2021_Zia et al_drought

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Microbiological Research 242 (2021) 126626
Available online 18 October 2020
0944-5013/© 2020 Elsevier GmbH. All rights reserved.
Plant survival under drought stress: Implications, adaptive responses, and 
integrated rhizosphere management strategy for stress mitigation 
Rabisa Zia a,b, Muhammad Shoib Nawaz a,b, Muhammad Jawad Siddique a,b, Sughra Hakim a,b, 
Asma Imran a,* 
a National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577 Jhang Road, Faisalabad, Pakistan 
b Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan 
A R T I C L E I N F O 
Keywords: 
Climate change 
Drought 
Soil water deficit 
Plant responses 
Yield losses 
Stress management strategies 
PGPR 
Osmoregulation 
A B S T R A C T 
In many regions of the world, the incidence and extent of drought spells are predicted to increase which will 
create considerable pressure on global agricultural yields. Most likely among all the abiotic stresses, drought has 
the strongest effect on soil biota and plants along with complex environmental effects on other ecological sys-
tems. Plants being sessile appears the least resilient where drought creates osmotic stress, limits nutrient mobility 
due to soil heterogeneity, and reduces nutrient access to plant roots. Drought tolerance is a complex quantitative 
trait controlled by many genes and is one of the difficult traits to study and characterize. Nevertheless, existing 
studies on drought have indicated the mechanisms of drought resistance in plants on the morphological, phys-
iological, and molecular basis and strategies have been devised to cope with the drought stress such as mass 
screening, breeding, marker-assisted selection, exogenous application of hormones or osmoprotectants and or 
engineering for drought resistance. These strategies have largely ignored the role of the rhizosphere in the plant’s 
drought response. Studies have shown that soil microbes have a substantial role in modulation of plant response 
towards biotic and abiotic stress including drought. This response is complex and involves alteration in host root 
system architecture through hormones, osmoregulation, signaling through reactive oxygen species (ROS), in-
duction of systemic tolerance (IST), production of large chain extracellular polysaccharides (EPS), and tran-
scriptional regulation of host stress response genes. 
This review focuses on the integrated rhizosphere management strategy for drought stress mitigation in plants 
with a special focus on rhizosphere management. This combinatorial approach may include rhizosphere engi-
neering by addition of drought-tolerant bacteria, nanoparticles, liquid nano clay (LNC), nutrients, organic 
matter, along with plant-modification with next-generation genome editing tool (e.g., CRISPR/Cas9) for quickly 
addressing emerging challenges in agriculture. Furthermore, large volumes of rainwater and wastewater 
generated daily can be smartly recycled and reused for agriculture. Farmers and other stakeholders will get a 
proper knowledge-exchange and an ideal road map to utilize available technologies effectively and to translate 
the measures into successful plant-water stress management. The proposed approach is cost-effective, eco- 
friendly, user-friendly, and will impart long-lasting benefits on agriculture and ecosystem and reduce vulnera-
bility to climate change. 
1. Introduction 
Population growth, climate change, and food shortage are some of 
the threatening current issues for the world community. The rate of 
population growth is higher in tropical countries than in the countries in 
the temperate regions. Drought, salinity, and extreme temperatures are 
the most significant and interrelated (Vinocur and Altman, 2005) factors 
limiting plant growth and global crop productivity (Jaleel et al., 2009), 
especially in tropical countries. Apart from the intricate relationship 
with high temperature and soil salinity, drought is associated with the 
oxidative stress and mechanical hindrance to root penetration in 
water-deficient hard soils (Wilkinson and Davies, 2010). These factors 
collectively affect the chemical composition, morphology, and physio-
logical functioning of the plant ultimately reducing the yield (Brenchley 
* Corresponding author at: Microbial Ecology Lab, Soil and Environmental Biotechnology Division, NIBGE, Pakistan. 
E-mail addresses: asmaaslam2001@gmail.com, asmaaslam2001@yahoo.com (A. Imran). 
Contents lists available at ScienceDirect 
Microbiological Research 
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https://doi.org/10.1016/j.micres.2020.126626 
Received 8 July 2020; Received in revised form 6 October 2020; Accepted 10 October 2020 
mailto:asmaaslam2001@gmail.com
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Microbiological Research 242 (2021) 126626
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et al., 2012). The consequences of drought depend on stress severity and 
the life stage of the plant facing stress. According to a recent report, 50 % 
of the crop losses are the consequence of abiotic stresses in which 10 % is 
attributed to drought, 20 % to heat stress while the remainder is the 
result of other abiotic stresses (Kajla et al., 2015). Annual dry seasons in 
the tropics significantly increase the chances of drought development 
and subsequent bush fires. Periods of heat can significantly worsen the 
drought conditions by hastening the evaporation of water vapor. The 
drought can be moderate –to- short term or very severe and of prolonged 
duration. The severity of the drought is continuously increasing 
throughout the world and will remain so in the future (Bates et al., 2008) 
which will cause agriculture decline in more than half of arable land by 
2050 (Kasim et al., 2013). The world population which will hit to 9 
billion by 2050 will need a continuous supply of food (Gatehouse et al., 
2011), so, it is imperative to find solutions to grow plants with limited 
water in those areas which are water-deprived (Gregersen et al., 2013). 
Drought is an event of prolonged shortages in the water supply, 
whether atmospheric (below-average precipitation), surface water, or 
groundwater. Meteorologists define drought as the prolonged scarcity of 
water with less than average precipitation. Agricultural drought occurs 
when there is an absence of significant rainfall which limits water 
availability for plant growth and development. This lack of water alters 
the physiology of plants (Gaspar et al., 2002) and disrupt the normal 
functioning. Hydrological drought is a condition when the water re-
serves fall below e locally significant threshold. As the drought persists, 
it gradually worsens the surroundings, and impacts on the local popu-
lation increase. Plants display several signals in their response to 
drought. These signaling pathways are broadly classified into hydraulic 
signals and chemical signals. Both can be initiated by a high vapor 
pressure deficit within the shoots (Tardieu and Davies, 1992). Hydraulic 
signals deal with internal changes in water relations that move from one 
plant part to another e.g. roots to leaves. Chemical signals involve hor-
mones and their transport from one plant portion to the other. The 
metabolic and hormonal responses are alike in and different stresses, but 
ion transport mechanisms are different (Munns, 2002). 
2. Implications of drought on plant 
Plants under water stress usually exhibit poor growth, reduced leaf 
water contents, low turgor pressure (Tahi et al., 2007), and low tran-
spiration rate (Özenç, 2008). Prolonged drought impairs many plant 
cellular functions like protein synthesis, nitrogen assimilation, and cell 
membrane activity (Saneokaet al., 2004). Fig. 1 presents an overview of 
various morphological, physiological, and molecular effects of drought 
stress on plants which have been discussed in detail herein. 
2.1. Seed Germination and early vegetative growth 
Germination starts when the seed imbibes water. In water deficit 
conditions, seeds do not imbibe enough water which ultimately de-
creases the rate of germination and reduces overall plant number per 
unit area (Jajarmi, 2009). Drought further causes significant fluctuation 
in gene expression patterns that eventually affect the seedling vigor 
(Zhang et al., 2009). Plant growth is the function of meristematic cell 
division followed by cell elongation. Interruption of water supply to 
meristematic cells severely inhibits cell elongation ultimately reducing 
cell division and plant growth (Nonami, 1998). Overall, drought nega-
tively affects various growth parameters such as plant height, root 
length, shoot and root fresh and dry weight, number of leaves, tillers, 
plant leaf, and root area, but few parameters e.g., root length and shoot 
length usually follow the phenomena of hydrotropism (Anjum et al., 
2011). Low turgor pressure decreases the number of leaves and overall 
leaf area whereas, low water supply reduces the photosynthetic activity 
of plants (Rucker et al., 1995) and this effect becomes prominent for 
plant height, leaf surface area, and biomass when the plant is exposed to 
moderate to severe drought (Hammad and Ali, 2014) because water 
deficit at early vegetative stage inhibits cell expansion causing a 
reduction in leaf area and internode elongation and decreased plant 
height (Waraich and Ahmad, 2010). 
Fig. 1. Effect of drought stress on morphological, biochemical and physiological functioning of the plant. 
R. Zia et al. 
Microbiological Research 242 (2021) 126626
3
2.2. Stomatal conductance and photosynthesis 
Soil water scarcity leads to increased synthesis of plant hormone 
abscisic acid (ABA) in the roots (Thompson et al., 2007) which is 
transported to shoots via xylem vessels (Dodd et al., 2008). When ABA 
arrives at guard cells it leads to stomatal closure through a branched 
signaling cascades (Acharya and Assmann, 2009; Sarwat and Tuteja, 
2017) this whole process is known as ABA long-distance signaling 
(Sauter et al., 2001). ABA-mediated stomatal closure is the foremost 
response of the plant to drought stress. Usually, stomata are closed to 
reduce the transpiration rate, which in turn reduces the gas exchange. 
