CONTENTS

1. Introduction
2. Review of literature
2.1 Definition of Drought
2.2 Drought Stress on Rice
2.3 Drought Instigators
2.3.1 Global warming
2.3.2 Erratic Rainfall
2.3.3 Changes in the Pattern of Monsoon
2.4 Drought Stress on Rice Plant
2.5 Drought-induced morphological changes
2.5.1 Effect on seed germination
2.5.2 Effect on leaf traits
2.5.3 Effect on root traits
2.5.4 Root system architecture under drought Stress
2.5.5 Physiological response under drought stress
2.5.6 Effect on leaf photosynthesis
2.5.7 Effect on nutrient assimilation
2.5.8 Effect on water relation and membrane function
2.5.9 Effect on biochemical characteristics
2.5.10 Osmolyte accumulation
2.5.11 Emergence of ROS
2.6 Role of antioxidants under drought stress
2.7 Role of plant hormones in drought stress Management
2.8 Plant strategies to counter drought stress
2.8.1 Drought avoidance
2.8.2 Drought escape
2.8.3 Drought tolerance
2.9 Molecular mechanism of drought Tolerance
2.10 Recent advances
2.10.1 QTLs linked drought Tolerance
2.10.2 Genes and transgenic approaches
2.10.3 Micro RNA approach in Drought Tolerance
2.10.4 Breeding approaches to improve Drought Tolerance
2.10.5 Marker assisted selection for drought Tolerance
2.10.6 Harnessing ‘OMICS ’approaches
2.10.6.1 Genomics
2.10.6.2 Transcriptomics
 2.10.6.3 Proteomics
2.10.6.4 Metabolics
2.10.6.5 Phenomics
2.11 Molecular Response and drought stress management
2.12 Nano Biotechnology and it’s intervention
3.MATERIALS AND METHODS
4.RESULT AND DISCUSSION
5.CONCLUSION
6.REFERENCES
INTRODUCTION
Rice (Oryza sativa L.), is a crop of Gramineae (Poaceae) family is a vital human food crop in
the world, and the 2ndmost important cereal crop after wheat in the world utilized exclusively as
human staple food about four billion Population worldwide depends on rice for their survival
(Datta, 2004). It’s crucial to boost rice production to match the growing population. Rice,
primarily grown in warm climates, directly nourishes more individuals than any other crop.
Increasing its cultivation is essential to meet rising food demands .It is also the predominant
staple food for a significant portion of the world population, particularly in Asian regions.(Samal
et al., 2018) Asia ranks highly in rice production and consumption. More than one-third of the
world’s population consumes rice, which can account for up to 80% of daily caloric intake for
most people, particularly in Asia. The creation of high yielding rice varieties with a high level of
tolerance and resilience to both biotic and abiotic challenges is a requirement in the drive to
achieve self-sufficiency in rice production. Several environmental factors are blamed for the
large yield disparity. Stress may result from biotic causes like the prevalence of pests, insects,
and diseases or abiotic factors such heavy metal toxicity, floods, salinity, drought, high
temperatures, and air pollution, among others. The rice crops, play a vital source of sustenance
for the farmers, who were constantly struggling due to two significant hurdles: a lack of
phosphorus and the ever-looming threat of drought.
Drought posed a significant obstacle for rice cultivation, especially in Asia where rice is a vital
crop. The scarcity of water in rainfed environments led to a drastic decrease in yields. The deep
root development and enhanced water uptake from deeper soil layers could help against drought
stress. Through molecular breeding approaches, scientists identified specific genes responsible
for drought tolerance, known as DRO1, Dro2, Dro3, Dro4, and Dro5. These genes were mapped
on different chromosomes, such as 9, 7, and 4&7, and played a crucial role in root growth.
Global climate change and increasing world Population Enriched with drought stress are making
situation more serious day by day to cope with the ever-growing food, feed and shelter needs of
human Population. Among the cereal crops, rice is particularly more sensitive to water stress
especially at critical growth stages such as panicle initiation, anthesis and grain filling (Yang et
al., 2008). Drought stress induces a range of physiological alterations in plants, such as
diminished levels of photosynthetically active radiation (PAR), reduced photosynthetic and
transpiration rates, decreased stomatal conductance, pigment degradation, and a decline in
relative water content (RWC), all contributing to a decrease in water use efficiency (WUE) and
impaired growth. Plants retrot to water stress is more sophisticated that includes adaptation of
various mechanisms when they encounter drought stress at various growth stages.
Drought stress induced by Polyethylene Glycol (PEG) brings drought stress on the plant vital
difference from the control carries to Augment with increase in solute probable. PEG-6000 has
long been used as a consistent indicator under laboratory for checking the drought tolerant
genotypes. Drought screening applying some seed technological parameters has been set up to be
quite functional in a number of crops. Under laboratory situations. This method can be further
expanded to test drought tolerance in other genotypes Even the behavior of genotypes within a
species is also different when exposed to water stress. There has been reports of water stress
affecting seed germination and early seedling growth that are potentially the most critical stages
of rice (Ahmad et al., 2009).
To produce drought tolerant genotypes, it is vital to understand how plants fight against drought
stress. Drought stress has two types: terminal and intermittent. The shortage of water accessible
to plants activates terminal drought stress, which causes stress if it persists for an extended
period of time and can result in plant death. In contrast, intermittent drought stress causes plant
growth to be slowed during periods of insufficient rainfall (Oladosu et al., 2019). The capacity of
a plant to thrive in the cytoplasm with low moisture levels is known as DT. DT mechanisms
include cellular adjustment, morphological and physiological adaptations, and are gene-directed
(Sahebi et al., 2018). Morphological adaptations include increased root length and thickness, a
delay in leaf senescence, and physiological adaptations such as stomatal closure, a condensed
transpiration rate, the relationship between parents' flowering and mature stages, and biomass
and yield partitioning. Improvements in chlorophyll content (CC), harvest index, and osmotic
potential are required for cellular DT adjustment. Understanding how roots respond to drought
stress is critical for improving DT in rice (Kim et al., 2020).Drought stress has become an
important aspect of plant research, and improving DT in plants is a difficult undertaking due to
the intricacy of these features. Genetic heterogeneity among rice cultivars is a significant factor
in the production of resistant cultivars because they react differently to drought stress. Genotypes
with the highest DT are frequently used to explore DT and provide the best source of DT genes
for the production of tolerant crop cultivars. To design drought-tolerant cultivars, it is vital to
understand the mechanisms by which plants deal with drought stress. DT mechanism in rice has
been widely investigated, which can assist to understand causes of drought stress and improve
DT.
2.REVIEW OF LITERATURE
2.1Definition of drought
Droughts Tangible across diverse global regions, including high rainfall areas, upholding
prolonged Water scarcity over extensive time frames, spanning from years to seasons or months.
Various factors, such as humidity, wind patterns, temperature fluctuations, and geographic
characteristics, influence the variability of the primary water source, namely precipitation, across
the affected areas.(Mishra et al.,2020)Drought can be defined as the scarcity of irrigation or
rainfall over a prolonged period, leading to diminished soil moisture and harm to plants. Drought
stress occurs when a plant loses more water than its roots can absorb, resulting in decreased
water content that disrupts normal plant functions. Typically, water is the primary limiting factor
for plant growth.(Brownet et al.,2018)
2.2 Drought Stress on Rice
Drought stands out as the foremost environmental challenge facing global agricultural
infrastructure and aincredibleattempt is being concerned to progress crop yields in the face of
rising water dearth. Drought impacts various aspects of plant life, including photosynthesis, crop
yield, pigment levels, membrane health, osmotic balance, water relations, and overall growth.
(Benjamin and Nielsen 2006).Regions prone to drought and lacking irrigation systems have seen
slower development compared to areas with better water management or consistent rainfall due
to challenges and high expenses associated with adopting advanced technologies. As a result,
rice production in non-irrigated and drought-prone regions is steadily declining worldwide. The
prevalence of drought is increasing in various areas, and by 2050, it is predicted to affect over
half of all cultivable lands severely. The global food grain production needs to double by 2050 in
order to satisfy the increasing demands of the growing population. (Tilman et al., 2002), which is
going to achieve Nine billion by that time .Abiotic stresses has a primary barrier in our endeavor
for sustainable food production, as they can reduce potential yields by up to 70% in crop plants.

