Abiotic Stress in Cotton: Insights into Plant Responses and Biotechnological Solutions

Abstract

Climate change has rapidly increased incidences of frequent extreme abiotic stresses, such as heat, drought, salinity, and waterlogging. Each of these stressors negatively affects the cotton crop (Gossypium spp.) and results in significant yield decreases. Every stressful event causes specific changes in the metabolism and physiology of plants, which are linked to complex molecular alterations. Understanding the molecular mechanisms that regulate a plant’s response to stress is essential to developing stress-resistant cotton varieties that can withstand various stress factors. Gene expressions in response to multiple stresses have been studied and mapped. These genes include ion transporters and heat shock proteins, which are vital to allowing adaptive responses. These approaches showed the ability to employ advanced genome sequencing and multi-omics techniques to identify dynamic gene expression patterns and elucidate intricate regulatory networks. Using genetic variation in combination with molecular techniques, it would be possible to generate stress-resilient cotton varieties that would enable sustainable cotton output in the face of abiotic stresses. Here, we reviewed the effects of major abiotic stressors on cotton plants, such as heat, salinity, drought, heavy metals, and waterlogging. We also examine the vast network of proteins, genes, and stress-sensitive signaling pathways that help cotton tolerate abiotic stress.

1. Introduction

Cotton (Gossypium spp.) is a vital fiber crop grown worldwide. However, it has faced significant challenges due to climate change and the increasing frequency of abiotic stress, impacting its yield and quality [1,2,3]. Abiotic stress has significantly reduced global cotton crop production, leading to substantial declines in yield and quality. Since cotton is a crucial crop for textile production and edible oil extraction, any reduction in its yield due to abiotic stress can have significant global economic impacts [4,5,6].

Critical abiotic stresses are considered the primary factors limiting cotton productivity. These stressors include various environmental conditions, such as extreme temperatures (both high and low), drought, salinity, heavy metals, and radiation, which disrupt cotton plants’ normal physiological and metabolic processes [7]. Also, various biotic factors, including pathogens, weeds, pests, and abiotic factors, including temperature, humidity, and drought, are involved in reducing cotton yield, significantly reducing cotton production. Various biotic factors directly affect cotton production and cause significant reductions in cotton crop yield, estimated to be up to 10 to 30%. In comparison, abiotic factors are even worse than biotic stresses and could cause a 50% reduction. Cotton yield has shown up to 50–60% yield losses due to drought and biotic stresses [8]. At higher temperatures (≥36 °C), the most significant decrease in carbon fixation occurs, and the plant struggles with an unpredictable growth habit, becoming susceptible to drought stress [9,10,11,12]. According to recent studies, high temperatures are one of the main abiotic factors that negatively affect cotton yields by impairing the photosynthetic process and membrane thermostability, which impacts plant metabolism [13,14,15]. Higher temperatures can denature proteins and alter enzyme activity; in cotton, high-temperature stress can impair the activity of critical enzymes like superoxide dismutase (SOD) and catalase (CAT), which are crucial for managing oxidative stress and maintaining metabolic processes [16,17]. Drought stress leads to a reduction in turgor pressure, which has a direct or indirect impact on photosynthesis and the metabolism of carbohydrates [18,19]. Drought reduces boll retention and leaf size in cotton plants. Breeding stress-tolerant cotton varieties, improving agricultural practices, and developing innovative technologies are necessary to ensure sustainable cotton production under challenging ecological stressors. These approaches can enhance the crop’s resilience to extreme conditions such as drought, salt, and high temperatures, ultimately improving yield and quality [20].

Breeding approaches are traditionally used to improve cotton stress resilience and yield. Considering the evolving multi-stress scenarios, cotton varieties with improved stress tolerance are highly desirable. Combining modern molecular tools and breeding would provide potential solutions for developing abiotic stress-resilient cotton varieties. Here, we reviewed recent advances in the abiotic stress physiology of cotton and its impacts on yield and quality by summarizing relevant studies on drought, heat, salinity, heavy metals, and waterlogging under changing climates. We further reviewed the potential of biotechnological approaches to developing stress-tolerant varieties, such as RNA interference (RNAi), multi-omics, transgenic approaches, and genome editing. The combined use of biotechnological tools enables a thorough understanding of the biochemical and metabolic changes in the cotton plant during stress encounters. It may allow for the development of faster stress-adapting cotton varieties. Thus, investing in research and development to understand the genetic basis of stress tolerance will also contribute to the long-term sustainability of cotton farming.

2. Abiotic Stressors Impact on Cotton Plants

The cotton plant faces various environmental stressors in the changing climate that can significantly affect its productivity. Understanding the impacts of each stressor on cotton growth is essential for enhancing stress tolerance and developing effective mitigation strategies. The physiological changes induced by abiotic stressors offer valuable insights into the underlying molecular mechanisms that govern cotton’s response to environmental challenges. Physiological changes provide a comprehensive insight into the intricate molecular mechanisms within cotton plants under abiotic stress [19,21,22]. Various physiological and biochemical changes in cotton in response to environmental stressors are summarized in Table 1 and Table 2, which provides a detailed summary of these physiological and biochemical responses, highlighting how different abiotic stressors, such as drought, salinity, and heat stress, affect cotton crops.