Both endogenously synthesized and exogenously applied ABA (to the 
soil or given to the plant xylem) could induce stomatal closure (Wil-
kinson and Davies, 2002). Transpiration inhibition activity of stomata is 
observed to be reduced when ABA is removed from the xylem vessel 
(Zhang and Davies, 1991). 
Closure of stomata reduces the stomatal conductance which is an 
adaptive (escape) mechanism to reduce water loss but it ultimately re-
duces photosynthesis. Drought affects the photosynthetic pigments, 
cause an imbalance in the ratio of chlorophyll and carotenoids (Anjum 
et al., 2011) and reduce cell division (Farooq et al., 2008b). 
Drought-induced decrease in photosynthetic activity and growth de-
pends upon the plant type and developmental stages (Chaves et al., 
2009). Severe drought denatures the proteins involved in photosynthesis 
(Anjum et al., 2011; Li et al., 2017) e.g., lipid peroxidation of chloroplast 
membranes, denaturation of chloroplasts proteins, the decline in pig-
ments due to ROS (Marcińska et al., 2013). CO2 assimilation rate de-
creases in plants with increasing water deficit stress (Waraich and 
Ahmad, 2010) and reduces the photosynthetic rate (Ali et al., 2007). 
Besides a significant decrease in chlorophyll contents (Manivannan 
et al., 2007; Guerfel et al., 2009), accessory pigments e.g., carotenoids 
which protect the photosystems from oxidative damage are also reduced 
during the drought (Jaleel et al., 2007; Simkin et al., 2008). 
2.3. Plant water relations 
Leaf relative water content (RWC) and cell membrane stability 
(CMS) are the measures of water deficit stress tolerance in plants. 
Drought stress usually correlates with oxidative and osmotic stresses 
leading to ion imbalance that further leads to drastic changes in cell 
membrane structure and various other cellular functions (Bernardo 
et al., 2019). Reduced RWC and CMS have been reported in plants grown 
under water deficit conditions, where the reduction gradually increases 
with increasing the severity of stress (Hammad and Ali, 2014). Drought 
induces the degradation of membrane lipids leading to the damaged cell 
membranes. Membranes often become more permeable to allow more 
electrolyte leakage from the cell (Petrov et al., 2018). More than 40 % 
reduction of relative water contents have been reported in different 
plants under stress (Nayyar and Gupta, 2006; Slabbert and Krüger, 2014; 
Efeoğlu et al., 2009). 
2.4. Reactive oxygen species (ROS) 
Water deficit in the rhizosphere leads to an imbalance in the utili-
zation of carbon resources, reduced production of adenosine triphos-
phate, and enhanced generation of reactive oxygen species. ROS (O2− , 
H2O2, and OH) are natural by-products produced in a low amounts as a 
result of normal metabolism and cell signaling. ROS production is trig-
gered and dramatically acclimated during stress (Apel and Hirt, 2004) 
which is one of the obvious and earliest responses of plants to stress. 
Extracellular ROS are mainly produced by plasma membrane-localized 
NADPH oxidases, whereas intracellular ROS are produced in multiple 
organelles. The ROS form a sophisticated cellular signaling network, 
with the accumulation of apoplastic ROS an early hallmark of stomatal 
movement. ROS cause multiple damaging effects to the plants known as 
oxidative damage that further acts as a secondary messenger to activate 
defense mechanism in plants. In addition to organelles, the plasma 
membrane together with the apoplast is the main site for ROS generation 
in response to endogenous signals and exogenous environmental stim-
uli. Stress-induced ROS are highly reactive and toxic to proteins, lipids, 
and nucleic acid which ultimately cause cellular damage and death. 
They cause lipid peroxidation, which increases the amount of malon-
dialdehyde (Møller et al., 2007); an indicator of membrane damage in 
the tissues under stress. Acclimation to the stress enables the plant 
antioxidant defense mechanism to quench ROS before they cause dam-
age to the plant facing drought stress (Selote et al., 2004; Selote and 
Khanna-Chopra, 2010). On the other hand, increased production of ROS 
during stress also act as signals for the activation of stress response 
pathways (Baxter et al., 2014). 
2.5. Plant yield 
Multiple factors determine the quantity and quality of crop yield. It is 
difficult to explain how plants integrate all processes associated with 
crop yield, but it is for sure that all the yield determining factors are 
affected by drought stress. A reduction in water availability reduces 
photosynthesis leading to fewer resources available to plants for in-
vestment into reproduction and flowers. Drought reduces flower size 
and number, pollen, and pollen viability (Waser and Price, 2016), the 
amount and quality of nectar affecting overall pollination (Carvell et al., 
2016). Pollination is directly related to the fruit and seed yield. Grain 
yield decreases up to 14 % under moderate while >40 % under severe 
drought (Hammad and Ali, 2014). Stomatal closure in water deficiency 
leads to a reduced gas exchange rate that further results in reduced size 
of both source and sink, assimilate production, translocation, and 
biomass partitioning in plants (Waraich andAhmad, 2010). The number 
of panicles, the number of grains per plant, and grain weight are three 
important parameters that define the yield in cereals. All these are 
directly related to water availability (Akram et al., 2014). Water deficit 
occurring at an early stage caused a yield decline of 27 % of the final 
yield compared to normal conditions (Hunsigi and Krishna, 1998). The 
water deficit at the reproductive stage is even more critical. At the 
reproductive stage, water deficit not only reduces the number of pani-
cles per plant, or the number of grains per panicles but the weight of 
grains. Water deficiency at three weeks after pollination does not reduce 
the panicles/plant, or grains/panicle but reduce the weight of grains. So, 
an increase in water use efficiency (WUE) and sink strength can reduce 
the effects of water deficit (Yoo et al., 2010; Khakwani et al., 2012). 
Drought reduces photosynthetic rate while the photo transpiration 
process is increased simultaneously, so reduced grains weight could be 
the result of impairment of the physiological and biochemical process. 
2.6. Seed quality 
Seed quality and nutritional value (protein, carbohydrates, lipids, 
minerals, and antioxidants) are significantly affected by drought stress. 
The overall effects may vary depending upon the timing, intensity, and 
crop species but the quality of the end product is mostly compromised. 
Protein concentration and amount of antioxidant increases while car-
bohydrates, lipids, feed value, and sensory traits are declines under 
stress (Wang and Frei, 2011). Proteins account for 10–30 % of the wheat 
grains dry weight but further increased under water deficit stress (Triboi 
and Triboi-Blondel, 2002), but grain yield is reduced if water deficiency 
persists throughout the growing season (Ozturk and Aydin, 2004). 
Exposure of stress at the grain filling stage favors the deposition of 
proteins in the grains over carbohydrates translocation (Fernandez-Fi-
gares et al., 2000; Pleijel et al., 1999). Water deficit stress affects the 
amylose and amylopectin ratio and reduces the amount of amylose in 
the seeds (Singh et al., 2008). In contrast, lipid concentration of oilseeds 
is reported to decrease (Flagella et al., 2010; Palese et al., 2010) 
along-with polyunsaturated fatty acids (Taarit et al., 2010) under 
drought. Minerals are the important components of seeds that are 
R. Zia et al. 
Microbiological Research 242 (2021) 126626
4
usually transported through the water to the plants. They perform their 
role in different processes and translocated to the seeds in a definite 
amount at the end. Soil water deficit hinders their translocation that 
ultimately causes impairment of different processes due to the defi-
ciency of essential minerals e.g., K, N, P, Mg, S, Fe, Zn, and Cu (Oktem, 
2008). 
3. Drought-mitigation responses of plant 
Plants display a variety of physiological and biochemical responses 
at cellular and whole-organism levels towards prevailing drought stress, 
thus making it a complex phenomenon. These responses are classified 
into three groups; drought escape, drought avoidance, and drought 
tolerance (Levitt, 1972). Some plants escape drought (DE) by 
completing their life cycle before the actual water deficit takes place, 
ascribed to their fast phenological development. This is endowed by the 
plant’s ability to reserve nutrients in some organs and circulate them for 
the production of yield. Drought avoidance (DA) strategy is to retain 
enough water either through modifications in the root structure 
(thickness, depth, and mass) for efficient water absorption or by 
lowering evapotranspiration by closing stomata, leaf curling, and 
shedding aged leaves. Drought tolerance (DT) maintains the ability to 
grow, flower, and display economic yield under compromised resources 
(water, nutrients, minerals). As in dry environments plants are subjected 
to random droughts, it is generally impossible for plants to escape from 
such adverse conditions. Drought tolerance enables plants to sustain a 
certain level of physiological activity through the regulation and 
fine-tuning of thousands of genes and various metabolic pathways to 
minimize the resulting damage. This involves the maintenance of turgor 
pressure through osmotic adjustment, an increase in elasticity in the 
cells, a decrease in cell size by protoplasmic resistance. 