2.3Drought instigators
2.3.1 Global warming
Climate change is causing widespread disruption in ecosystems reliant on agriculture, with
temperatures consistently increasing since 1906 by more than 0.9 degrees Celsius. This upward
trend is leading to the depletion of water reservoirs, diminishing irrigation supplies for
Agriculture. Moreover, precipitation is decreasing in numerous rain-fed agricultural regions due
to the effects of global warming. If temperatures continue to climb as projected, with an increase
of around 2°C by the end of the century, roughly one-fifth of the world's population could
confront significant water shortages

2.3.2 Erratic rainfall

Erratic precipitation and drought are often seen as enduring natural disasters, especially in areas
where rainfall levels dip below average. The impact is more severe in regions depending solely
on rainfall for farming compared to those using irrigation from canals, rivers, and water
channels. The yearly distribution of rainfall plays a vital role in worsening water scarcity during
droughts in rain-fed areas. Human actions like industrialization, deforestation, and urbanization
greatly affect rainfall patterns and water availability for plants, worsening drought conditions.
Global drought conditions are illustrated in Figure, displaying the 9-month Standardized
Precipitation Index (SPI) updated monthly with data from the previous month. The Global
Precipitation Climatology Centre (GPCC) monthly precipitation dataset (from 1901 to the
present) is obtained from worldwide station data.

2.3.3 Changes in the pattern of monsoon

The monsoon season contributes substantially to precipitation in many regions worldwide, and
its occurrence is closely linked to temperature fluctuations. If current trajectories persist, summer
rainfall in rain-dependent areas is projected to decrease by 70% by the beginning of the twenty-
first century .This will inevitably exert a negative impact on agricultural output. Astonishingly,
over half of the global population faces food insecurity due to significant seasonal variations in
rainfall caused by shifts in monsoon patterns. Monsoonal downpours have had and will continue
to influence moisture levels in the rhizosphere, thereby impacting crop yields through changes in
rainfall intensity, frequency, and duration in certain regions of the world . Hence, there is a
necessity for a transition in agricultural methodologies to adapt to evolving monsoon weather
patterns, prioritizing sustainable crop production. Options such as crop planning and
management are crucial for mitigating the effects of fluctuating monsoon patterns, which
alternate between inadequate and excessive rainfall, and vice versa.

2.4Drought stress effect on plant

Fig. showing different types of stress and its impact on rice plant (Anjum et al., 2011)

tolerance refers to a plant’s ability to achieve its highest economic output even when facing
restricted water availability (Rollins et al, 2013). This trait is multifaceted, contingent upon the
interplay of various morphological, biochemical, and physiological reactions.
2.5 Morphological alterations due to drought stress
These morphological characteristics comprise leaf size reduction, stomatal decrease, cuticle
formation on leaf surface, and thickening of leaf cell walls, among others. Drought stress impacts
mitosis, cell elongation, and expansion, leading to diminished growth and yield attributes.
(Hussain et al., 2008). Drought stress leads to diminished plant size, decreased lifespan, and a
decrease in leaf count per plant owing to reduced soil water potential. This environmental factor
triggers a reduction in leaf surface area, thereby negatively impacting crop plants through
diminished production of both fresh and dry biomass found that shoot length turned down in
crop plants by increasing the severity and duration of drought stress as reported earlier (Farooq et
al., 2009).the capacity of a plant to finish its life cycle prior to the onset of significant soil water
deficiencies'.(Kumar et al.2016) as ‘the ability of plants to maintain relatively high tissue water
potential despite a shortage of soil moisture.Drought stress causes changes in length, plant
height, biomass, and leaf area, which are linked to leaf senescence in different crops.;
( Upadhyaya et al., 2008, 2012, 2016). Phenotypic plasticity refers to the ability of a specific
genotype to exhibit varied phenotypes in reaction to fluctuating environmental conditions. The
plasticity observed in root and shoot morphological characteristics is advantageous for enhancing
drought stress resilience in rice. (Kadam et al., 2017).Drought resilience is described to be as
‘the capacity of plants to withstand low tissue water contents . Drought negatively impacts crop
growth factors and leads to decreased yield. The extent of damage depends on the severity,
duration, and growth phase of the plant. Adverse effects manifest through alterations in the
plant’s physical, physiological, biochemical process.

2.5.1 Effects of drought stress on seed germination and seedling growth

When rice plants experience water stress, their initial appearance changes. (Farooq et al,
2012).Due to water constraint, severe reductions in seedling development and germination are
seen during drought stress. . Unlike some other crops, rice is extremely sensitive to drought
conditions during the germination and early seedling growth stage. Seed germination needs
appropriate temperature and soil humidity. Drought inhibits water intake, which weakens
seedlings, and has a detrimental effect on the germination process.(Vibhuti et al, 2015). Drought
stress disrupts the water balance, affects metabolic processes at the cell level, impairs membrane
transport, and reduces ATP synthesis and respiration, resulting in poor seed germination. (Kadam
et al, 2017). Several studies have shown that water stress causes declines in plant height, leaf
area, and biomass.

2.5.2Effects of drought stress on leaf traits

Drought stress inhibits leaf growth as a result of low water potential. (Zhu et al, 2020).
Disrupted water movement from xylem to another cell, including reduced turgor pressure due to
water constraint, leading to poor cell growth and reduced leaf area in crops.(Hussain et al, 2018).
The drought alters the bodily structure and the internal structure of leaves. These changes include
smaller leaves, fewer stomata, a thicker cell wall, cutinization on the leaf surface, and
insufficient growth of the conducting system. (Rollins et al. 2013).Leaf curling and the onset of
incipient senescence are two other major features observed under intense drought.(Anjum et al.
2011). Several leaf traits have been used to screen drought-tolerant cultivars, including enhanced
flag leaf area, leaf surface index, leaf relative water content, and leaf pigment content(.Farooq et
al. 2009)

2.5.3 Effects of drought stress on root traits

The root properties of plants are critical for increasing production under drought conditions.
Crop performance under water stress is defined by the composition and production of rice root
systems. Rice output under water stress can be anticipated using mass of roots (dry) and length
(Comas et al., 2013). Root development characteristics show diverse and varied responses to
water stress. noticed an increase in rice root length during drought stress due to an increase in
abscisic acid content in the roots. Rice varieties having a deep and abundant root system are
more drought-tolerant (Mishra et al., 2019; Kim et al., 2020). In the case of rice, variants
possessing profound root system and coarse roots, Drought resistance is dependent on the ability
to produce multiple branches and a high root-to-shoot ratio . The morpho-physiological features
of rice roots play an important role in influencing shoot growth and overall grain yield under
drought stress.

2.5.4 Root System Architecture under Drought Stress

Roots are the principal plant organs that detect changes in soil conditions and play an important
role in responding to water stress (Ghosh, D.et al.2014 )There is substantial evidence that
changing the root structure of cereal crops cultivated in water and nutrient deficient conditions
can increase production by improving their ability to acquire soil resources .A study of rice root
systems under drought stress found a significant connection between root diameter and depth and
plant vigour (Yoshida, S et al.,1982) Drought resistance is achieved in many highland japonica
rice types by broad and deep root systems, but indica subspecies often reduce their development
cycle (Champoux et al,.1995) Root architecture is widely recognised as a critical component of
the root system's ability to obtain soil resources. Rice roots differ genetically in terms of
thickness and penetrating ability. Rice, in comparison to other cereal crops, is poorly adapted to
water scarcity. Rice absorbs little or no soil water at 60 cm depth .The deep roots system found in
upland rice varieties is thought to be beneficial at maintaining yield during droughts Rain-fed
lowland rice experiences variable soil water conditions, and certain rice genotypes adjust by
boosting root development before and during drought. Rain-fed rice can also penetrate hardpan,
which is essential for developing a deep root system and adapting to drought stress .Root
structure, function, and movement are all affected by soil moisture content, and root density in
the subsurface horizon dictates how the roots respond to drought stress. A deep root system for
obtaining moisture from soil profiles has been proven to be favourable for short-statured plants
such as rice, wheat, and beans, as well as plants that grow in limited water situations .Plants can
meet moisture demand with root length densities ranging from 0.5 to 1 cm−3 However, in dry
soil, a larger root surface area density is required to overcome hydraulic resistance. To increase
the surface area for water uptake, soil with low moisture promotes higher allocation of the
assimilates to the roots changes carbon assimilation in the roots, leading to accelerated growth
towards the water-sufficient soil layers .Severe water stress in rice leads to economic yield
reductions of 48-94% during the reproductive stage and 60% during the grain-filling stage(Uga
et al., 2013). The drying of the soil surface layer may cause the roots to seek moisture deep
within the soil profile. Breeding for plants with low root length density in shallow layers of soil
and high root length density in medium and deep layers has been deemed an effective water
management approach. The root system’s hierarchical structure may improve hydraulic lift,
which aids in water uptake from deep soil profiles (Doussan, C et al., 2006 )When deep root
systems can boost crop output, large-diameter xylem vessels may improve the axial hydraulic
conductivity of roots growing in deep soil.

