsis thaliana. Front. Plant Sci. 2017, 8, 59. [CrossRef] [PubMed]
70. Funar-Timofei, S.; Borota, A.; Crisan, L. Combined molecular docking and QSAR study of fused heterocyclic
herbicide inhibitors of D1 protein in photosystem II of plants. Mol. Divers. 2018, 21, 437–454. [CrossRef]
[PubMed]
71. Sardare, M.D.; Admane, S.V. A review on plant without soil. Int. J. Res. Eng. Technol. 2013, 2, 299–304.
[CrossRef]
72. Bilova, T.; Lukasheva, E.; Brauch, D.; Greifenhagen, U.; Paudel, G.; Tarakhovskaya, E.; Frolova, N.;
Mittasch, J.; Balcke, G.U.; Tissier, A.; et al. A Snapshot of the Plant Glycated Proteome: Structural, Functional,
and Mechanistic Aspects. J. Biol. Chem. 2016, 291, 7621–7636. [CrossRef] [PubMed]
73. Simon, C.; Brad, H.; Maclin, D.; Bo, X.; Asmini, A.; Sam, H.; Lucy, A.; Vanessa, C.; Monique, S.; Sigfredo, F.;
et al. Protocol: Optimising hydroponic growth systems for nutritional and physiological analysis of
Arabidopsis thaliana and other plants. Plant Methods 2013, 9, 4. [CrossRef]
74. Jacomini, E.; Bertani, A.; Mapelli, S. Accumulation of polyethylene glycol 6000 and its effects on water
content and carbohydrate level in water-stressed tomato plants. Can. J. Bot. 1988, 66, 970–973. [CrossRef]
75. Blum, A. Osmotic adjustment is a prime drought stress adaptive engine in support of plant production:
Osmotic adjustment and plant production. Plant Cell Environ. 2017, 40, 4–10. [CrossRef] [PubMed]
76. Van der Weele, C.M.; Spollen, W.G.; Sharp, R.E.; Baskin, T.I. Growth of Arabidopsis thaliana seedlings under
water deficit studied by control of water potential in nutrient-agar media. J. Exp. Bot. 2000, 51, 1555–1562.
[CrossRef] [PubMed]
77. Smith, R.H.; Bhaskaran, S.; Miller, F.R. Screening for drought tolerance in Sorghum using cell culture. In
Vitro Cell Dev. Biol. 1985, 21, 541. [CrossRef]
78. Chen, T.; Fluhr, R. Singlet Oxygen Plays an Essential Role in the Root’s Response to Osmotic Stress. Plant
Physiol. 2018, 177, 1717–1727. [CrossRef] [PubMed]
79. He, Y.; Wu, J.; Lv, B.; Li, J.; Gao, Z.; Xu, W.; Baluška, F.; Shi, W.; Shaw, P.C.; Zhang, J. Involvement of 14-3-3
protein GRF9 in root growth and response under polyethylene glycol-induced water stress. J. Exp. Bot. 2015,
66, 2271–2281. [CrossRef] [PubMed]
80. Yang, C.J.; Zhang, X.K.; Zou, C.S.; Cheng, Y.; Zhen, P.Y.; Li, G.Y. Effects of drought simulated by PEG-6000
on germination and seedling growth of rapeseed (Brassica napus L.). Chin.J.Oil Crop Sci. 1998, 29, 425–430.
[CrossRef]
Int. J. Mol. Sci. 2018, 19, 4089 20 of 25

81. Channaoui, S.; Kahkahi, R.E.; Charafi, J.; Mazouz, H.; Fechtali, M.E.; Nabloussi, A. Germination and Seedling
Growth of a Set of Rapeseed (Brassica napus) Varieties under Drought Stress Conditions. Int. J. Environ.
Agric. Biotechnol. 2017, 2. [CrossRef]
82. Hamayun, M.; Khan, S.A.; Shinwari, Z.K.; Khan, A.L.; Ahmad, N.; Lee, I.-J. Effect of polyethylene glycol
induced drought stress on physio-hormonal attributes of soybean. Abstractsofpapers 2010, 42, 977–986.