Drought tolerance is a cost-intensive phenomenon, as a considerable 
quantity of energy is spent to cope with it. Different plants exhibit 
different drought-tolerate mechanisms while plants may combine mul-
tiple adaptation strategies to survive. Few plants accumulate compatible 
solutes called osmoprotectants (e.g., polyols, sulfonium compounds, 
sugars, amino acids, etc.) into the organelles while others show elevated 
ABA level, or antioxidants activity or induce defense mechanisms 
(Chaves et al., 2003; Reddy et al., 2004). All of these pathways ulti-
mately lead to the activation of stress-responsive genes. These drought 
alleviating strategies usually coincide with each other and plants may 
employ combinations for optimal response. Hence the optimized plant 
productivity in response to plant acclimation under drought is a cu-
mulative response of all these strategies. Under challenging field con-
ditions where various biotic and abiotic stresses are working at the same 
time, more complex plant responses come into play so, various 
stress-relieving pathways overlap (Ohashi et al., 2006). In the following 
section, mechanisms of drought tolerance at different levels are 
presented. 
3.1. Osmoprotection and osmotic adjustment 
One of the most common stress tolerance strategies in plants is the 
overproduction of different types of compatible organic solutes called 
"osmolytes”. These osmolytes (e.g., proline, glycine-betaine, mannitol, 
sorbitol, etc.) are usually non-toxic, small non-reactive molecules protect 
plants from stress through different means such as contribution towards 
osmotic adjustment, detoxification of reactive oxygen species, stabili-
zation of membranes, and native structures of enzymes and proteins 
(Munns, 2002). Among the compatible solutes, proline is the most 
important cytosolute. Its synthesis is leaves at low water potential is 
caused by the combination of increased biosynthesis and slow oxidation 
in mitochondria. Glycinebetaine is one of the most extensively studied 
compatible solute in plants. Fig. 2 describes the synthesis of 
glycine-betaine and its role in drought stress alleviation in plants. 
Similarly, trehalose is a non-reducing disaccharide of glucose that 
functions as a compatible solute in the stabilization of biological struc-
tures under abiotic stress. 
Some inorganic ions such as Cl− , Na+, Ca+ and K+ are also accu-
mulated along the osmolytes in the vacuole and cytosol (Yuan et al., 
2014). Silicon increases root endodermal silicification and improve the 
cell water balance (Savant et al., 1999; Xia, 2001). These are also known 
as compatible solutes and known to be accumulated in the cytosol of the 
cell under drought stress to maintain the osmotic balance of the cell 
(Hoekstra et al., 2001). Biological membranes are the first target of 
many abiotic stresses. It is generally accepted that the maintenance of 
integrity and stability of membranes under water stress is a major 
component of drought tolerance in plants. The osmoprotectants also 
protect the membrane from the damaging effect of stress by maintaining 
hydrophilic interaction between the lipids and proteins (Tamura et al., 
2003). They also act as free-radical scavengers that protect DNA from 
ROS-damage. Plants also maintain CMS through osmotic adjustment via 
these solutes which not only work as osmoprotectantsbut also scavenge 
the ROS under osmotic stress (Mahajan and Tuteja, 2005). Osmolytes 
maintain the structure of macromolecules, give stability to various 
proteins, and also act as a pH buffer (Szabados and Savoure, 2010; 
Trovato et al., 2008). 
3.2. Antioxidant defense 
Plants are equipped with antioxidant defense system consisting of 
enzymatic (superoxide dismutase, polyphenol oxidase, peroxidase, 
ascorbate peroxidase, catalase, and glutathione reductase) as well as 
non-enzymatic (β-carotene, ascorbate, α-tocopherol, glutathione and 
carotenoid) antioxidants that work with each other to protect the plant 
from the damaging effects of ROS (Zhu et al., 2009; You and Chan, 
2015). In drought stresses, the role of both components becomes 
important and plant needs a higher concentration of these to mitigate 
the effects caused by drought. Antioxidant enzymes are highly efficient 
Fig. 2. Schematic illustration of glycine-betaine (GB) synthesis from choline 
(synthesized from S- adenosyl methionine); choline is converted to betain 
aldehyde by choline monooxygenases (CMO), which is finally converted to GB 
by betaine aldehyde dehydrogenase enzyme (BADH) and interaction of GB with 
plant hormones (GA: gibberellic acid, CK: cytokinin, IAA: indole acetic acid). 
Redrawn from (Kurepin et al., 2017). 
R. Zia et al. 
Microbiological Research 242 (2021) 126626
5
at scavenging ROS, that’s why most studies of drought tolerance report 
upregulation of these enzymes (Farooq et al., 2008a). Various enzymatic 
antioxidants are involved in the scavenging of ROS in the glutathione 
cycle in cell cytosol and stroma of various organelles (Fazeli et al., 2007) 
and show a correlation with the drought tolerance in the plants (Anjum 
et al., 2011). Fig. 3 summarizes ROS generation in various plant or-
ganelles during abiotic stress and its scavenging by enzymatic pathways. 
Besides traditional antioxidants, pieces of evidence indicate that 
soluble sugars (e.g., disaccharides, raffinose, fructans, etc.) have a dual 
role concerning ROS. Other mechanisms such as leaf movement and 
curling, photosynthetic apparatus rearrangement, may also represent an 
attempt to avoid the over-reduction of ROS by balancing the amount of 
energy absorbed by the plant with the availability of CO2 (Mittler, 
2002). 
3.3. Plant growth regulators 
Plant growth regulators, when applied externally, and phytohor-
mones, when produced internally, are substances that influence the 
physiological processes of plants at very low concentrations. Under 
drought stress, endogenous auxins, gibberellins, and cytokinin usually 
decrease, while that of abscisic acid and ethylene increases. Auxins 
(IAA) induce new root formation by breaking the root apical dominance 
induced by cytokinin thus have an indirect key role. Indole-3-butyric 
acid synthetase is also drought-inducible in Arabidopsis (Ludwig-Mül-
ler and Schuller, 2007). Drought rhizogenesis is an adaptive strategy 
that occurs during drought stress in some plant species and it is known to 
be controlled by gibberellic acid (Vartanian et al., 1994). 
Abscisic acid (ABA) is a ubiquitous growth inhibitor in flowering 
plants and is generally recognized as a stress hormone that regulates 
gene expression and acts as a signal for the initiation of processes 
involved in adaptation to drought and other environmental stresses. It 
alters the relative growth rates of various plant parts such as an increase 
in the root-to-shoot dry weight ratio, inhibition of leaf area develop-
ment, and production of prolific and deeper roots (Sharp et al., 1994). It 
triggers the occurrence of a complex series of events leading to stomatal 
closure, which is an important water-conservation response (Tran et al., 
2007) and controls the rate of transpiration. 
Similarly, the role of ethylene, salicylic acid, and polyamines during 
drought stress is obvious. Ethylene is a growth inhibitory hormone that 
determines the onset of natural senescence and mediates the drought- 
induced senescence. Polyamines are known to have a profound 
influence on plant growth and development. Studies suggested that rice 
has a great capacity to enhance polyamine biosynthesis, particularly 
spermidine and spermine in free form and putrescence in insoluble- 
conjugated form, in leaves earlier in response to drought stress (Yang 
et al., 2007). 
3.4. Plant response at omics levels 
The expression of various genes directly involved in the drought 
tolerance process or those which regulate the expression of genes 
directly involved has been studied at the molecular level (Lang and Buu, 
2008; Pastori et al., 2000; Zhang et al., 2003; Xiao et al., 2009). Gene 
expression may be triggered directly by the stress conditions or result 
from secondary stresses and/or injury responses. Nonetheless, it is well 
established that drought tolerance is a complex phenomenon involving 
the concerted action of many genes e.g., aquaporins, seed proteins, heat 
shock proteins, dehydrins, membrane stabilizing proteins, late 
embryogenic abundant proteins. Although the exact mechanism is yet 
unknown over-expression of most of these genes and their proteins their 
expression is positively correlated with the drought tolerance in plants 
(Taji et al., 2002; Umezawa et al., 2004). Table 1 summarizes various 
genes conferring drought tolerance to different plants. 