2.5.5 Physiological responses to drought stress

Drought stress has a deleterious impact on various physiological processes, and plants react to
drought to acclimatise in harsh conditions. Prior to beginning a breeding programme,
physiological factors and processes must be optimised to increase yield under drought conditions
(Dash et al., 2018). Water scarcity has a wide range of negative effects on rice physiological
characteristics, including declines in net photosynthetic rate, transpiration level, stomatal
conductance, water use efficiency, internal CO2 concentration, photosystem II (PSII) activity, in
relation water content, and membrane stability index.

2.5.6 Effects of drought stress on leaf photosynthesis

Photosynthesis is a critical metabolic activity that determines crop growth and production, and it
is influenced by water scarcity/drought stress. Water stress alters the usual rate of photosynthesis
as well as the gas exchange properties in plants (Zhu et al., 2020). In water-limited environments,
stomata contract, limiting carbon dioxide inflow to leaves and pushing additional electrons for
the generation of reactive oxygen species (Farooq et al., 2009). Several factors contribute to
photosynthesis decline, including stomatal closure, decreased turgor pressure, reduced leaf gas
exchange, and decreased CO2 uptake, all of which damage the photosynthetic apparatus (Farooq
et al, 2009). Photosynthetic capacity of leaves and the availability of water to root zones are
critical variables in reducing output in sensitive rice genotypes under drought stress conditions
(Zhu et al., 2020). During drought stress, there is an inequality between light collection and
usage, as well as a reduction and degradation in Rubisco activity, pigments, and photosynthetic
system (Farooq et al., 2009), all of which contribute to photosynthesis reductions. Water stress
disrupts the regular functioning of PSI and PSII. PSII function is critical in the reduction reaction
and the creation of ATP. Several investigations have been carried out in vivo and showed that
drought causes significant negative decrease in centres for oxygen evolution as well as
photosystem, leading to inhibition of the electron transport chain andPSII was subsequently
inactivated. Plant pigments, including chlorophyll, are essential precursors of photosynthesis,
primarily for receiving light and producing reducing powers . Water deficit reduces the ability of
mesophyll cells to utilise the carbon dioxide they contain. As a result, the amount of living
chlorophyll decreases (Sarwar et al, 2013). Water-stressed rice plants show a decrease in
chlorophyll and the highest quantum generation of PSII (Fv/Fm). Carotenoids are crucial
photoprotective components that work as a precursor in directing plant development signals
under stress situations. Therefore, particular attention is currently being taken Plant biologists
use breeding and genetic manipulation to increase the carotenoid content of plants (Ashraf and
Harris, 2013)

2.5.7 Effect of drought on nutrient assimilation

Water scarcity reduces soil nutrient mobility and root nutrient translocation, resulting in lower
ion content across plant tissues. Potassium uptake is impeded in water-stressed situations due to
diminished mobility, delayed transpiration, and reduced root membrane transporter efficiency.
Drought-stressed plants, such as Malushupehensis, have lower potassium levels. Triticum durum
genotypes with high potassium level are resistant, but those with high sodium content are
sensitive. Dehydration lowers the expression of genes associated with potassium transporters.
Drought also has an impact on photosynthetic activities by limiting CO2 conductivity through
stomatal and mesophyll limits, which reduce Rubisco activity and other important enzymes.
Water scarcity alters canopy architecture, influencing gas exchange, water relations, and
vegetative development. The number and weight of maize kernels decrease with extended water
stress. Water availability has a considerable impact on Chlorophyll content, a key measure of
photosynthetic activity, resulting in decreased plant yield.

2.5.8 Effects of drought stress on water relations and membrane functions

The link between plants and water can be illustrated in a variety of ways, including leaf water
potential and relative water content (RWC) (Farooq et al., 2009). Water utilisation efficiency is
considered a significant factor in determining plant production potential under water stress
circumstances. It can be viewed as a strategy for boosting crop production during drought
(Mishra et al., 2019). RWC is an important property of water relations in plants and is regarded
as the best integrated measurement of plant water status, representing fluctuations in water
potential and turgor potential (Gupta et al., 2020). Water stress is one of several factors that
negatively affect the RWC, turgor pressure, and transpiration in many crops. . The relationship
between plants and water can be represented in a variety of ways, including leaf water potential
and relative water content (RWC). Water utilisation efficiency is regarded as a critical aspect in
evaluating plant output capacity under water stress conditions. It can be considered as a strategy
for increasing agricultural production during droughts (Mishra et al.,2019). RWC is a critical
feature of plant water relations and is recognised as the finest comprehensive measurement of
plant water status, including changes in water potential and turgor potential (Gupta et al., 2020).
Water stress is one of the variables that reduce RWC, turgor pressure, and transpiration in many
crops.

2.5.9 Effects of drought stress on biochemical characteristics

Under drought stress, plants attempt to preserve cell turgor by accumulating organic and
inorganic solutes that diminish osmotic potential. The accumulation of osmoprotectants such as
proline, glycinebetaine, and soluble sugar allows plants to alter their osmotic conditions (Kumar
et al, 2016). Drought resistance is improved through protein content and profile, as well as
increased antioxidant activity for scavenging reactive oxygen species (Pandey and Shukla,
2015). Tissue- and time-specific expression of drought-responsive characteristics, such as
abscisic acid and ethylene phytohormone pathways, increases drought response while without
reducing yield .

2.5.10 Osmolyte accumulation under drought stress

Osmoregulation is the primary process in plants, and a decrease in turgor leads to the
accumulation of osmoprotectants. Under water stress, the accumulation of various osmolytes
such as proline, soluble sugar, phenolic and total free amino acids increases, and it plays a
significant role in plant drought tolerance (Anjum et al, 2017). Proline is a proteinogenic five-
carbon amino acid that serves as an osmolyte in plants . Kemble and Macpherson (1954)
discovered the presence of free proline in rye grasses under water deficiency stress. Proline build
up rises in drought conditions when compared to well-watered situations in all rice
cultivars .Higher proline accumulation is frequently connected with drought resistance, and it
helps to maintain leaf turgor and Stomatal conductance has improved (Kumar et al., 2016). Thus,
proline content can serve as a biochemical marker for drought screening in plants. The structural
unit that supplies energy to support plant biomass is known as carbohydrates/soluble sugar.
Under abiotic stress, three forms of water-soluble carbohydrates play an important role in stress
tolerance: disaccharides, oligosaccharides, and fructans . Soluble sugars are critical for balancing
several physiological activities, including photosynthesis and mitochondrial respiration (Gill and
Tuteja, 2010a). Sugars have varied roles in plants, which adopt a variety of sugar-based
techniques to cope with environmental stress (). Mannitol, sorbitol, and trehalose levels are
critical for plant development and metabolic activity. Drought induces build up of soluble sugars
that even works as osmoprotectants in unfavourable conditions and protects the plants to some
extent (Kumar et al., 2016).
2.5.11 Emergence of ROS during periods of drought stress

Fig.-Drought stress disrupts the balance between reactive oxygen species and antioxidants in
plants , resulting oxidative stress