83. Tardieu, F. Drought perception by plants do cells of droughted plants experience water stress?
Plant Growth Regul. 1996, 20, 93–104. [CrossRef]
84. Raghavendra, A.S.; Gonugunta, V.K.; Christmann, A.; Grill, E. ABA perception and signalling.
Trends Plant Sci. 2010, 15, 395–401. [CrossRef] [PubMed]
85. Govender, M.; Govender, P.; Weiersbye, I.; Witkowski, E.; Ahmed, F. Review of commonly used remote
sensing and ground-based technologies to measure plant water stress. Water SA 2009, 35. [CrossRef]
86. Donovan, L.; Linton, M.; Richards, J. Predawn plant water potential does not necessarily equilibrate with
soil water potential under well-watered conditions. Oecologia 2001, 129, 328–335. [CrossRef] [PubMed]
87. Soltys-Kalina, D.; Plich, J.; Strzelczyk-Zyta, D.; Sliwka, J.; Marczewski, W. The effect of drought stress on
the leaf relative water content and tuber yield of a half-sib family of ‘Katahdin’-derived potato cultivars.
Breed. Sci. 2016, 66, 328–331. [CrossRef] [PubMed]
88. De Silva, M.A.; Jifon, J.L.; da Silva, J.A.G.; Sharma, V. Use of physiological parameters as fast tools to screen
for drought tolerance in sugarcane. Braz. J. Plant Physiol. 2007, 19, 193–201. [CrossRef]
89. Novak, V.A.; Osmolovskaya, N. Phytomonitoring in plant physiology: Organization, arrangement, and
possibilities. J. Plant Physiol. 1997, 44, 121–128.
90. Sobeih, W.Y. Long-distance signals regulating stomatal conductance and leaf growth in tomato (Lycopersicon
esculentum) plants subjected to partial root-zone drying. J. Exp. Bot. 2004, 55, 2353–2363. [CrossRef]
[PubMed]
91. Damour, G.; Simonneau, T.; Cochard, H.; Urban, L. An overview of models of stomatal conductance at the
leaf level: Models of stomatal conductance. Plant Cell Environ. 2010, 33, 1419–1438. [CrossRef] [PubMed]
92. Burkhardt, J.; Kaiser, H.; Goldbach, H.; Kappen, L. Measurements of electrical leaf surface conductance
reveal re-condensation of transpired water vapour on leaf surfaces. Plant Cell Environ. 1999, 22, 189–196.
[CrossRef]
93. Monteith, J.; Campbell, G.; Potter, E. Theory and performance of a dynamic diffusion porometer. Agric.
Forest Meteorol. 1988, 44, 27–38. [CrossRef]
94. Dbira, S.; Al Hassan, M.; Gramazio, P.; Ferchichi, A.; Vicente, O.; Prohens, J.; Boscaiu, M. Variable Levels
of Tolerance to Water Stress (Drought) and Associated Biochemical Markers in Tunisian Barley Landraces.
Molecules 2018, 23, 613. [CrossRef] [PubMed]
95. Fathi, A.; Tari, D.B. Effect of Drought Stress and its Mechanism in Plants. Int. J. Life Sci. 2016, 10, 1–6.
[CrossRef]
96. Guo, P.; Baum, M.; Grando, S.; Ceccarelli, S.; Bai, G.; Li, R.; von Korff, M.; Varshney, R.K.; Graner, A.;
Valkoun, J. Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes
in response to drought stress during the reproductive stage. J. Exp. Bot. 2009, 60, 3531–3544. [CrossRef]
[PubMed]
97. López-Jurado, J.; Balao, F.; Mateos-Naranjo, E. Deciphering the ecophysiological traits involved during water
stress acclimation and recovery of the threatened wild carnation, Dianthus inoxianus. Plant Physiol. Biochem.
2016, 109, 397–405. [CrossRef] [PubMed]
98. Chen, Y.-E.; Liu, W.-J.; Su, Y.-Q.; Cui, J.-M.; Zhang, Z.-W.; Yuan, M.; Zhang, H.-Y.; Yuan, S. Different response
of photosystem II to short and long-term drought stress in Arabidopsis thaliana. Physiol. Plant. 2016, 158,
225–235. [CrossRef] [PubMed]
99. Klughammer, C.; Schreiber, U. Complementary PS II quantum yields calculated from simple fluorescence
parameters measured by PAM fluorometry and the Saturation Pulse method. PAM Appl. Notes 2008, 1, 27–35.