Multiple studies indicate the high expression of different genes such 
as high expression of 9-cis-epoxycarotenoid dioxygenase genes in Arabi-
dopsis (Saito et al., 2004), CYP707A3 (Umezawa et al., 2004), tran-
scriptional factors & protein kinases genes (Kulik et al., 2011; Liu et al., 
2015), OsbZIP46 gene (Lindemose et al., 2013) Os-SIPP2C1 coding gene 
(Guo et al., 2013), catalase gene (Ye et al., 2011), AP37 gene (Park et al., 
2006), OsNAC5, OsNAC6, OsNAC9 and OsNAC10 root-specific expres-
sion in rice (Oh et al., 2009; Nakashima et al., 2007; Jeong et al., 2010; 
Redillas et al., 2012; Jeong et al., 2013), NAC gene (Saad et al., 2013), 
lipid transfer proteins gene (Blein et al., 2002) and RAB 21(Trujillo 
et al., 2008) under drought stress. 
Drought stress imposes osmotic and temperature stress which ulti-
mately denatures cellular proteins. Plants under stress increase the 
number of specific proteins such as HSPs and LEA proteins to maintain 
the integrity of other proteins to keep the normal functionality of cells 
(Wang et al., 2004). Membrane stabilizing proteins and LEA increase the 
water binding capacity by creating a protective environment for other 
proteins or structures, referred to as dehydrins. They also play a major 
role in the sequestration of ions that are concentrated during cellular 
dehydration (Gorantla et al., 2007). At the proteomic level, LEA 
Fig. 3. Schematic of ROS generation in various plant organ-
elles during abiotic stress and its scavenging by enzymatic 
pathways. In apoplast, ROS is generated by cell membrane 
bound NADPH oxidase, cell wall peroxidase, leading to cellular 
damage like lipid peroxidation, sugar oxidation Singlet oxygen 
(O2*-) and hydrogen peroxide (H2O2) are synthesized from 
electron transport chain (ETC) in mitochondria, and during 
photosynthesis in chloroplast through superoxide dismutase 
(SOD). In peroxisomes, H2O2 is synthesized during the con-
version of glycolate to glyoxalate via glycolate oxidase enzyme 
(GO) and also through SOD. Scavenging of ROS is carried out 
by different enzymes like ascorbate peroxidase (APX), catalase 
(CAT), peroxiredoxins (PRX), glutathione peroxidase (GPx) 
and dehydroascorbate reductase (DHAR) and also through 
ascorbic acid, glutathione and flavonoids etc. through non- 
enzymatic scavenging. Redrawn from Khedia et al., 2019. 
R. Zia et al.Microbiological Research 242 (2021) 126626
6
Table 1 
Stress responsive genes conferring drought tolerance in plants. 
Gene/name Plant Species Pathway 
involved/ 
activated 
Function Reference 
AHK1/ histidine kinase 1 Arabidopsis 
thaliana 
ABA responsive 
gene 
positive regulator of osmosensing and 
drought tolerance 
(Tran et al., 2007) 
SnRK2/ Sucrose non-fermenting 1-related 
protein kinase 2 
Gossypium 
hirsutum 
ABA responsive 
gene 
imparts cellular adaptation in response to 
dehydration stress. 
(Kulik et al., 2011) 
NCED3/ 9-cis-epoxycarotenoid dioxygenase Arabidopsis 
thaliana 
ABA responsive 
gene 
key enzyme of ABA biosynthesis (Takahashi et al., 2018) 
AtABCG25/ ATP-binding cassette (ABC) 
transporter 
Arabidopsis 
Thaliana 
ABA responsive 
gene 
drought tolerance (Yoo et al., 2010) 
OSCA1/Hyperosmolality-gated calcium- 
permeable channel 1 
Arabidopsis 
thaliana 
ABA responsive 
gene 
membrane protein mediating osmotic stress 
responses 
(Yuan et al., 2014) 
OsNAC5/6/9/10/ O. sativa abiotic stress- 
responsive NAC-type transcription factor5/ 
6/9/10 
Oryza sativa ABA responsive 
genes 
drought avoiance (Nakashima et al., 2007; Jeong et al., 
2010; Redillas et al., 2012; Jeong et al., 
2013) 
SNAC1, stress-responsive NAC transcription 
factor 1 
Oryza sativa ABA responsive 
gene 
drought avoidance and activation of 
transcriptional regulation of various other 
genes 
(Saad et al., 2013) 
TaNAC69/ T. aestivum NAC transcription factor 
69 
Triticum 
aestivum 
ABA responsive 
gene 
drought avoidance and activation of 
transcriptional regulation of various other 
genes 
(Xue et al., 2011) 
ZFP252/ TFIIIA-type zinc finger protein gene Oryza sativa Not identified drought avoidance and activation of 
transcriptional regulation of various other 
genes 
(Xu et al., 2008) 
Zat10/ Zinc-finger transcription factor 10 Arabidopsis 
thaliana 
Not identified drought avoidance and activation of 
transcriptional regulation of various other 
genes 
(Xiao et al., 2009) 
OsMYB2/ O. sativa MYB transcrption factor Oryza sativa Not identified drought avoidance and activation of 
transcriptional regulation of various other 
genes 
(Yang et al., 2012) 
TaPIMP1/ T. aestivum putative integral 
membrane protein 1 
Triticum 
aestivum 
Not identified drought avoidance and activation of 
transcriptional regulation of various other 
genes 
(Zhang et al., 2012) 
StMYB1R-1/ S. tuberosum MYB-related 
transcription factor 1 
Solenum 
tuberosum 
Not identified drought avoidance and activation of 
transcriptional regulation of various other 
genes 
(Shin et al., 2011) 
Fig. 4. An overview (roadmap) of integrated management strategy for improving water stress tolerance in plant under field. 
R. Zia et al. 
Microbiological Research 242 (2021) 126626
7
proteins, mRNA-binding proteins, antioxidants, enzymes for biosyn-
thesis of compatible solutes, and water channel proteins are directly or 
indirectly responsible for plant protection under drought stress (Bray, 
2002). Transcription factors, phosphatases, kinases (Xiong et al., 2002) 
perform their role in the metabolism and signaling process during both 
stressed and normal conditions and responsible for the induction of 
stress-responsive genes (Abe et al., 2003). 
Various chemical signals transduced under drought stress activate an 
array of genes, leading to the synthesis of proteins and metabolites, 
conferring drought tolerance in many plant species. 
4. Management strategies to improve drought tolerance 
Drought stress effects can be managed by the production of the most 
appropriate plant genotype together with the adjustment of agronomic 
practices (soil management, plant density, etc.) and rhizosphere man-
agement. All these strategies are of paramount importance and have 
their pros and cons. Many of these approaches have been discussed in 
detail in different reviews. To get a comprehensive outcome, we have 
suggested a roadmap for overcoming the water shortage problem in 
agriculture. Fig. 4 describes an integrated approach for the management 
of water using a combination of available technologies mentioned and 
discussed below. 
4.1. Rhizosphere enrichment 
4.1.1. Soil organic matter (SOM) 
Soil life plays a major role in many natural processes that determine 
nutrient and water availability for agricultural productivity. The trans-
formation and movement of materials within soil organic matter (SOM) 
pools is a dynamic process influenced by climate, soil type, vegetation, 
and soil organisms. In natural humid and sub-humid ecosystems without 
human disturbance, the living and non-living components are in dy-
namic equilibrium with each other. This equilibrium creates almost 
closed-cycle transfers of nutrients between the soil and the vegetation 
adapted to such site conditions, resulting in almost perfect physical and 
hydric conditions for plant growth, i.e. a cool microclimate, increased 
evapotranspiration, good rooting conditions with good porosity, and 
sufficient soil moisture. This facilitates water infiltration and prevents 
erosion and runoff. In human-managed systems, the soil biological ac-
tivity is influenced by the land use system, plant types, and management 
practices. Temperature is a key factor controlling the rate of decompo-
sition of plant residues. The relatively faster rate of decomposition 
induced by the continuous heat in the tropics and low water implies that 
high equilibrium levels of organic matter are difficult to achieve in 
tropical agro-ecosystems. Hence, large annual rates of organic inputs are 
needed to maintain an adequate labile soil organic matter pool in 
cultivated soils. 