During aerobic metabolism, reactive oxygen species (ROS) are an inherent by product. However,
various biotic and abiotic stimuli frequently trigger excessive ROS production, which damages
cells and kills plants (Gill and Tuteja, 2010b). Photosynthesis and respiration generate ROS in
different parts of the cytosol. Under unfavourable conditions, the over-reduction of
mitochondrial and chloroplast electron transport chains results in an excess of ROS generation
(Melandri et al., 2020). Drought can also produce an imbalance in ROS production and
quenching in rice, resulting in oxidative damage and a negative impact on the plant’s life cycle
(Gill and Tuteja., 2010). leakage to O2 reduces photosynthesis and generates ROS via the
Mehler reaction. Drought can cause excessive production of superoxide radical (O2·), hydrogen
peroxide , and hydroxyl radical (OH·) through the photo-respiratory pathway (Gill and Tuteja,
2010a). These are highly toxic radicals that damage different cell components during drought
stress, including lipid peroxidation, protein and membrane degradation, and ultimately lead to
cellular death. As a result, the most effective strategy to improve drought tolerance in rice is to
reduce ROS overproduction or boost antioxidant activity in rice organs. Figure depicts the route
of ROS formation, the hazardous consequences of oxidative stress, cell damage that causes plant
death, and various antioxidative systems that scavenge ROS.
Fig. Schematic representation of reactive oxygen species damage and aioxidant protection of rice
plants under drought stress gill and Tuteja., 2010

2.6 Role of antioxidants under drought stress

Plants have antioxidant defense system as a shield against oxidative damage (Gill and Tuteja,
2010b). It comprises enzymatic and non-enzymatic antioxidants. The enzymatic antioxidants are
superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase
(APX), monodehy-droascorbate reductase (MDHAR), dehydroascorbatereductase (DHAR) and
glutathione reductase (GR), etc. The non-enzymatic antioxidants are ascorbate (AsA) and
glutathione. Increased expression of the antioxidativesystem can progress tolerance against
drought stress and can be a strategy against oxidative stress and boost drought tolerance in rice.
(Mishra and Panda, 2017). SOD is the fundamental defence line against oxidative stress and can
be found in practically all cellular components of all animals (Gill and Tuteja, 2010b). Drought
stress increases SOD levels in a variety of plants, including rice, pea, wheat, sunflower, bean,
and sweet potato . CAT is a heme-containing enzyme that catalyses the dismutation of H2O2 to
H2O and O2 (Gill and Tuteja, 2010b). It is found in peroxisomes, mitochondria, and other
cellular structures. CAT activity might rise or decrease depending on the degree of the stress
(Gill and Tuteja, 2010b). However, the decrease in CAT activity is A typical reaction to a variety
of stressors (Qureshi et al, 2018). This decrease in CAT is thought to be caused by the inhibition
of enzyme active sites during stressful situations. However, under drought stress, CAT activity is
variable and can increase, decrease, or remain constant (Mishra and Pandaet al . 2017)GPX is an
enzyme made up of 40-50 kDa monomers. These monomers use hydrogen peroxide to oxidise
various substrates (Gill and Tuteja, 2010b). GPX is an excellent ROS scavenger and creates
related chemicals such as lignin, guaiacol, and pyrogallol, which serve as electron donors for
scavenging. Ascorbate peroxidase (APX), catalase (CAT), dehydroascorbate reductase (DHAR),
glutathione reductase (GR), guaiacol peroxidase (GPX), monodehydroascorbate reductase
(MDHAR), and superoxide dismutase (SOD). Hydrogen peroxide is both intra- and extracellular.
Many studies show that GPX levels rise in drought-stressed plants such as wheat and rice
(Mishra and Panda, 2017). The increase in GPX activity in rice under drought stress has been
widely examined and validated as a suitable screening tool for tolerance features. Until now, the
AsA-GSH (Halliwell-Asada) pathway has played a critical role in antioxidant associations. It
takes place in the chloroplast and provides photoprotection by neutralising hydrogen peroxide
(Noctor and Foyer, 1998). The AsA-GSH cycle consists of the following steps: H2O2 is
decreased by APX, with AsA acting as an electron donor. Later, the oxidised AsA is reduced with
the help of reduced GSH. GR uses NADPH to produce reduced GSH from oxidised glutathione
(GSSG). These enzymes are critical to plants' stress tolerance. For rice plants, the elevated
activity of these enzymes are regarded one of the extremely significant defensive mechanisms
against oxidative stress amid drought circumstances.(Gill and Tuteja, 2010b).Furthermore,
increased activity of the AsA-GSH cycle correlates with higher levels of ascorbic acid and GSH
in rice plants under drought stress (Sharma and Dubey, 2005;Bhattacharje and Dey, 2018). Many
studies have proven the importance of this pathway in rice water deficit management
(Bhattacharje and Dey, 2018; Qureshi et al, 2018). Melandri et al. (2020) investigated the
biomarker for yield loss in rice under drought stress and proposed that an integrated network of
antioxidant enzymes, specifically the AsA-GSH cycle, demonstrates resistance and can reduce
grain yield losses under drought stress. AsA plays a crucial role in scavenging H2O2 via the
AsA-GSH cycle. (Gill and Tuteja, 2010b). The oxidation of AsA consists of two steps: the
production of monodehydroascorbate (MDHA) followed by dehydroascorbate (DHA). Increased
ascorbic acid concentration is strongly linked to oxidative tolerance in rice and is thus widely
utilised as a screening indicator for drought tolerance in rice plants (Upadhyaya and Panda,
2019). GSH protects against drought stress and ROS overproduction (Bhattacharje and Dey,
2018). AsA oxidation occurs in two steps: monodehydroascorbate (MDHA) is produced first,
followed by dehydroascorbate. Increased ascorbic acid concentration is strongly associated with
oxidative tolerance in rice and is thus commonly used as a screening indication for drought
tolerance in rice plants (Upadhyaya and Panda, 2019). GSH protects against drought stress and
excessive ROS generation .

2.7 Role of Plant Hormone In Drought Stress Management In Rice

Phytohormones particularly ABA, play an important role in integrating drought stress signals and
controlling downstream stress responses. ABA levels are continuously adjusted by plants in order
to cope with abiotic stresses including drought, salinity and cold (Li et al., 2017). Plants use
various tricks to handle tough conditions like lack of water, too much salt, or cold weather. When
there’s not enough water, some plant hormones like cytokinins decrease, making the plant close
up its stomata to conserve water. This can lead to leaves aging and falling off, which helps the
plant keep more water. Some researchers found that tweaking certain hormones can make plants
better at handling drought and salty soil. For example, in tobacco plants, adding a gene called
JERF3 makes them tougher against drought and salt produce less damaging molecules known as

ROS. Another hormone, auxin, helps plants produce new roots, which is critical for drought
survival. When there isn't enough water, plants produce less auxin and more other hormones such
as ABA and ethylene. In rice, scientists discovered that several auxin-responsive genes become
more active under drought. However, too much of another hormone called JA can reduce rice's
ability to withstand drought. However, introducing extra auxin can help rice plants produce seeds
even in hot and dry weather.

2.8 Plant strategies to counter drought stress

DT is the plant’s ability to produce greater yield during drought stress (Moonmoon and Islam
et.al 2017). DT is a polygenic characteristic whose effects are determined by the interaction and
relationship of several morphological, biochemical, and physiological responses (Mitra, 2001).
Rice primarily responds to drought stress by closing stomata, leaf rolling, and increased abscisic
acid (ABA) synthesis (Price et al., 2002a). DT in rice is generally determined by water use
efficiency, which allows for the least amount of water required for osmotic adjustment, allowing
plants to retain turgor and protect meristem tissues (Nguyen et al., 2004).

2.8.1 Drought escape

Escape from drought. It entails completing the plant life cycle before the intensity of the drought
worsens (Kumar et al., 2017).It comprises two mechanisms: rapid developmental flexibility and
phonological development. Plants can produce flowers with less vegetative growth, allowing
them to produce seeds on a limited water supply. Plants with developmental plasticity can
generate more vegetative growth, blooms, and seeds during a high rainfall season (Kumar et al.,
2008).