100. Ruhle, T.; Reiter, B.; Leister, D. Chlorophyll Fluorescence Video Imaging: A Versatile Tool for Identifying
Factors Related to Photosynthesis. Front. Plant Sci. 2018, 9, 55. [CrossRef] [PubMed]
101. Krause, G.H. Photoinhibition of photosynthesis. An evaluation of damaging and protective mechanisms.
Physiol. Plant. 1988, 74, 566–574. [CrossRef]
102. La Rocca, N.; Pupillo, P.; Puppi, G.; Rascio, N. Erythronium dens-canis L. (Liliaceae): An unusual case of
change of leaf mottling. Plant Physiol. Biochem. 2014, 74, 108–117. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2018, 19, 4089 21 of 25

103. Jefferies, R.A. Drought and chlorophyll fluorescence in field-grown potato (Solanum tuberosum). Physiol.
Plant. 1994, 90, 93–97. [CrossRef]
104. Wingler, A.; Marès, M.; Pourtau, N. Spatial Patterns and Metabolic Regulation of Photosynthetic Parameters
during Leaf Senescence. New Phytol. 2004, 161, 781–789. [CrossRef]
105. Kauser, R.; Athar, H.U.R.; Ashraf, M.; Roubina, K.; Habib, A. Chlorophyll fluorescence: A potential indicator
for rapid assessment of water stress tolerance in Canola (Brassica napus L.). Pak. J. Bot. 2006, 38, 1501–1509.
106. Shinozaki, K.; Yamaguchi-Shinozaki, K. Gene networks involved in drought stress response and tolerance.
J. Exp. Bot. 2006, 58, 221–227. [CrossRef] [PubMed]
107. Daszkowska-Golec, A.; Szarejko, I. Open or Close the Gate—Stomata Action under the Control of
Phytohormones in Drought Stress Conditions. Front. Plant Sci. 2013, 4. [CrossRef] [PubMed]
108. Seki, M.; Umezawa, T.; Urano, K.; Shinozaki, K. Regulatory metabolic networks in drought stress responses.
Curr. Opin. Plant Biol. 2007, 10, 296–302. [CrossRef] [PubMed]
109. Wilkinson, S.; Davies, W.J. Drought, ozone, ABA and ethylene: New insights from cell to plant to community.
Plant Cell Environ. 2010, 33, 510–525. [CrossRef] [PubMed]
110. Munemasa, S.; Oda, K.; Watanabe-Sugimoto, M.; Nakamura, Y.; Shimoishi, Y.; Murata, Y.
The coronatine-insensitive 1 Mutation Reveals the Hormonal Signaling Interaction between Abscisic Acid
and Methyl Jasmonate in Arabidopsis Guard Cells. Specific Impairment of Ion Channel Activation and
Second Messenger Production. Plant Physiol. 2007, 143, 1398–1407. [CrossRef] [PubMed]
111. Dalal, M.; Tayal, D.; Chinnusamy, V.; Bansal, K.C. Abiotic stress and ABA-inducible Group 4 LEA from
Brassica napus plays a key role in salt and drought tolerance. J. Biotechnol. 2009, 139, 137–145. [CrossRef]
[PubMed]
112. Desikan, R.; Last, K.; Harrett-Williams, R.; Tagliavia, C.; Harter, K.; Hooley, R.; Hancock, J.T.; Neill, S.J.
Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis.
Plant J. 2006, 47, 907–916. [CrossRef] [PubMed]
113. Fraire-Velazquez, S.; Emmanuel, V. Abiotic Stress in Plants and Metabolic Responses. In Abiotic Stress—Plant
Responses and Applications in Agriculture; InTech: London, UK, 2013; ISBN 978-953-51-1024-8.