Any application of animal manure, slurry, or the carbon-rich waste 
can improve the SOM. Biochar is one of the strategies to improve the 
nutrient status of soil which improves the water and nutrient retaining 
ability of the soil (Fazal and Bano, 2016). Due to high porosity, biochar 
is a promising candidate as bacterial inoculant carrier, with nutrients 
naturally derived from the biomass, high water, and nutrient retention 
properties, which favor microbial growth. It alleviates drought and os-
motic stress (Thomas et al., 2013), increases growth (Kammann and 
Graber, 2015), and activity of antioxidants (Wang et al., 2014; Atkinson 
et al., 2010; Gaskin et al., 2008). It supports and enhances the microbial 
activity in the soil resulting in improved soil geochemistry (Quilliam 
et al., 2013; Saarnio et al., 2013). 
Compost is another way to augment soil fertility and soil quality as it 
increases SOM, particularly in sandy areas where water and nutrient 
holding capacity of the soil is low (Lakhdar et al., 2009). In this way 
composts also improve the soil structure (Tejada et al., 2009) porosity 
and hydraulic conductivity (Aggelides and Londra, 2000) water holding 
capacity, and aggregation (Curtis and Claassen, 2005; Sodhi et al., 
2009). The addition of nutrients from compost enhances the fertility of 
the soil but the effect of compost available water usually depends upon 
the type of soil, type of compost, and frequency of treatment with 
compost (Weber et al., 2007). Compost from poultry, food waste, or 
dairy waste improves soil water contents after application (Zebarth 
et al., 1999; Johnson et al., 2009). 
4.1.2. Phyto-hormones 
The drought stress tolerance mechanism is an intricate process in 
plants. The plant produces multiple hormones under the normal or 
stressed conditions to perform an important role in the signaling process 
and coordinate various processes in the plants specifically involved in 
water deficit stress (Zhou et al., 2013). Salicylic acid (SA), a phenolic 
compound, multifunctional in plant growth, and a mediator ofsystemic 
acquired resistance (SAR) during pathogen invasion. Recently, SA has 
been reported to be a major player in plant abiotic and biotic stress 
responses and symbiotic relationships (Pandey and Chakraborty, 2015). 
SA confers drought tolerance in plants (Hussain et al., 2008) by 
increasing the rate of photosynthesis, membrane stability, leaf area, 
relative water content, and activity of various enzymes (Hayat et al., 
2007). SA pretreatment is related to higher proline content under 
drought stress as a consequence of the up-regulation of 
pyrroline-5-carboxylate synthase (P5CSA and P5CSB) which is involved 
in the synthesis of proline and down-regulation of proline dehydroge-
nase gene (PDH) which promotes cell death. These results signify the 
role of SA under drought stress (Lee et al., 2019). H. vulgare treated with 
SA (500 μM) under drought stress resulted in enhanced CO2 assimila-
tion, resulting in elevated stomatal conductance and ultimately 
enhanced plant dry weight (Habibi, 2012). SA applied to plants expe-
riencing drought stress can regulate enzymatic and non-enzymatic en-
tities of the ascorbic acid-glutathione pathway and glyoxalase system 
thereby reducing oxidative stress in plants (Alam et al., 2013). SA 
application to leaves significantly augmented the antioxidant defense 
system is Z. mays as compared to controls (Saruhan et al., 2012). SA (0.5 
mM) application to wheat reduced membrane lipid peroxidation and 
leaf wilting with an increase in plant height and dry weight (Kang et al., 
2014a). It has been observed that SA levels are not related to 
SA-biosynthetic enzymes like isochorismate synthase but with 
ortho-hydroxy-cinnamic acid, which is crucial for inducing drought 
tolerance in O. sativa (Pál et al., 2014). 
Jasmonic acid (JA) plays its role in plant developmental processes as 
well as tolerance to drought stress (Cheong and Do Choi, 2003) by 
increasing the activity of antioxidants (Bao et al., 2009). Closing of 
stomata to reduce water loss is the typical phenomenon exhibited by 
plants (Acharya and Assmann, 2009) controlled by JA and its precursor 
12-OPDA (Savchenko et al., 2014). The synthesis of 12-OPDA is gov-
erned by 13-Lipoxygenase LOX6, which plays a remarkable role in 
alleviating drought stress in plants by closing stomata without being 
affected by ABA. 
Cytokinin produced as a result of drought stress, prevents leaves 
senescence (Peleg and Blumwald, 2011) but its biosynthesis is limited 
under drought stress (Pospí̌silová et al., 2000). Ethylene induces sto-
matal closure in plants (Desikan et al., 2006), while it also increases the 
expression of SodERF3 gene responsible for drought tolerance in plants 
(Trujillo et al., 2008). 
4.1.3. Nanoparticles and liquid nano clay (LNC) 
Nanoparticles (NPs) are highly reactive molecules with nanometer- 
size possessing high surface area, customized pore size, and the ability 
to target specific organelles of the plant cell (Siddiqui et al., 2015). NPs 
are involved in drought tolerance by increasing the activities of anti-
oxidant. However, the use of nanotechnology remains untested for 
implementation into sustainable agriculture (Saxena et al., 2016). 
The application of nanoparticles increased germination and seedling 
growth, physiological activities including photosynthesis and nitrogen 
metabolism, leaf activities of CAT, POX and APX, chlorophyll contents, 
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Microbiological Research 242 (2021) 126626
8
protein, carbohydrate contents, and yield, and also positive changes in 
gene expression indicating their potential use in crop improvement. 
NPs-mediated stress alleviation mechanism includes up-regulation of 
antioxidants, co-precipitation, and immobilization of toxic metals, 
improved nutrient uptake, and partitioning of the nutrient ions. Nano-
particles enhance the water stress tolerance via enhancing root hy-
draulic conductance and water uptake in plants, increasing relative 
water content, membrane stability, and showing a differential abun-
dance of proteins involved in oxidation-reduction, ROS detoxification, 
stress signaling, hormonal pathways, pigment contents, and osmolytes 
(Ashkavand et al., 2015). The mobility of the nanoparticles is very high, 
which leads to rapid transport of the nutrient to all parts of the plant. 
Usually, metal oxides were used in different studies such as nano-
particles of TiO2, increasing the activities of SOD (Laware and Raskar, 
2014; Pei et al., 2010), ZnO nanoparticles improved the growth of 
seedlings and nutrient use efficiency (NUE) of plants under drought 
(Sedghi et al., 2013). The application of silicon nanoparticles was also 
reported to mitigate the water-deficit stress in wheat (Baybordi, 2005). 
In a recent report, Fe-NPs were applied to wheat under drought and 
cadmium stress which enhanced the photosynthesis rate and decreased 
oxidative stress (Adrees et al., 2020). In another study, only ZnO-NPs 
were applied to sorghum and as a result, enhanced nutrient acquisi-
tion during drought stress was observed (Dimkpa et al., 2019). Chitosan 
is a naturally existing non-toxic, biodegradable and eco-friendly poly-
mer derived from the N-deacetylation of chitin. Chitosan nanoparticles 
have been applied to mitigate drought stress as foliar and soil applica-
tion (90 ppm) in wheat (Behboudi et al., 2019) with significant results. 
Liquid nano clay (LNC) is a highly advanced soil recovery technol-
ogy. A Norwegian scientist Kristian Morten Olesen has patented a pro-
cess to mix nano-particles of clay with water and bind them to sand 
particles to condition desert soil. He has been working on LNC since 
2005. The LNC treatment gives sand particles a nanostructured clay 
coating. The treatment completely changes the physical qualities of the 
sand particles, allowing them to bind water. This changes sandy soil of 
poor quality into a high yield soil of good quality. Appropriate fertilizers 
must be applied. LNC application saves ½-2/3 of the crop water 
requirement along with a significant yield increase on anything planted 
in soil. LNC technology is turning desert to green land, lowers the sur-
face temperature around 15 ◦C, and reduces CO2 emissions by 15− 17 
tons per hectare. This is a game-changer technology and will turn deserts 
into fertile lands once it becomes cheaper for the farmers. 
4.1.4. Balanced nutrition 
When the supply of nutrients in the soil is ample, crops are more 
likely to grow well. The efficiency of fertilizer use is high when SOM is 
restored. Fertilizers can help maintain the revolving fund of nutrients in 
the soil by increasing crop yield. Balanced fertilization leads to building 
up soil health, while imbalanced fertilization leads to soil mining and its 
sickness. It is well documented that the unbalanced availability of nu-
trients not only produces low and poor-quality yield but can also lead to 
mining of soil nutrient reserves which results in short supply. Applica-
tion of P-fertilizer show a significant positive effect on root biomass, 
improved water-extracting capacity from the soil, increased leaf relative 
water content, net photosynthetic rate, and maximal quantum efficiency 
of PSII under drought stress conditions (Tariq et al., 2018). 