2.8.2 Drought Avoidance

Drought resistance is defined as the plant’s ability to preserve significantly larger tissue water
potential in the face of water constraint (Kumar et al., 2017). In a period of high evaporative
requirement and rising soil water scarcity, plant tissue has two options for maintaining a higher
water level: reduce water loss or preserve the source of water. Rice cultivars that can retain water
via ABA production or have broad root systems can reduce drought-related yield losses (Singh et
al., 2012)These plants typically have a deep root system capable of branching and soil
infiltration, bigger root to shoot ratio, effective stomatal closure, and increased cuticular wax
(Wang et al.,2017)
 fig. Plant response to drought escape ,avoidance and tolerance
2.8.3 Drought Tolerance
The plant’s ability to live in tissues with lower water content is known as DT (Fleury et al., 2010;
Zhang et al., 2018). In rice, DT is a complex process regulated by multiple genetic determinants
and intricate morphophysiological mechanisms (Li and Xu, 2007), which include turgor pressure
retention by osmotic management, enhanced cell flexibility, reduced cell size, and DT via
protoplasmic resistance. The plant’s reaction to tissue water potential determines its degree of
DT (Mitra, 2001), and secondary features such as osmotic adjustment, leaf rolling, and stomatal
conductance are utilised as DT selection criteria (Kato et al., 2006).To trigger the DT
mechanism, the plant must detect an imbalance between water loss and availability. This sense is
transformed into a measure of cellular stress. Plants have evolved a complicated chain of signals
that go through multiple primary and secondary signalling pathways. The combination of
hormonal signals, metabolites such as ROS, proteins, and osmolytes is required for the varied
expression of genetic factors. The plant may produce these chemicals itself or actively add them
in response to cell injury (Hu and Xiong, 2014). In response to DT, signalling cascades are
activated, causing additional alterations in gene expression, which ultimately leads to DT. Figure
2 shows how plants respond to drought stress. A decrease in cytoplasmic osmotic potential
results from an increase in Solutes (organic or inorganic) help to sustain turgor pressure during
stress. The degree of osmotic adjustment is determined by stress. The aggregation of proline,
glycine betaine, and solutes causes osmotic adjustment, which facilitates water absorption.
Proline is a widely studied osmolyte due to its importance in stress reduction under adverse
settings (Liang et al., 2013). Proline build up is an important predictor of DT that can develop
DT.

2.9 Molecular mechanism of drought tolerance

Environmental drought stimuli are detected by membrane sensors that have yet to be fully
portrayed, and the signals are then transmitted down through various signal transduction
pathways, resulting in the outflow of drought-responsive qualities with appropriate gene
functions and drought tolerance (Dash et al, 2018; Oladosu et al, 2019). Drought is a complex
phenomenon (Zargar et al, 2011; Kumar et al., 2016). As a result, hybridisation and selection
procedures cannot provide exact findings in terms of drought tolerance. However, using DNA
markers in molecular investigations can improve the method and provide more accurate results.
Furthermore, these molecular markers can be useful for screening drought-tolerant germplasms
from a large sample and employing them to improve crops. Several research have been
performed to find several quantitative trait loci (QTLs) related with specific traits.(Kumar et al,
2016; Barik et al, 2019; Upadhyaya and Panda, 2019). DNA studies using marker-based
phenotyping are the principal approaches used to differentiate genes involved in drought
resilience in rice. Despite advances, only a few features have been practically accepted as
drought-resistant (Zargar et al, 2011; Prakash et al, 2019). In this approach, molecular breeding
can help to improve crop varieties and yield assortments, resulting in productive harvests that are
safe and have high agronomic legitimacy. Recent Advances

2.10.1 QTLs linked to drought tolerance in rice

Genes and transgenic approaches for drought tolerance in rice
Following drought stress in rice, several distinct types of genes are variably expressed, with
around 5000 genes upregulated and 6000 down regulated of the genes and activities related with
drought tolerance in rice. These genes are divided into three key categories: membrane transport
genes, signalling genes, and transcriptional regulatory genes (Upadhyaya and Panda, 2019; Kim
et al, 2020). Their expression regulates the majority of the biochemical, physiological, and
molecular pathways during drought stress in rice. Many distinct genes/transcription factors, as
revealed exhibit differential expression in rice and are employed for transgenic plants in response
to drought stress. The majority of drought-regulated genes use both ABA-independent and ABA-
independent regulation mechanisms (Du et al, 2018; Gupta et al, 2020). Several genes are also
linked to osmoregulation and late embryogenesis abundant (LEA) proteins, which confer water
deficit tolerance in rice (Dash et al, 2018; Upadhyaya and Panda, 2019). The gene DRO1
increases root elongation and deeper rooting in transgenic rice (Uga et al., 2013). Overexpression
of OsDREB2B, CYP735A, and OsDREB1F in rice increases root morphological responses
during drought stress (Kim et al, 2020). Huang et al. (2018) reported that the DREB2-like gene
OsDRAP1 confers drought resistance in rice. Increased grain yield in rice during drought is
critical and can be achieved through transgenic approaches that introduce genes such as
OsNAC5 (Hu et al, 2006), OsLEA3-1 (Xiao et al, 2007), OsbZIP71 (Liu et al, 2014),
OsWRKY47 (Raineri et al, 2015), OsbZIP46 (Tang et al, 2012), and OsNAC10 (Jeong et al,
2010). Better water usage efficiency, accumulation of osmolytes, better antioxidant enzyme
activity, and enhanced photosynthesis are seen in transgenic rice by interrogating genes such as
EDT1/HDG11 (Yu et al., 2013), AtDREB1A (Ravikumar et al. 2014), OsMIOX(Duan et al,
2012) OsCPK9 enhances drought tolerance in transgenics by improving stomatal closure and
osmoregulation (Wei et al, 2014). Overexpression of OsDREB2A improves transgenic plant
survival under extreme drought and salinity situations (Cui et al, 2011). In rice, CDPK7 and
CIPK03/CIPK12 govern a number of regulatory proteins, signal transduction pathways, and
protein kinases. OsITPK2 is responsible for reduced inositol triphosphate and ROS homeostasis
in rice during drought stress (Du et al, 2011). The WRKYgenes contribute significantly to plant
development by regulating to drought stress (Sahebi et al, 2018). Several genes have been
explored for drought resistance in rice using transgenic techniques in a laboratory or glasshouse
setting. However, these genes need to be tested.

2.10.2 Approaching drought tolerance through functional genomics

The purpose of functional genomics is to understand how the genome functions to produce a
whole plant, as well as to comprehend the information conserved in the genes that comprise the
genome. Second, using that information for crop genetic improvement is beneficial (Jiang et al.,
2015)The full genome sequence of rice (International Rice Genome Sequencing Project 2005
provides a foundation for functional genomic technologies (microarray, express sequence tags
(EST), etc.) by knowing the expression and sequence of thousands of genes. These genes are
then evaluated using RNA expression profiling to identify possible candidate genes associated
with drought stress. Using expression profiling, (Kawasaki et al., 2001) Some potential rice
genes have been identified. Rice genomes has advanced significantly in recent years. Several
functional genomic techniques have been used in rice, including macro and micro arrays
(Kawasaki et al., 2001). Using functional genomics, we produced an abundance of ESTs from
cDNA libraries and discovered 589 genes involved in drought stress. These ESTs also helped to
identify drought QTLs and candidate genes. The availability of a huge number of ESTs and
microarrays, together with bioinformatics, will indicate the function of rice possible genes
associated with drought resistance ( Langridge et al., 2006). Proteomics is a wide and expanding
field of study that focuses on protein structure and function. Fortunately, our understanding of
the rice proteome is highly advanced in contrast to other crops and Even said, proteomics
research on drought remain in their early stages .More than 2000 proteins were identified as
being implicated in drought stress. Following this, they were able to identify 42 proteins based
on their effect on drought and infer their roles in detail. (Malik et .al 2017) conducted another
rice investigation, this time focusing on a protein actin depolymerizing factor. They discovered a
significant rise in ADF concentration in drought-tolerant plants, indicating the relevance of this
protein in response to drought stress. (Rabello et al., 2008).Mass spectroscopy was used to
identify 22 proteins that may be involved with drought tolerance. Micro RNAs are a new family
of tiny single-stranded non-coding RNAs that regulate post-transcriptional gene expression by
targeting mRNAs for cleavage or translational repression (Zhao et al., 2007). They employed an
oligonucleotide microarray in rice to examine the expression profile of micro RNA in drought
stress and discovered two micro RNAs that were induced.