114. Krasensky, J.; Jonak, C. Drought, salt, and temperature stress-induced metabolic rearrangements and
regulatory networks. J. Exp. Bot. 2012, 63, 1593–1608. [CrossRef] [PubMed]
115. Valadez-Bustos, M.G.; Aguado-Santacruz, GA.; Tiessen-Favier, A.; Robledo-Paz, A.; Munoz-Orozco, A.;
Rascon-Cruz, Q.; Santacruz-Varela, A. A reliable method for spectrophotometric determination of glycine
betaine in cell suspension and other systems. Anal. Biochem. 2016, 498, 47–52. [CrossRef] [PubMed]
116. Alimohammadi, M.; Lahiani, M.H.; Khodakovskaya, M.V. Genetic reduction of inositol triphosphate
increases tolerance of tomato plants to oxidative stress. Planta 2015, 242, 123–135. [CrossRef] [PubMed]
117. Birkemeyer, C.; Osmolovskaya, N.; Kuchaeva, L.; Tarakhovskaya, E. Distribution of natural ingredients
suggests a complex network of metabolic transport between source and sink tissues in the brown alga Fucus
vesiculosus. Planta 2018. [CrossRef] [PubMed]
118. Tarakhovskaya, E.; Lemesheva, V.; Bilova, T.; Birkemeyer, C. Early Embryogenesis of Brown Alga Fucus
vesiculosus L. is Characterized by Significant Changes in Carbon and Energy Metabolism. Molecules 2017,
22, 1509. [CrossRef] [PubMed]
119. Milkovska-Stamenova, S.; Schmidt, R.; Frolov, A.; Birkemeyer, C. GC-MS Method for the Quantitation of
Carbohydrate Intermediates in Glycation Systems. J. Agric. Food Chem. 2015, 63, 5911–5919. [CrossRef]
[PubMed]
120. Ghasemzadeh, A.; Jaafar, H.Z.; Rahmat, A. Synthesis of phenolics and flavonoids in ginger (Zingiber
officinale Roscoe) and their effects on photosynthesis rate. Int. J. Mol. Sci. 2010, 11, 4539–4555. [CrossRef]
[PubMed]
121. Ma, D.; Sun, D.; Wang, C.; Li, Y.; Guo, T. Expression of flavonoid biosynthesis genes and accumulation of
flavonoid in wheat leaves in response to drought stress. Plant Physiol. Biochem. 2014, 80, 60–66. [CrossRef]
[PubMed]
122. Bechtold, U.; Rabbani, N.; Mullineaux, P.M.; Thornalley, P.J. Quantitative measurement of specific biomarkers
for protein oxidation, nitration and glycation in Arabidopsis leaves. Plant J. 2009, 59, 661–671. [CrossRef]
[PubMed]
Int. J. Mol. Sci. 2018, 19, 4089 22 of 25

123. Bilova, T.; Paudel, G.; Shilyaev, N.; Schmidt, R.; Brauch, D.; Tarakhovskaya, E.; Milrud, S.; Smolikova, G.;
Tissier, A.; Vogt, T.; et al. Global proteomic analysis of advanced glycation end products in the Arabidopsis
proteome provides evidence for age-related glycation hot spots. J. Biol. Chem. 2017, 292, 15758–15776.
[CrossRef] [PubMed]
124. Frolov, A.; Schmidt, R.; Spiller, S.; Greifenhagen, U.; Hoffmann, R. Arginine-derived advanced glycation end
products generated in peptide-glucose mixtures during boiling. J. Agric. Food Chem. 2014, 62, 3626–3635.
[CrossRef] [PubMed]
125. Greifenhagen, U.; Nguyen, V.D.; Moschner, J.; Giannis, A.; Frolov, A.; Hoffmann, R. Sensitive and site-specific
identification of carboxymethylated and carboxyethylated peptides in tryptic digests of proteins and human
plasma. J. Proteome Res. 2015, 14, 768–777. [CrossRef] [PubMed]
126. Greifenhagen, U.; Frolov, A.; Hoffmann, R. Oxidative degradation of N(ε)-fructosylamine-substituted
peptides in heated aqueous systems. Amino Acids 2015, 47, 1065–1076. [CrossRef] [PubMed]
127. Schmidt, R.; Böhme, D.; Singer, D.; Frolov, A. Specific tandem mass spectrometric detection of AGE-modified
arginine residues in peptides. J. Mass Spectrom. 2015, 50, 613–624. [CrossRef] [PubMed]
128. Soboleva, A.; Vikhnina, M.; Grishina, T.; Frolov, A. Probing Protein Glycation by Chromatography and Mass
Spectrometry: Analysis of Glycation Adducts. Int. J. Mol. Sci. 2017, 18, 2557. [CrossRef] [PubMed]
129. Fedorova, M.; Frolov, A.; Hoffmann, R. Fragmentation behavior of Amadori-peptides obtained
by non-enzymatic glycosylation of lysine residues with ADP-ribose in tandem mass spectrometry.