Nitrogen supply might affect plant drought tolerance through regu-
lation of root water uptake. It has also been demonstrated that ammo-
nium nutrition enhanced drought tolerance in rice when compared with 
nitrate nutrition (Guo et al., 2007), which is associated with the regu-
lation of aquaporin expression. The N form and the levels of N available 
affect root water uptake where high nitrate significantly increased the 
nitrate uptake rate, as well as root water uptake rate. Evidence showed 
that the effect of nitrateon growth inhibition under drought stress was 
associated with pH based ABA redistribution (Ding et al., 2018). Zhao 
et al., 2020 show that the responses of nutrient resorption and allocation 
to N enrichment and drought are highly species-specific. 
Several inorganic nutrient ions have been known to work as stress 
relievers such as Zn, N, P, K (Aown et al., 2012; Gevrek and Atasoy, 
2012) and Se (Nawaz et al., 2013). Priming of plant seeds with silicon 
protected the plant from the lethal consequences of drought stress (Ali 
et al., 2013) by improving antioxidant scavenging potential and 
conferring stability to the membranes (Gong et al., 2005; Pei et al., 
2010). Silicon has a well-documented role in drought stress which works 
by improving leaf surface area, leaf water status, and ultimately 
improved plant biomass (Gong et al., 2003). Water deficit stress causes a 
deficiency of potassium ions in the plants (Cakmak, 2005). Foliar 
application of potassium on wheat under drought stress at various stages 
particularly at the reproductive stage improves uptake of N, P, K, and Ca 
but decreases the sodium (Na) uptake (Raza et al., 2013). Application of 
boron (B) also improves its capacity to tolerate stress and enhanced 
growth parameters (Abdel-Motagally and El-Zohri, 2018). 
The role of Zn in stress amelioration has also been reported (Cakmak, 
2000) where the application of 23 Kg/ha foliar spray of Zn enhanced 
yield up to 16 % (Bagci et al., 2007) under drought. Soil application of 
NPK along with the foliar application of zinc (Zn), boron (B), and 
manganese (Mn) increase grain yield as well as micronutrient concen-
tration of the grain under drought. The rate of photosynthesis, pollen 
viability, the number of fertile spikes, the number of grains per spike, 
and water use efficiency, are increased by late foliar application of these 
micronutrients. This indicates that by increasing foliar application of Zn, 
B, and Mn at booting to anthesis can reduce the harmful effects of the 
drought that often occur during the late stages of cereal production 
(Karim and Rahman, 2015). 
4.2. Effective water management 
In the current situation, the world is facing various issues related to 
water like water scarcity, water pollution, etc. which render the use of 
water bodies unfit for agriculture. Water governance is receiving special 
attention for its role in formulating and implementing solutions to the 
world’s critical water challenges. The scenario is even worse in devel-
oping countries where the combined effect of anthropogenic activities, 
growing demand for resources, and population explosion. So it is 
imperative to use alternate water for agriculture rather than depending 
upon already depleting the freshwater resources. 
4.2.1. Rainwater harvesting (RWH) 
Drylands have low crop yields because not only rainfall is irregular or 
insufficient but also because the significant proportion of rainfall (up to 
40 %) may disappear as instantaneous runoff. To minimize the impact of 
drought, the soil needs to capture the rainwater that falls on it, store as 
much of that for later use. The capacity of soil to retain and release water 
depends on a broad range of factors such as soil texture, soil depth, ar-
chitecture, organic matter, and biological activity. Practices that main-
tain soil moisture contents can be categorized into three groups. i) those 
that increase water infiltration, ii) those that manage soil evaporation, 
iii) those that increase soil moisture storage capacity. All these factors 
are directly related to SOM which influences the physical conditions of 
the soil in several ways. SOM indirectly contributes to the soil porosity 
and directly to the stability of soil aggregate through the bonding or 
adhesion properties of the organic matter. OM mixed with the mineral 
soil material has a considerable effect on increasing the soil moisture- 
holding capacity. 
Rainwater harvesting has two components i.e., collection of rain-
water for surface storage and recharge to groundwater aquifers. 
Managed aquifer recharge (MAR) is a process by which the groundwater 
reservoir is augmented at a rate exceeding the rate of natural recharge. 
Any man-made structure that facilitates augmentation of groundwater is 
MAR or an Artificial Recharge system. It raises groundwater levels, 
improves the availability of water in wells/ tube-wells during the lean 
period, improves the quality of existing groundwater through dilution, 
saves energy in the lifting of groundwater- one-meter rise in water level 
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Microbiological Research 242 (2021) 126626
9
saves about 0.40 kW h of electricity, improves vegetation cover, reduces 
soil erosion due to reduced runoff and improves health/living conditions 
in rural areas. Rainwater harvesting has been made mandatory in 11 
Indian states for recharging the groundwater reserves. 
4.2.2. Wastewater treatment (WWT) 
Another approach to fetch water for agriculture is to use recycled 
sewerage (waste) water. Since more than half of the world’s water re-
sources are polluted. Treatment of such water provides an alternative for 
agricultural use and frees up freshwater resources for human and animal 
use. Moreover, treated water is usually rich in nutrients like N, P, and K 
as well as the inorganic matter which is necessary for soil fertility and 
plant growth (Ricart and Rico, 2019). Water can be cleaned by various 
techniques including membrane filtration, activated sludge method, and 
bioreactors. However, these processes are expensive, not suitable for 
largescale application and the quality of treated water does not match 
with wastewater treatment standards (Suhani et al., 2020). 
The Israeli government has developed a comprehensive precise and 
unique policy regarding integrated water resource management 
(IWRM). Their water management is highly centralized which works on 
the "close market principle", together with the construction of desali-
nation plants along the Mediterranean coast. The wastewater manage-
ment is an integral part of IWRM as wastewater (effluents) are 
considered as a legitimate water resource. As a result of IWRM, Israel 
holds a world record of 85 % reclaimed effluents reuse in agriculture and 
is a world leader in the development and the production of efficient 
water-saving irrigation systems. Out of a total of ~ 510 million cubic 
meters (MCM) of sewage produced in Israel yearly, 97 % of the sewage is 
collected and about 85 % of it is reused (Golan, 2016). The water 
recycling strategy of Israel signifies the need for strong legislation and 
administration of a country in the context of water conservation and 
reuse. 
4.3. Plant modification using genome editing tools 
Amongst the available genome-editing platforms, the clustered 
regularly interspaced short palindromic repeat-Cas (CRISPR/Cas) sys-
tem has emerged as a revolutionary tool for its simplicity, adaptability, 
flexibility, and wide applicability (Shi et al., 2017). The CRISPR-Cas9 
system works as an endonuclease which induces double-stranded breaks 
at a specific site in the genome and such sites are repaired by 
non-homologous end-joining method (NHEJ) (Wang et al., 2017). The 
system has been tested in a number of crops like maize, soybean, rice, 
and wheat (Zhou et al., 2014). 
Owing to the complex nature of drought stress, the usage of genome 
editing towards drought tolerance has been demonstrated only recently. 
Natural tolerance to drought could be demonstrated by genome-editing 
approaches to target drought-sensitive (S) or negatively regulating genes 
that control abiotic stresses (Osakabe et al., 2016a, 2016b). As the first 
proof of concept study, plasma membrane proton (H+) ATPases 
CRISPR/Cas9 systemwas used to introduce novel alleles in the gene 
encoding OPEN STOMATA 2 (OST2), a prominent plasma membrane H 
+ ATPase responsible for stomatal response in Arabidopsis (Osakabe 
et al., 2016a, 2016b). Ethylene resistance has been induced in maize by 
altering the expression of the ARGOS8 gene, improving grain yield 
under drought conditions (Barnawal et al., 2017). A transcription factor 
PtoMYB170 mutated by CRISPR/Cas9, conferred drought tolerance in 
Arabidopsis (Li et al., 2017). Along with this, zinc-finger nucleases (ZFN), 
transcription activator-like effector nucleases (TALEN) have been 
employed for targeted gene modifications in plants (Xu et al., 2008; Xue 
et al., 2011). 
4.4. Rhizosphere engineering with plant growth-promoting rhizobacteria 
(PGPR) 
Plant Growth Promoting Rhizobacteria (PGPR) are an integral part of 
rhizospheric soil or plants roots where they live and perform various 
growth-promoting activities such as solubilization of unavailable P 
(Saharan and Nehra, 2011), Zn, Ca, and K, production of organic acids, 
atmospheric nitrogen fixation, production of various phytohormones, 
production of 1-aminocyclopropane-1-carboxylate deaminase, chelation 
of iron and other metals, quorum sensing, biocontrol activity against 
phytopathogens, insects and induce systematic resistance against biotic 
and abiotic pathogens (Aslam, 2011). These bacteria have the root 
colonizing and biofilm-forming ability which enables them to attach 
onto the root and directly influence the plant growth (Bhattacharyya 
and Jha, 2012). 