2.10.3 microRNA in drought stress tolerance in rice

MicroRNAs (miRNAs) are tiny noncoding regulatory RNAs that influence the expression of
genes during abiotic stress (Singh et al, 2018) Table 3 lists the reported miRNA-mediated genes
and their functions in rice during drought stress. Several miRNAs also improve rice drought
tolerance by influencing gene expression ( Singh et al, 2018). The expression of miR393,
miR319, and miR397 in response to drought stress was first reported in Arabidopsis (Sunkar and
Zhu, 2004), and miR393 also controls tiller number increment, early flowering, and auxin
sensitivity in rice by regulating transcriptional factors OsAUX1 and OsTIR1 (Xia et al, 2012).
There are 30 miRNAs found in rice of which 11 are down-regulated and 8 are up-regulated
during droughts. (Zhou et al, 2010). Mutum et al (2016) also reported 71 novel miRNAs from
Nagina 22, a drought tolerant check variety.( Balyan et al. 2017) discovered that miR398
influences the production of Cu/ZnSODs, which modulates SOD activity during drought stress in
rice. MiR160 and miR167 regulate the expression of the ARF gene during drought stress, which
in turn regulates the early auxin response. Increased stomatal closure and decreased stomatal
density via ROS homeostatic genes and, eventually, DST-amiRNA improve drought tolerance
(Faisal et al., 2017). Overexpression of the UDP-glucose-4-epimerase gene regulates root
development, cell wall synthesis, and carbohydrate metabolism, which is mediated by Osa-
miR169-3p and Osa-miR166e-3p.

(Cheah et al. (2015). S identified ten miRNAs (miR531, miR827, miR8175, miR977, miR6300,
miR1861, miR440, miR9773, miR3982, and miR1876) that are altered by drought stress in
traditional rice landraces. These miRNAs can be targeted for genetic manipulation to enhance
drought resistance. As a result, miRNAs govern many of the drought tolerance responses, which
may aid in the development of drought-tolerant rice varieties.

2.10.4 Breeding approaches for drought tolerance in rice

Natural rice genotypic variation can be investigated to uncover novel genotypes with drought-
tolerant features of interest, as well as linked genes and loci. These novel genotypes can be used
in standard breeding programmes to generate drought-tolerant rice varieties using marker-
assisted selection. The breeding programme is intended to produce high yield lines with better
quality criteria, and then to release the cultivars for farming. Previous research has focused on
breeding drought-tolerant rice genotypes (Singh et al, 2016; Vikram et al, 2016; Dixit et al, 2017)
However, the success rate has been significantly lower than expected due to the difficulties in
locating suitable donors with a greater tolerance level, as well as the environment-specific
character. The majority of marker-assisted breeding procedures for the production of drought-
tolerant rice varieties were carried out at the International Rice Research Institute during the
previous decade (Kumar et al, 2016;). Using marker-assisted breeding procedures, numerous
QTLs for drought tolerance in rice are introduced into leading cultivars. (Singh et al, 2016).They
effectively included QTLs such as qDTY9.1, qDTY2.2, qDTY10.1, and qDTY4.1 in the high-
yielding variety IR64 using a marker-assisted backcrossing strategy (Singh et al, 2016).
effectively produced the drought-tolerant elite Malaysian rice cultivar MR219 by pyramiding
three QTLs, qDTY2.2, qDTY3.1, and qDTY12.1.( Dixit et al. 2017) produced TDK1, a rice
variety with excellent yield under drought, by incorporating three QTLs. Drought has only
gained importance as a limitation, and no effective measures have yet been developed to develop
drought-tolerant rice cultivars. The majority of high-yielding cultivars, including as Swarna,
Samba-mahsuri, and IR36, which were previously recommended for cultivation in irrigated
regions, were utilised in a drought breeding programme. Farmers who plant these kinds in
rainfed ecosystems during frequent drought spells experience large losses in rice production
since the aforementioned high-yielding varieties cannot withstand numerous droughts . As a
result, in the future, greater attention will be required to improve special rice varieties with high
yield during drought and adaptation to a wide variety of harsh climatic circumstances.
2.10.4 Marker-Assisted Selection (MAS) for Drought Tolerance
The MAS technique has the potential to speed breeding efforts for drought tolerance, which is a
complicated genetic and physiological issue. A significant number of genes
are believed to be implicated in drought tolerance. These genes are involved in signal
transduction, osmotic correction, and transcriptional control. Drought tolerance is significantly
influenced by several QTLs. Research has linked the QTL on chromosome 9 to spikelet fertility
under stress, as well as root and shoot attributes (Courtois et al., 2000). Plant breeders use MAS,
a DNA-based marker, for three purposes: (a) accumulating beneficial alleles through generations,
and (b) identifying desirable individuals from separated breeding lines based on the complete
genome (c) Break undesired linkage sites to introduce beneficial alleles or parts of the allelic
composition. Modern breeding procedures utilise several names such as marker-assisted
selection (MAS), marker-assisted pedigree selection (MAPS), genomic selection (GS) or
genome-wide selection (GWS), marker-assisted recurrent selection (MARS), and marker-
assisted backcrossing. MABC is the most extensively used and effective strategy among those
described above(Miah G et al. 2017) When a single gene controls the expression of a desirable
characteristic or is responsible for a high percentage of phenotypic traits, it is possible to transfer
a specific region from one parent to the other.MABC has been shown to significantly improve
such traits.Many plant breeders use MABC because it is more cost-effective and faster than
traditional phenotypic approaches. Using MAS in backcrossing (BC) offers three key benefits:
Selecting a connected maker to an allele from a donor cultivar at a locus closer to the target gene
can improve the accuracy and effectiveness of selection for complex phenotypic traits, while also
allowing for introgression. MAS allows for the transfer of a desirable gene from a donor parent
to a recipient parent while maintaining the recipient parent's essential characteristics.
Conventional breeding methods require additional selfings in each backcross generation, which
can be excessively low for most breeding purposes. MAS is an effective method for boosting
selection gain, especially in phenotypic selection., (Knapp et al.2017 ) Quantitative genetics
theory argues that the effectiveness of MAS is inversely related to the heritability of desirable
qualities. Knapp's hypothesis for estimating genotype selection by MAS defines the parameter
for estimate of the cost-effectiveness of MAS for phenotypic selection. Plant breeders using
conventional phenotypic selection may need to test 1.0 to 16.7 times more breeding lines than
those using MAS to select superior genotypes, depending on trait heritability, selection pressure,
and genotypic superiority.Using MAS can drastically reduce the time and resources needed to
achieve a selection aim for low to moderate heritability features.The selection intensity is strong
MAS is the most precise, environmentally safe, quick, and cost-effective technology for
developing drought-resistant rice cultivars. Genetic engineering has enabled the identification,
cloning, and introgression of several QTLs in rice to create drought-resistant variants. Analytical
and theoretical research suggest that combining phenotypic and molecular information leads to
the most effective selection of quantitative traits. DNA markers help identify superior genotypes
in early generations, reducing the number of examined progenies and speeding up breeding
programmes. An overview of the approach used in developing drought tolerant rice
varieties(Francia, E et al.2005)

2.10.6 Harnessing OMICS Strategies for Improving Rice Resilience to Abiotic Stress
Omics is a study that examines how biological information functions and interacts across diverse
life forms. This field studies genes, transcripts, proteins, metabolites, and lipids.
(Langridge and Fleury et al.2011) define interactions (interactomics) and phenotypes
(phenomics). Crop yield is limited by environmental stressors such as water scarcity, harsh
temperatures, and high salt levels in soil or water. (Nogoy et al. 2016) identify four types of
stresses: drought, heat, cold, and salinity.