J. Mass Spectrom. 2010, 45, 664–669. [CrossRef] [PubMed]
130. Jacobson, E.L.; Cervantes-Laurean, D.; Jacobson, M.K. Glycation of proteins by ADP-ribose. Mol. Cell. Biochem.
1994, 138, 207–212. [CrossRef] [PubMed]
131. Ashraf, M.; Foolad, M.R. Roles of glycine betaine and proline in improving plant abiotic stress resistance.
Environ. Exp. Bot. 2007, 59, 206–216. [CrossRef]
132. Giri, J. Glycinebetaine and abiotic stress tolerance in plants. Plant Signal Behav 2011, 6, 1746–1751. [CrossRef]
[PubMed]
133. Templer, S.E.; Ammon, A.; Pscheidt, D.; Ciobotea, O.; Schuy, C.; McCollum, C.; Sonnewald, U.; Hanemann, A.;
Förster, J.; Ordon, F.; et al. Metabolite profiling of barley flag leaves under drought and combined heat and
drought stress reveals metabolic QTLs for metabolites associated with antioxidant defense. J. Exp. Bot. 2017,
68, 1697–1713. [CrossRef] [PubMed]
134. Doczi, R.; Csanaki, C.Z.; Banfalvi, Z. Expression and promoter activity of the desiccation-specific Solanum
tuberosum gene, StDS2. Plant Cell Environ. 2002, 25, 1197–1203. [CrossRef]
135. Muvunyi, B.P.; Yan, Q.; Wu, F.; Min, X.; Yan, Z.Z.; Kanzana, G.; Wang, Y.; Zhang, J. Mining Late
Embryogenesis Abundant (LEA) Family Genes in Cleistogenes songorica, a Xerophyte Perennial Desert
Plant. Int. J. Mol. Sci. 2018, 19, 3430. [CrossRef] [PubMed]
136. Park, C.J.; Seo, Y.S. Heat Shock Proteins: A Review of the Molecular Chaperones for Plant Immunity.
Plant Pathol. J. 2015, 31, 323–333. [CrossRef] [PubMed]
137. Wang, W.; Vinocur, B.; Shoseyov, O.; Altman, A. Role of plant heat-shock proteins and molecular chaperones
in the abiotic stress response. Trends Plant Sci. 2004, 9, 244–252. [CrossRef] [PubMed]
138. Yang, Y.; Dong, C.; Yang, S.; Li, X.; Sun, X.; Yang, Y. Physiological and Proteomic Adaptation of the Alpine
Grass Stipa purpurea to a Drought Gradient. PLoS ONE 2015, 10, e0117475. [CrossRef] [PubMed]
139. Xiang, J.; Chen, X.; Hu, W.; Xiang, Y.; Yan, M.; Wang, J. Overexpressing heat-shock protein OsHSP50.2
improves drought tolerance in rice. Plant Cell Rep. 2018, 37, 1585–1595. [CrossRef] [PubMed]
140. Kovacs, D.; Kalmar, E.; Torok, Z.; Tompa, P. Chaperone Activity of ERD10 and ERD14, Two Disordered
Stress-Related Plant Proteins. Plant Physiol. 2008, 147, 381–390. [CrossRef] [PubMed]
141. Dure, L., III; Greenway, S.C.; Galau, G.A. Developmental biochemistry of cottonseed embryogenesis and
germination: Changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein
synthesis. Biochemistry 1981, 20, 4162–4168. [CrossRef] [PubMed]
142. Hatanaka, R.; Hagiwara-Komoda, Y.; Furuki, T.; Kanamori, Y.; Fujita, M.; Cornette, R.; Sakurai, M.; Okuda, T.;
Kikawada, T. An abundant LEA protein in the anhydrobiotic midge, PvLEA4, acts as a molecular shield by
limiting growth of aggregating protein particles. Insect Biochem. Mol. Biol. 2013, 43, 1055–1067. [CrossRef]
[PubMed]