PGPR potentially offer better assistance to plants growing under 
water deficit conditions and modulate plant systems to cope with stress 
conditions (de Vries et al., 2020). Plants shape and select their microbial 
communities (Berendsen et al., 2012) and selection is stronger around 
the roots predisposed to various roots processes which is the major 
residential site for the microflora. Studies so far suggest that root exu-
dates are the ultimate designers of the rhizospheric microbiome. Plug 
and play with root exudates could alter the microflora according to plant 
requirements, it can provide a key to a new research arena for main-
taining global food security (Williams and Vries, 2020). 
Studies have shown that PGPR adapted to desert or native to stressed 
areas help plant fight and survive various stresses more efficiently 
(Sandhya et al., 2010; Lim and Kim, 2013) and induce drought stress 
tolerance by improving the morphological and physiological features of 
plants (Nawaz, 2019; Zia, 2018; Jawad, 2020). Inoculation with Bacillus 
subtilis alleviated drought stress in maize and improved several 
morphological and physiological characteristics (Belimov et al., 2009). 
Phyllobacterium rassicacearum strain was also reported to alleviate 
drought and improve the water status of oilseed rape (Bresson et al., 
2014). Bacillus cereus, Bacillus subtilis, and Serratia sp. were also reported 
to induce drought tolerance in cucumber (Wang et al., 2012b). Fig. 5 
elaborates numerous effects and pathways of PGPR mediated growth 
promotion and alleviation of drought stress in plants Fig. 5. 
4.4.1. PGPR-mediated amelioration of drought stress 
Several mechanisms have been reported on the PGPR-mediated 
stress tolerance in plants which include the production of phytohor-
mones, stimulation of antioxidant enzymes, accumulation of osmopro-
tectants, and inorganic ions, and the most important the activity of ACC- 
deaminase (Barnawal et al., 2017; Chakraborty et al., 2013). The 
increment in the synthesis of ethylene from its immediate precursor, 
ACC, secreted by plants as root exudates, has been found in almost all 
plants growing under stress conditions. Ethylene is a plant hormone 
involved in the regulation of various physiological processes of plants, 
but the stress-induced ethylene production in plants inflicts a significant 
reduction in plant growth and development and if not monitored 
properly could result in plant death (Iqbal et al., 2017). The major 
mechanisms utilized by PGP bacteria to reduce the stress includes 
lowering the level of ethylene via hydrolyzing 1-aminocyclopropane-1--
carboxylic acid (ACC) by the enzyme ACC deaminase. ACC is the im-
mediate precursor of the hormone ethylene in plants. It is widely 
reported that certain PGPR possess ACC deaminase enzyme that can 
degrade ACC to ammonia and α-ketobutyrate, thus reducing the level of 
ethylene inside the plants (Shakir et al., 2012; Raghuwanshi and Prasad, 
2018). Apart from ACC-deaminase activity, PGPR-mediated drought 
tolerance is a collective response of many bacterial traits as detailed 
below. 
4.4.2. Improvement in morphological characteristics 
Azospirillum brasilense inoculation to the wheat under water stress 
showed an improved number of coleoptiles, fresh weight, and leaf water 
status (Alvarez et al., 1996). Azospirillum inoculation alleviated drought 
stress in maize, Arabidopsis and wheat, through the production of 
abscisic acid to regulate the signaling of the root cells (Dodd et al., 2010) 
and was positively correlated with the stress tolerance because 
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Microbiological Research 242 (2021) 126626
10
inoculation improved plant biomass, the number of grains and yield 
compared to the non-inoculated plants (Dodd et al., 2010). Rhizobium 
leguminosarum, R. phaseoli (Hussain et al., 2014), B. thuringiensis, Pseu-
domonas sp., and Azotobacter chrocoocum (El-Afry, 2012) have been re-
ported to protect the wheat from the devastating effects of the stress and 
improved fresh and dry mass, water relations, and anatomical features 
of wheat. Recently, a new bioprospecting pipeline has been employed to 
screen PGPR capable of alleviating drought stress in wheat and maize 
seedlings. Seedling vigor was significantly improved in both maize and 
wheat seedlings treated with the PGPR with a drought stress period of 7 
days. The ability of the strains to mitigate drought stress advocates their 
potential use to help plants cope with water deficit (Jochum et al., 
2019). 
As discussed earlier, the exogenous application of plant hormones/ 
regulators has a significant role in plant drought tolerance. Likewise, the 
inoculation of phytohormone-producing PGPR have a significant impact 
on plant tolerance towards drought stress. For instance, the role of IAA- 
producing bacteria for root length elongation and proliferation is well- 
established (Hanif et al., 2020). Bacteria mediated changes in the elas-
ticity of the root cell membranes is one of the first steps towards 
enhanced tolerance to water deficiency. Longer roots help the plant to 
explore more soil to get nutrients and water from the surrounding 
environment. PGPB with both IAA- and ACC-deaminase-producing ac-
tivity will control the excess ethylene production level and thereby 
lessen the ethylene feedback inhibition of IAA biosynthesis. This is 
because a large portion of the additional ACC produced due to a cu-
mulative effect of plant and bacterial IAA is cleaved by bacterial ACC 
deaminase. Therefore, the overall result of this cross-talk well defines 
the role of IAA to enhance plant growth promotion under stressful 
conditions in the presence of ACC deaminase activity (Saikia et al., 
2018). 
4.4.2.1. Improvement of physiological characteristics. There are multiple 
roles played by the PGPR which differ among the bacterial species 
depending upon the part of the plant from where these bacteria are 
isolated and the type of strain. Induction of stress tolerance (IST) is one 
of the important roles played by the PGPR under different stress 
conditions. Although, the exact mechanism of IST remains unknown due 
to the complex and diverse nature of interactions between plants,mi-
crobes, and the environment. PGPR-induced drought tolerance impacts 
on the physiological parameters of plants under stress (Garcia-Gutierrez 
et al., 2013; Khan et al., 2017; Verma et al., 2016). PGPR-inoculation 
improves water content in drought-stressed wheat by widening xylem 
vessels and enhancing hydraulic conductance of the roots and increased 
membrane integrity (Çakmakçı et al., 2017; Furlan et al., 2017; Pereyra 
et al., 2012). Inoculation with Burkholderia phytofirmans exhibited 
drought tolerance and improved various physiological characteristics of 
wheat such as rate of photosynthesis, pigment content, water status of 
the plant leaves, water use efficiency (WUE), and photosynthetic rate 
(Naveed et al., 2014; Chakraborty et al., 2013). Pseudomonas inoculation 
in maize grown under drought stress improved relative water contents 
by 23 % compared with well-watered plants (Rezazadeh et al., 2019). 
4.4.2.2. Biochemical adjustment. The production of phytohormones is 
the plant mechanism against abiotic stresses (Frankenberger and 
Arshad, 1995), but PGPR also can produce these hormones extracellu-
larly which helps the plant to overcome various stresses (Barnawal et al., 
2017; Lastochkina et al., 2017). Studies have shown the production of 
phytohormones by bacteria, such as Indole acetic acid (IAA). IAA is a 
widely reported phytohormone in plants and bacteria. It causes changes 
in the root morphology of plants by initiating cell division in the root 
cells making them use water and nutrients efficiently. Azospirillum has 
also been reported to increase the number of root hairs and phospho-
lipids in the roots by producing IAA that leads to efficient uptake of 
water to the plant leaves, these characters are directly co-related with 
the tolerance of stress by plants (Arzanesh et al., 2011; Paul et al., 2008). 
A. brasilense inoculation gave better tolerance against drought tolerance 
by maintaining high relative water content of leaves and seeds with 
increased concentration of mineral elements (K, Ca, and Mg) compared 
to the non-inoculated plants (Egamberdieva and Kucharova, 2009). 
Several research studies report the accumulation of osmolytes in the 
plants as a result of PGPR inoculation (Dimkpa et al., 2009; Paul et al., 
2008). Proline is the osmolyte possessing the properties of chaperons, 
work as osmoprotectants against osmotic stress in plants (Shakirova 
Fig. 5. PGPR-Mediated alleviation of drought stress in plants. 
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Microbiological Research 242 (2021) 126626
11
et al., 2012). Plant growth-promoting rhizobacteria mediated proline 
accumulation in wheat under drought and salinity stress has also been 
reported (Nawaz, 2019; Nawaz et al., 2020a, b). Studies also reported 
that PGPR mediated antioxidant activities scavenge ROS (Gusain et al., 
2015; Lastochkina et al., 2017; Ullah and Bano, 2015). Amelioration of 
osmotic stress by the inoculation of PGPB have been found in many 
plants including wheat (Kang et al., 2014b; Kasim et al., 2013). 