2.10.6.1 Genomics
Genomic research is the study of an organism's entire genetic makeup. Genomic science include
DNA sequencing, recombinant DNA, and genome function analysis. The genetic code forms the
foundation of all biological life. Sequence of the primary Studying a genome requires
understanding the DNA code and gene expression pathways under various circumstances. The
International Rice Genome Sequencing Project (2005) successfully sequenced the entire rice
genome (O. sativa L.) (Sasaki, 2005). Rice is the only plant genome that has been fully
sequenced after Arabidopsis (Bolger et al., 2014). Plant adaptation begins with perceiving the
stress. Plants start responding to abiotic stress. Plants have various receptors that detect stress.
The initial perception of stress triggers a signal transduction process. The activation of genes that
respond to stress.The primary function of secondary messengers in signal transduction cascades.
Stress-induced genes produce molecules with two separate roles. They encode proteins involved
in signal transduction and plant stress responses. Stress-tolerance genes produce enzymes and
proteins important for osmolyte biosynthesis and ROS detoxification. These pathways are tightly
controlled, and the output of a single genePlants possess highly controlled pathways where one
gene can act as a receptor or activator for other genes, regulating gene expression patterns
(Hasegawa et al. 2000) found that different abiotic stressors can activate genes through
comparable signalling pathways.

2.10.6.2 Transcriptomics
A transcriptome refers to the entire set of RNA molecules expressed by a single cell or
population. There are several types of RNA, including messenger RNA (mRNA), transfer RNA
(tRNA), ribosomal ribonucleic acid (rRNA), microRNA (miRNA), and RNA interference.There
are various types of RNA, including InRNA, sRNA, nRNA, snRNA, and piRNA.
Transcriptomics involves studying transcriptome data (McGettigan, 2013). RNA-Seq combines
high-throughput sequencing and computational approaches. This method detects and quantities
transcripts in an RNA extract. Microarrays are a hybridization-based technology. This approach
can only quantify known genes and has a limited range of application. RNA-Seq can analyse
previously unidentified genes.(Lowe et al., 2017)Plant cells respond to stress signals by
activating specific genes first. Transcription factors are signal transduction proteins that regulate
the activity of RNA polymerase II. Thus, during stress situations, the cell patterns of
transcription (Khong et al. 2008) found that scriptome components contribute to stress
tolerance.Transcriptomic study of stress-exposed plants identifies key molecules for adaptability.
(Muthuramalingam et al. 2017) validated transcriptome information and identified significant
stress-responsive genes.

2.10.6.3 Proteomics
After genomes and transcriptomics, the next stage in studying biological systems is the analysis
of cell complex protein composition. The genome of a cell is more stable than its proteome.
Because the same genes have distinct patterns of expression under varied situations.(Dhingra et
al. 2005); Proteomics is the analysis of all proteins in a cell, organ, or organism at a specific
moment. Proteomic approaches can be used for a variety of studies, including proteome
profiling, differential expression analysis, post-translational modification identification, and
protein profiling.(Chandramouli and Qian, et al. 2009).It has been demonstrated that there is no
direct association between the cell transcriptome and proteome.Since mRNA does not always
convert into proteins. The cell proteome is determined by gene transcription levels and mRNA
translation, depending on the physiological condition.(Buckingham, 2003)Cells produce diverse
sets of proteins during development, differentiation, and the cell cycle, as well as in response to
stressors. Furthermore, post-translational protein changes provide complexity to the proteome.
Barkla (2016) suggests that specific proteins can serve as indicators for specific illnesses .

2.10.6.4 Metabolomics
The “omics” sciences focus on the chemical processes of metabolites as a key research area.
Metabolomics is the study of quantifying biological chemicals produced or destroyed in
organisms. Typically, the representation of all.The “metabolome profile” refers to the collection
of metabolites produced by cellular processes within a biological unit (Kusano et al., 2015).
Transcriptomics and proteomics investigations reveal the gene products produced in cells. These
data aid scientists in understanding one element of cellular activity. Metabolite profiling gives a
static snapshot of a cell’s physiological state. Integrating transcriptomic, proteomic, and
metabolomic findings into a single pipeline can lead to improved outcomes(Arbona et al.,
2013).To cover all metabolites, numerous analytical techniques are used, including separation
and detection with mass spectrometry (MS).Gas chromatography-mass spectrometry is the most
commonly utilised technology for plant metabolomics research. This approach primarily targets
volatiles and principal metabolites following derivatization. Thin layer chromatography is
suitable for a wide range of chemicals, particularly secondary metabolites without previous
derivatization . Capillary electrophoresis-MS identifies polar and charged substances based on
charge-to-mass ratio. Nuclear magnetic resonance spectroscopy differs from MS-based analytical
techniques that rely on atomic interactions (Obata and Fernie, 2012).Plants adapt to
environmental challenges by altering their transcriptional and post-translational patterns, as well
as metabolite synthesis and accumulation. Plants undergo physiological adaptations to adapt to
harsh circumstances (Verslues et al).

2.10.6.5 Phenomics
Phenomics is a high-throughput examination of an organism’s morphological, physiological, and
biochemical properties, including the association of genetic, epigenetic, and environmental
factors (Deshmukh et al., 2014). Plant phenomics is the study of plant growth, performance, and
characteristics. Phenotyping technical advancements have Genetic markers are often used to
predict phenotypes from germplasm collections. This strategy is known as “genetic symptoms”
(Deshmukh et al., 2014). This screening examines the correlation amongst phenotypic traits and
biotic or abiotic factors influencing the phenotype. Tolerant plants exhibit unique traits in
genomes, transcriptomics, and proteomics.Omics and metabolomics. Integrating phenomics with
other omics methods can provide valuable insights into how cell biochemical and biophysical
processes result in a specific phenotype. Plant phenotypes are complicated due to genetic
interactions with environmental variables (Bilder et al., 2009).Crop enhancement of response to
climate conditions can be studied using automated, high-throughput phenotyping machines.
Phenomics has revealed significant insights into background variation, trait relationships, and
plant growth behaviours (Brown et al., 2014). Analysing phenotyping systems allows researchers
to link multi-parametric data to genetic variability. Automated imaging methods provide non-
destructive phenotypic data for quantitative investigations, including growth, tolerance,
resistance, physiology, and yield (Li et al., 2014). This technology can evaluate numerous
physiological traits for a single plant. Automated imaging offers advantages such as high speed
and accuracy, as well as the ability to scan temperature profiles, measure photosynthetic and
growth rates, and understand root physiology (Finkel, 2009).

2.11 Molecular responses and drought stress management in rice

Drought promotes a variety of chemical responses in rice. Molecular research into drought stress
has shown significant changes in gene expression, encouraging the identification and analysis of
their regulatory components. This understanding drives the development of novel techniques to
improving plant resilience, with detailed studies covering the complex signalling systems of
drought stress in numerous plant species, including rice. Studies on drought-induced gene
expression patterns in plants revealed that most drought-inducible genes respond to ABA
treatment, while others do not ( Duet al., 2018).Rice has both ABA-independent and ABA-
dependent regulatory networks for gene expression, which influence drought escape
mechanisms. Research on ABA-inducible genes in model plants has showed that numerous
genes require protein biosynthesis to be stimulated by ABA, indicating the presence of at least
two different pathways linking endogenous ABA production to gene expression under stress
circumstances. Two of the four signal transduction pathways found are ABA-dependent (I and
II), whereas the other two are ABA-independent (III and IV). One of the ABA-dependent
processes (I) requires protein synthesis, whilst another ABA-dependent pathway (I) involves the
abscisic acid response element.pathway (II). Within the ABA-independent pathways,
drought/dehydration responsive elements (DRE) regulate genes in response to drought, salt, and
cold stress in one pathway (IV), while another ABA-independent pathway responds to drought
and salt stress but not cold.(Rabello et al. 2008)Several drought-responsive genes have been
found in the roots of upland rice (Oryza sativa L.). These genes govern signalling pathways such
as Ca-dependent protein kinase and ethylene-responsive factors, as well as CO2 metabolism,
oxidative damage reduction, cellular osmoregulation, and ionic balance. Transcriptomic and
proteomic investigations spanning many plant species, including rice, have shown numerous
pathways associated with drought-responsive gene expression (Jogaiah et al., 2012; Nahar et al.,
2016)