143. Kovacs, D.; Agoston, B.; Tompa, P. Disordered plant LEA proteins as molecular chaperones.
Plant Signal. Behav. 2008, 3, 710–713. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2018, 19, 4089 23 of 25

144. Magwanga, R.O.; Lu, P.; Kirungu, J.N.; Lu, H.; Wang, X.; Cai, X.; Zhou, Z.; Zhang, Z.; Salih, H.; Wang, K.;
et al. Characterization of the late embryogenesis abundant (LEA) proteins family and their role in drought
stress tolerance in upland cotton. BMC Genet. 2018, 19, 1–6. [CrossRef] [PubMed]
145. Cruz de Carvalho, M.H. Drought stress and reactive oxygen species: Production, scavenging and signaling.
Plant Signal. Behav. 2008, 3, 156–165. [CrossRef] [PubMed]
146. Møller, I.M.; Jensen, P.E.; Hansson, A. Oxidative modifications to cellular components in plants. Annu. Rev.
Plant Biol. 2007, 58, 459–481. [CrossRef] [PubMed]
147. Ma, F.; Chen, X.-B.; Sang, M.; Wang, P.; Zhang, J.-P.; Li, L.-B.; Kuang, T.-Y. Singlet oxygen formation and
chlorophyll. Photosynth. Res. 2009, 100, 19–28. [CrossRef] [PubMed]
148. Møller, I.M. Plant mitochondria and oxidative stress: Electron transport, NADPH turnover, and metabolism
of reactive oxygen species. Annu. Rev. Plant Biol. 2001, 52, 561–591. [CrossRef] [PubMed]
149. Foyer, C.H. Reactive oxygen species, oxidative signaling and the regulation of photosynthesis.
Environ. Exp. Bot. 2018, 154, 134–142. [CrossRef] [PubMed]
150. Pospisil, P. Molecular mechanisms of production and scavenging of reactive oxygen species by photosystem
II. Biochim. Biophys. Acta 2012, 1817, 218–231. [CrossRef] [PubMed]
151. Zulfugarov, I.S.; Tovuu, A.; Eu, Y.-J.; Dogsom, B.; Poudyal, R.S.; Nath, K.; Hall, M.; Banerjee, M.; Yoon, U.C.;
Moon, Y.-H.; et al. Production of superoxide from Photosystem II in a rice (Oryza sativa L.) mutant lacking
PsbS. BMC Plant Biol. 2014, 14, 242. [CrossRef] [PubMed]
152. Velasquez, M.; Santander, I.P.; Contreras, D.R.; Yanez, J.; Zaror, C.; Salazar, R.A.; Pérez-Moya, M.;
Mansilla, H.D. Oxidative degradation of sulfathiazole by Fenton and photo-Fenton reactions. J. Environ. Sci.
Health Part A 2014, 49, 661–670. [CrossRef] [PubMed]
153. Noctor, G.; Veljovic-Jovanovic, S.; Driscoll, S.; Novitskaya, L.; Foyer, C.H. Drought and oxidative load in
the leaves of C3 plants: A predominant role for photorespiration? Ann Bot. 2002, 89, 841–850. [CrossRef]
[PubMed]
154. Atkin, O.K.; Macherel, D. The crucial role of plant mitochondria in orchestrating drought tolerance. Ann.
Bot. 2009, 103, 581–597. [CrossRef] [PubMed]
155. Moore, K.; Roberts, L.J. Measurement of Lipid Peroxidation. Free Radic. Res. 1998, 28, 659–671. [CrossRef]
[PubMed]
156. Del Rio, D.; Stewart, A.J.; Pellegrini, N. A review of recent studies on malondialdehyde as toxic molecule and
biological marker of oxidative stress. Nutr. Metab. Cardiovasc. Dis. 2005, 15, 316–328. [CrossRef] [PubMed]
157. Davey, M.W.; Stals, E.; Panis, B.; Keulemans, J.; Swennen, R.L. High-throughput determination of
malondialdehyde in plant tissues. Anal. Biochem. 2005, 347, 201–207. [CrossRef] [PubMed]
158. Rhee, S.G.; Chang, T.-S.; Jeong, W.; Kang, D. Methods for detection and measurement of hydrogen peroxide
inside and outside of cells. Mol. Cells 2010, 29, 539–549. [CrossRef] [PubMed]
159. Foyer, C.H.; Noctor, G. Ascorbate and glutathione: The heart of the redox hub. Plant Physiol. 2011, 155, 2–18.
[CrossRef] [PubMed]