Volatiles are induced when plants are exposed to different stresses 
(Holopainen and Gershenzon, 2010; Loreto and Schnitzler, 2010). This 
stress-induced volatiles serve as signals for developing priming and 
systemic responses within the same and in neighboring plants (Niine-
mets, 2010). PGPR-inoculation under drought stress results in enhanced 
plant biomass and five-fold higher survival under severe drought due to 
a significant reduction of volatile emissions and increased photosyn-
thesis (Timmusk et al., 2014). A volatile metabolite 2R, 3R-butanediol 
produced by root colonizing Pseudomonas sp. prevents water loss by 
stomatal closure and induce resistance to drought stress in Arabidopsis. 
This volatile 2R, 3R-butanediol is a major determinant in inducing 
resistance to drought through an SA-dependent mechanism (Cho et al., 
2008). 
Exopolysaccharides (EPS) of microbial origin with novel function-
ality, reproducible physicochemical properties, are an important class of 
polymeric materials. EPS are believed to protect bacterial cells from 
desiccation, produce biofilms, thus enhancing the chances of the cells of 
bacterial colonizing special ecological niches. In the rhizosphere, EPS is 
known to be useful to improve the moisture-holding capacity. The 
exopolysaccharides are attached to clay surfaces using cation bridges, 
hydrogen bonding, Van der Waals forces, and anion adsorption mech-
anisms. This organic product may further promote aggregate stability by 
reducing wettability and swelling. The stability of soils against erosion 
and improvement of the soil physical conditions for plant growth are 
closely related to aggregation. Aggregation is an important part of soil 
formation because it influences the soil behaviors in infiltration, aera-
tion, root penetration, and reducing runoff. Soil aggregation and water 
infiltration increases when EPS producing microorganisms are added to 
crop development. Bashan et al. (2004) reported the role of poly-
saccharides producing Azospirillum in soil aggregation. Table 2 outlines 
some studies regarding PGPR-mediated drought stress induction in 
cereals. 
4.4.2.3. PGPR- induced Omics response. Gene expression analysis is a 
powerful tool to comprehend the wide-ranging responses of an organism 
to its environment. Different mechanisms for the PGPR mediated 
drought tolerance have been reported at the molecular and proteomic 
level. PGPR improves the stability of plant cell membranes by activating 
the antioxidant defense system, enhancing drought tolerance in plants. 
Bacteria induce the expression of various genes and proteins that are 
directly or indirectly involved in conferring stress tolerance to the plants 
(Egamberdiyeva, 2007; Saravana kumar and Samiyappan, 2007; Dar-
danelli et al., 2008). Rhizobacteria inoculation in the wheat and cu-
cumber under drought, up-regulated the expression of cytosolic 
ascorbate peroxidase (cAPX), rubisco larger (rbcL) and smaller subunits 
(rbcS) (Wang et al., 2012a) and PIP2 gene (del Mar Alguacil et al., 2009). 
Endophytic bacteria, Pseudomonas fluorescens, were reported to upre-
gulate a 2-fold expression of 105 genes in Arabidopsis involved in various 
processes positively co-related with stress tolerance (Wang et al., 2005). 
Dehydrin proteins are also reported to be upregulated in wheat with 
PGPR inoculation under drought stress (Borovskii et al., 2002). 
Expression of "Pathogenesis-related proteins" (PRs) and “small heat 
shock proteins” (sHSPs) under biotic and abiotic stress has also been 
reported (Sarkar et al., 2009). Microarray analysis showed that a set of 
drought signaling response genes were down-regulated in the 
P. chlororaphis O6-inoculated A. thaliana plant while jasmonic 
acid-marker genes, VSP1 and pdf-1.2, salicylic acid regulated gene, PR-1, 
and the ethylene-response gene, HEL, were up-regulated in response to 
drought stress (Cho et al., 2013). Vargas et al., 2014 revealed using 
Illumina sequencing that the diazotrophic bacterium Gluconacetobacter 
diazotrophicus sp. PAL5 activated ABA-dependent signaling genes 
conferring drought resistance in sugarcane. 
5. Conclusions 
Water scarcity will remain a major threat to agricultural productivity 
under the current climate-change. The exploitation of climate-smart 
strategies for improving agriculture yield is the main approach to in-
crease the food production of the rapidly growing population. This re-
view summarizes how water stress negatively impacts plant growth and 
productivity by imparting nutritional and hormonal imbalance. A 
plethora of strategies are available, and currently being applied to 
induce plant drought tolerance but the molecular bases of the plant- 
microbiome interactions is largelynot explored yet. The microbiome 
also referred to as the second genome of the plant, is crucial for plant 
health. Recent advances in plant-microbe interactions research revealed 
that plants can shape their rhizosphere microbiome, as evidenced by the 
Table 2 
PGPR-mediated drought mitigation in different cereals. 
Cereals Developmental stage 
of plant at which 
Drought induced 
Yield loss PGPR-inoculated for stress mitigation Inoculation Effect on plants References 
Triticum 
aestivum 
seedling, 
pre-flowering, grain 
filling, whole life 
cycle 
57 % Azospirillum sp., Rhizobium 
leguminosarum (LR30), Mesorhizobium 
ciceri (CR-30 and CR-39), Rhizobium 
phaseoli (MR-2), Rhizobacteria, 
Bacillus thuringiensis AZP2, Bacillus 
amyloliquefaciens 5113 and Azospirillum 
brasilense NO40 
enhanced root growth, increased water 
uptake, biomass and drought tolerance 
index, higher photosynthetic activity, 
upregulation of stress related genes 
APX1, SAMS1, and HSP17.8 
(Fahad et al., 2017), ( 
Arzanesh et al., 2011),( 
Hussain et al., 2014),(Shakir 
et al., 2012), (Timmusk et al., 
2014), (Kasim et al., 2013) 
Hordeum 
vulgare 
seedling, 
pre-flowering, grain 
filling, whole life 
cycle 
17.3 % in 
winter barley 
yield, 33.6 % in 
spring barley 
Bacillus licheniformis RC02, Rhodobacter 
capsulatus RC04, Paenibacillus polymyxa 
RC05, Pseudomonas putida RC06, and 
Bacillus OSU-142 
increased root mass, enhanced uptake of 
Zn, Fe, Mn and N, 
(Anderson et al., 2018), 
(Cakmakci et al., 2007), 
Oryza 
sativa 
seedling, flowering 63− 87% Azoapirillum brasilense Az39, Consortia 
containing Pseudomonas jessenii, R62, 
Pseudomonas synxantha, R81 and A. 
nitroguajacolicus strainYB3, strain YB5 
PGPR mediated root growth and 
alleviation of drought stress, 
accumulated proline improved plant 
growth 
(Fahad et al., 2017), (Cassan 
et al., 2009), (Gusain et al., 
2015) 
Zea mays seedling, flowering 63− 82% Azospirillum lipoferum, Pseudomonas 
putida GAP-P45, Pseudomonas putida 
GAP-P45, Klebsiella variicola F2, 
Pseudomonas fluorescens YX2 and 
Raoultella planticola YL2 
alleviated drought stress by ABA, 
cytokinin, auxin, proline and gibberellins 
production which enhanced stress 
tolerance, plant biomass, relative water 
content and leaf water potential 
(Anderson et al., 2018), 
(Cohen et al., 2009),(Sandhya 
et al., 2010), (Ansary et al., 
2012), (Gou et al., 2015), 
R. Zia et al. 
Microbiological Research 242 (2021) 126626
12
fact that different plant species host-specific microbial communities 
when grown on the same soil. A better understanding of what makes a 
beneficial PGPR interaction with the plant would provide important 
insight to better handle microbes and exploit them for plant-benefit. 
This review opens a new chapter in the integrated management 
approach for drought stress management in plants. Depending upon the 
soil type, intensity of drought, and climate, different combinations/ap-
proaches can be adapted by manipulating rhizosphere microbiome, 
rhizosphere enrichment with OM, nutrients, phytohormones, the addi-
tion of liquid nano clay, nanoparticles, and better water management for 
obtaining the agricultural output without any yield compromise. 
Advanced genome editing tools could also help produce drought- 
tolerant cultivars but most of these studies are in their initial phases 
and require further validation for field trials. More research on 
biochemical and physiological aspects of stress will open up new fields 
to deal with food insecurity issues. Concerted future research is needed 
in terms of identification of the right kind of microbes for drought stress- 
tolerance and addressing the issue of delivery systems and field evalu-
ation of potential organisms. 
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