2.12 Nanobiotechnology and its intervention in drought stress management in rice

Various research on the impact of nanoparticles on plant growth and development have revealed
enormous promise for nanotechnology applications in agriculture. In the past decade, plant and
agricultural experts have shown great interest in the development of nanotechnological addresses
in agriculture to enhance productivity of crops at low costs as well as energy usage through the
application of nanomaterials in the form of synthetic fertiliser, nanopesticide, nanoherbicide,
nanosensor, and smart delivery methods for controlled release of agricultural chemical products
for biotic and abiotic stress management of crop plants. According to studies, the application of
silica nanoparticles improves plants’ tolerance to water deficit stress.( Ashkavand et al. 2015)
found that silica nanoparticles improved photosynthetic metrics, malondialdehyde, RWC, and
membrane electrolyte leakage in plants( Hattori et al. 2005) demonstrated that the application of
silicon can improve drought tolerance in crops. Thus, silicon nanoparticles could potentially be
employed to reduce drought stress in crop plants such as rice.( Pei et al. 2010) demonstrated that
sodium silicate might somewhat reduce the effects of drought stress in wheat.(Sedghi et al. 2013)
Zinc oxide nanoparticles have been shown to boost seed germination percentage and rate in
soybean plants under drought stress. Studies have shown that the use of composite
micronutrients can help plants cope with drought and salinity stress . (Davar et al. 2014)
proposed that foliar application of iron nanoparticles can reduce drought stress impacts on yield
components and oil % in Carthamustinctorious L. Foliar spraying of titanium nanoparticles to
crops has been shown to modify seed gluten and starch levels while alleviating the negative
effects of drought stress. Thus, the use of titanium dioxide nanoparticles may contribute to the
improvement of certain agronomic features in crop and zinc oxide nanoparticles have also been
shown to affect physiological and biochemical responses in O. Sativa L reported that ZnO NP at
a low dose promotes growth and antioxidant activities, and that a specific concentration of ZnO
NP can improve the qualitative and quantitative characteristics of rice plants. discovered that
ZnO NP improves Zn absorption and translocation in rice, which has a good influence on
growth, yield, and moisture stress tolerance. Drought stress reactions in crops in general, and
particularly in rice, can be regulated using nanobiotechnological intervention by emphasising
designing nanoparticles for improved positive impacts with less toxicity, so that nanomaterials
find their application not just in drought stress .However, various abiotic stress and nutrient
management strategies ensure higher rice output.

MATERIALS AND METHODS

32 types of Rice varieties were used in the experiment for screening based on germination and
Hydroponics lab setup system .The seeds were Available in the laboratory of the supervisor

Sl . No Genotype
1 185
2 206
3 210
4 236
5 238
6 241
7 246
8 247
 9 252
10 254
11 257
12 262
13 263
14 264
15 267
16 268
17 269
18 235-1
19 238-3
20 249-1
21 251-1
22 253-1
23 254-1
24 261-1
25 261-2
26 261-3
27 263-1
28 263-2
29 267-2
30 268-1
31 268-2
32 268-3

Germination of seeds

The seeds were washed thoroughly with distilled water. Twenty-five seeds of each variety were
taken.These seeds were then soaked in water and were imbibed for 24-48 hours and then the
seeds were placed in Petri dishes containing Normal tissue paper to allow them to germinate.

Transplanting it to hydroponic system

After the seed germination upto 4-5 cm all the seeds of each genotype were transferred To the
hydroponics system. The hydroponic system consisted of plastic tray with a Capacity to hold
upto 5 L of nutrient solution. Thermocol sheets having 32 holes and Knitted with plastic net that
was being used as a substrate in the hydroponics system Photoperiodism was maintained under
artificial alternative lightning.Those seedlings were allowed to grow upto 10-15 cm which took
around 15 days. Each alternative days the pH of the nutrient solution was checked which was
supposed To be maintained at 5.5.After 15 days of growth the length of each plant of every
variety Were measured. The next day , PEG treatment was given to all the 4 sets. Three
Different concentrations were taken for the experiment along with a control group in Each
set .After 7 days of treatment a visual observation was done for long root , short Root and Root
color(the roots color were not much distinguishable.) Right after 7 days Of treatment the
seedlings were taken out of the system and was dismanteled for Respective root and shoot length
and the Fresh weight was also measured simultaneously.

PEG TREATMENT
Four different Trays were used in this experiment, After 15 days the plantlets are transferred to
the Trays filled with poly-ethylene glycol with a concentration of control,5%,10%,15%
respectively with 4 different Trays and monitor regularly.Under hydroponics screening moisture
stress is induced artificially by using poly ethylene glycol (PEG) 6000 MW in various strengths
(Swapna and Shylaraj, 2017).

Collection of leaf sample

Rice plants of same genotype which are are alredy examined under Different concentration of
PEG are collected from field . 2-3 fresh young leaves from each genotype collected and placed
them immedietly to a test tube containing normal tap water which prevent leaf rolling. The
samples were then put in a ziplock bag and stored at -20°C refrigerator.

DNA Isolation

For DNA isolation from the leaves , TPE buffer method was used to obtain high-quality DNA
from the leaf samples. Tris-Phosphate-EDTA (TPE) is a commonly used buffer solution in
molecular biology for gel electrophoresis and gene expression studies. Its main purpose is to
solubilize DNA or RNA while protecting it from degradation.

To prepare 100ml.of TPE buffer, Take 70ml. of distled water of pH 8.0, add 10ml. of 1M Tris
Hcl of pH 9.5 ,7.4 gm of Kcl ,2ml.of 0.5M EDTA of pH 8.0 and making volume 100 ml . by
adding distled water .
For the DNA isolation process, By taking 2-4 cm long leaf tissue from the stored leaf samples
and crushed it carefully into a powdered form using liquid nitrogen with the help of micropestle .
Then, by adding 200 µl of TPE buffer to each leaf sample. The samples were then incubated at
maximum 65°C for 25-30 minutes with a shaking stab to the samples in every 5 mintues for 30
sec.-60 sec.depending on the tissue type. After incubation or water bath 500µl of water were
added to each tube to dilute the extract, and the mixture was vigorously mixed. Then the
samples were then centrifuged at 10,000 rpm for 15-16 minutes at room temperature, and the
supernatant was collected carefully with micropipette without disturbing pellets .The collected
supernatants contains DNA and were stored in a 1.5 ml. Centrifuged tube at -20° C .

Gel electrophoresis

Agarose gel electrophoresis is a technique used to separate nucleic acid fragments (DNA or
RNA) based on their sizes, an agarose gel is utilized. When an electrical current is applied to the
gel, negatively charged DNA/RNA migrates through the pores towards the positively charged
end of the gel, with smaller ones moving more quickly. The resulting bands can be visualized
using ultraviolet (UV) light.

Preparation of 1 % Agarose gel

For 1% agarose gel was prepared by dissolving 1 g of Agarose in 100 ml. 0.5 x TBE Buffer. The
solution was boiled till Transparent color visible .When the solution Cols down a bit 10µl (micro
litre )of Ethidium bromide was added to the solution and casted in a electrophoretic casting tray
and the Comb was placed at the end of the gel .They set the comb inside the casting tray and
slowly removed it after the gel solidified. The casting tray with the gel was then placed inside the
electrophoretic chamber with the wells facing the cathode. The electrophoretic tank was filled
with 0.5 X TBE buffer to submerge the gel. Then 10 µl of supernatants were dispatched to PCR
tubes and about 3 µl of Loading dye is used .Then added to the i the small wells of th gels by
using a micropipette. Then ran the gel until the tracking dye covered more than ¾ distance in the
gel.
 3% agarose gel was prepared in the similar manner for separation of PCR amplified product.

PCR

A B

C D
Fig.-representing the morphological changes in rice seedlings under variable PEG
concentrations after 7 days of treatment of Set-4.A:- control B:-5% ,PEG ,C:10%PEG and
D:15%PEG