160. Smirnoff, N. BOTANICAL BRIEFING: The Function and Metabolism of Ascorbic Acid in Plants. Ann. Bot.
1996, 78, 661–669. [CrossRef]
161. Noctor, G.; Foyer, C.H. Ascorbate and Glutathione: Keeping Active Oxygen under Control. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 1998, 49, 249–279. [CrossRef] [PubMed]
162. Meyer, A.J. The integration of glutathione homeostasis and redox signaling. J. Plant Physiol. 2008, 165,
1390–1403. [CrossRef] [PubMed]
163. Kang, G.Z.; Li, G.Z.; Liu, G.Q.; Xu, W.; Peng, X.Q.; Wang, C.Y.; Zhu, Y.J.; Guo, T.C. Exogenous salicylic acid
enhances wheat drought tolerance by influence on the expression of genes related to ascorbate-glutathione
cycle. Biol. Plant. 2013, 57, 718–724. [CrossRef]
164. Moon, H.-D.; Lee, M.-S.; Kim, S.-H.; Jeong, W.-J.; Choi, D.-W. Identification of a drought responsive gene
encoding a nuclear protein involved in drought and freezing stress tolerance in Arabidopsis. Biol. Plant 2016,
60, 105–112. [CrossRef]
165. Huang, T.; Jander, G. Abscisic acid-regulated protein degradation causes osmotic stress-induced
accumulation of branched-chain amino acids in Arabidopsis thaliana. Planta 2017, 246, 737–747. [CrossRef]
[PubMed]
166. Soleymani, A. Light response of barley (Hordeum vulgare L.) and corn (Zea mays L.) as affected by drought
stress, plant genotype and N fertilization. Biocatal. Agric. Biotechnol. 2017, 11, 1–8. [CrossRef]
Int. J. Mol. Sci. 2018, 19, 4089 24 of 25

167. Niu, G.-L.; Gou, W.; Han, X.-L.; Qin, C.; Zhang, L.-X.; Abomohra, A.E.-F.; Ashraf, M. Cloning and Functional
Analysis of Phosphoethanolamine Methyltransferase Promoter from Maize (Zea mays L.). Int. J. Mol. Sci.
2018, 19, 191. [CrossRef] [PubMed]
168. Lu, X.; Zhang, X.; Duan, H.; Lian, C.; Liu, C.; Yin, W.; Xia, X. Three stress-responsive NAC transcription factors
from Populus euphratica differentially regulate salt and drought tolerance in transgenic plants. Physiol. Plant.
2017, 162, 73–97. [CrossRef] [PubMed]
169. Bundig, C.; Vu, T.H.; Meise, P.; Seddig, S.; Schum, A.; Winkelmann, T. Variability in Osmotic Stress Tolerance
of Starch Potato Genotypes (Solanum tuberosum L.) as Revealed by an In Vitro Screening: Role of Proline,
Osmotic Adjustment and Drought Response in Pot Trials. J. Agron. Crop Sci. 2016, 203, 206–218. [CrossRef]
170. Bothe, A.; Westermeier, P.; Wosnitza, A.; Willner, E.; Schum, A.; Dehmer, K.J.; Hartmann, S. Drought tolerance
in perennial ryegrass (Lolium perenne L.) as assessed by two contrasting phenotyping systems. J. Agron.
Crop Sci. 2018, 204, 375–389. [CrossRef]
171. Landi, S.; Nurcato, R.; De Lillo, A.; Lentini, M.; Grillo, S.; Esposito, S. Glucose-6-phosphate dehydrogenase
plays a central role in the response of tomato (Solanum lycopersicum) plants to short and long-term drought.
Plant Physiol. Biochem. 2016, 105, 79–89. [CrossRef] [PubMed]
172. Defez, R.; Andreozzi, A.; Dickinson, M.; Charlton, A.; Tadini, L.; Pesaresi, P.; Bianco, C. Improved Drought
Stress Response in Alfalfa Plants Nodulated by an IAA Over-producing Rhizobium Strain. Front. Microbiol.
2017, 8. [CrossRef] [PubMed]
173. Vasques, A.R.; Pinto, G.; Dias, M.C.; Correia, C.M.; Moutinho-Pereira, J.M.; Vallejo, V.R.; Santos, C.; Keizer, J.J.
Physiological response to drought in seedlings of Pistacia lentiscus (mastic tree). New For. 2016, 47, 119–130.
[CrossRef]
174. Tatli, O.; Ozdemir, B.S.; Doganay, G.D. Time-dependent leaf proteome alterations of Brachypodium distachyon
in response to drought stress. Plant Mol. Biol. 2017, 94, 609–623. [CrossRef] [PubMed]
175. Diaz-Vivancos, P.; Faize, L.; Nicolás, E.; Clemente-Moreno, M.J.; Bru-Martinez, R.; Burgos, L.; Hernández, J.A.
Transformation of plum plants with a cytosolic ascorbate peroxidase transgene leads to enhanced water
stress tolerance. Ann. Bot. 2016, 117, 1121–1131. [CrossRef] [PubMed]
176. Li, X.; Yang, Y.; Yang, S.; Sun, X.; Yin, X.; Zhao, Y.; Yang, Y. Comparative proteomics analyses of intraspecific
differences in the response of Stipa purpurea to drought. Plant Divers. 2016, 38, 101–117. [CrossRef]
[PubMed]
177. Rampazzo, P.; Marcos, F.; Cipriano, M.; Marchiori, P.; Freitas, S.; Machado, E.; Nascimento, L.; Brocchi, M.;
Ribeiro, R. Rhizobacteria improve sugarcane growth and photosynthesis under well-watered conditions.
Ann. Appl. Biol. 2018, 172, 309–320. [CrossRef]
178. Tavakol, E.; Jakli, B.; Cakmak, I.; Dittert, K.; Karlovsky, P.; Pfohl, K.; Senbayram, M. Optimized potassium
nutrition improves plant-water-relations of barley under PEG-induced osmotic stress. Plant Soil 2018, 430,
23–35. [CrossRef]
179. Xiong, X.; Chang, L.; Khalid, M.; Zhang, J.; Huang, D. Alleviation of Drought Stress by Nitrogen Application
in Brassica campestris ssp. Chinensis L. Agronomy 2018, 8, 66. [CrossRef]
180. Pang, Y.; Chen, K.; Wang, X.; Xu, J.; Ali, J.; Li, Z. Recurrent selection breeding by dominant male sterility for
multiple abiotic stresses tolerant rice cultivars. Euphytica 2017, 213, 268. [CrossRef]
181. Redox imbalance contributed differently to membrane damage of cucumber leaves under water stress and
Fusarium infection. Plant Sci. 2018, 274, 171–180. [CrossRef] [PubMed]
182. Argyrokastritisa, I.G.; Papastylianoub, P.T.; Alexandrisa, S. Leaf Water Potential and Crop Water Stress Index
variation for full and deficit irrigated cotton in Mediterranean conditions. Agric. Agric. Sci. Procedia 2015, 4,
463–470. [CrossRef]
183. Pieczynski, M.; Marczewski, W.; Hennig, J.; Dolata, J.; Bielewicz, D.; Piontek, P.; Wyrzykowska, A.;
Krusiewicz, D.; Strzelczyk-Zyta, D.; Konopka-Postupolska, D.; et al. Down-regulation of CBP80 gene
expression as a strategy to engineer a drought-tolerant potato. Plant Biotechnol. J. 2013, 11, 459–469. [CrossRef]
[PubMed]
184. Socias, X.; Correia, M.J.; Chaves, M.; Medrano, H. The role of abscisic acid and water relations in drought
responses of subterranean clover. J. Exp. Bot. 1997, 48, 1281–1288. [CrossRef]
Int. J. Mol. Sci. 2018, 19, 4089 25 of 25

185. Bano, A.; Ullah, F.; Nosheen, A. Role of abscisic acid and drought stress on the activitiesof antioxidant
enzymes in wheat. Plant Soil Environ. 2012, 58, 181–185. [CrossRef]
186. Ullah, N.; Yüce, M.; Gökçe, Z.N.Ö.; Budak, H. Comparative metabolite profiling of drought stress in roots
and leaves of seven Triticeae species. BMC Genom. 2017, 18, 969. [CrossRef] [PubMed]
187. Weng, M.; Cui, L.; Liu, F.; Zhang, M.; Shan, L.; Yang, S.; Deng, X. Effects of drought stress on antioxidant
enzymes in seedlings of different wheat genotypes. Pak. J. Bot. 2015, 47, 49–56.

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