Significant changes determining the plant’s resilience to stress include alterations in photosynthesis, water use efficiency and altered oxidative stress pathways. In addition, modifications in root development, canopy, and bolls per plant are crucial in response to stress resilience. By exploring these responses, we can better understand the complex interactions between stress factors and cotton’s adaptive mechanisms, paving the way for more targeted approaches to improving stress tolerance in cotton. The following subsections discuss cotton responses to major abiotic stressors in detail.

2.1. Impact of Drought Stress on Cotton

Drought is a critical abiotic stress factor that significantly affects cotton production. The impact of water stress on yield depends on the length and duration of the drought. Water scarcity disrupts various physiological and biochemical processes in cotton plants, reducing growth and yield. Water plays a critical role in every stage of plant growth, from seed germination to maturation and overall morphology [23]. Water shortages or droughts can affect reproductive growth and reduce agricultural productivity, while excess water during flowering leads to vegetative dominance, causing flower and boll drop. Reduced cotton yield under drought conditions is primarily due to decreased boll numbers and, to a lesser extent, reduced boll weight, which is linked to increased square drop and boll abscission [24,25,26,27]. Therefore, adequate water availability from square formation to boll development is crucial for achieving higher yields. Drought stress also impacts fiber development and quality. Given the substantial demand for cotton fiber, enhancing crop tolerance to drought stress is of prime importance, requiring a profound understanding of the morphological and physiological basis for selecting and breeding drought-tolerant cotton varieties.

Drought stress reduces photosynthetic activity due to stomatal and non-stomatal factors [11,13,14,28,29,30,31]. The reduced relative water content and leaf water potential lead to lower photosynthetic rates, with complex effects involving stomatal closure and potential chlorophyll reduction [32,33]. Drought causes cotton plants to close their stomata, reducing gas exchange, transpiration, and nutrient uptake [34]. Water deficit impacts physiological processes such as stomatal conductance, CO₂ diffusion, and photosynthesis. Non-stomatal factors primarily cause the reduction in photosynthetic rate during severe drought, while during mild drought, stomatal limitation drives the decrease in photosynthetic rate [35]. A negative correlation between guard cell length and stomatal density on both leaf surfaces in cotton varieties suggests an architectural synchronization, either direct or indirect, between stomatal numbers and guard cell length under water stress [36].

Cotton plants have adaptive strategies for drought stress, such as modifying root architecture and activating stress response genes [37,38]. Leaves of preconditioned plants maintain turgor at lower water potential than well-watered counterparts, accompanied by reduced osmotic potential. The genes GhRD29A, GhDREB1A, GhP5CS, GhBADH, GhPIP, and GhTIP were upregulated in response to drought stress. The expression of EPF2, SDD1, and TMM genes, which regulate stomatal development, exhibited a distinct pattern in stomatal numbers and guard cell length in the drought-tolerant cotton variety under drought stress. Moreover, these findings suggest that the EPF2, SDD1, and TMM genes are potentially valuable targets for developing transgenic cotton varieties with enhanced drought tolerance [36]. GhMYB44 promotes stomatal closure, contributing to enhanced drought tolerance in cotton. Physiological analysis under drought stress revealed that GhMYB44 improves cotton’s drought resilience by facilitating the detoxification of reactive oxygen species (ROS) [39]. Understanding the impacts of abiotic stresses on cotton is essential for sustainable cultivation, and creating such cultivars through breeding programs is crucial for improving stress tolerance [40,41,42,43].

Table 1. Impact of abiotic stress on cotton physiological changes. Growth impacts are indicated based on the severity of the effect observed, ranging from negative (-) to positive (+).

Stress factor	Impact on Development and Traits	Impact	Citations
Heat stress	Leaf wilting	+	[44]
	Photosynthesis efficiency	-	[45]
	Fiber quality	-	[46]
	Leaf number	-	[47]
	Stomatal density	-	[48]
	Trichome density	+	[49]
	Flowering	-	[50]
	Boll size	-	[51]
	Root length	-	[45]
	Shoot length	-	[52]
	Premature boll opening	+	[53]
Drought	Leaf rolling	+	[54]
	Leaf growth	-	[55]
	Leaf area	-	[56]
	Fiber quality	-	[57]
	Root length	-	[58]
	Shoot length	-	[59]
	Stomatal density	+	[60,61]
	Trichome density	+	[62]
	Flowering	--	[63]
	Leaf number	--	[64]
	Boll size	-	[65]
	Premature boll opening	+	[63]
Salinity	Root length	-	[66,67]
	Leaf necrosis	+	[68]
	Nutrient absorption	-	[25]
	Fiber quality	-	[69]
	Stomatal density	+	[70]
	Boll size	-	[71]
	Premature boll opening	+	[72]
	Leaf rolling	+	[73]
	Leaf area	-	[74]
	Shoot length	-	[75]
	Trichome density	-	[76]
	Flowering delay	+	[77]
	Leaf number	-	[78]
Heavy metal toxicity	Shoot length	-	[79]
	Leaf chlorosis, necrosis	+	[80]
	Leaf rolling	+	[81]
	Leaf area	-	[80]
	Fiber quality	-	[82]
	Root length	-	[79]
	Stomatal density	+	[80]
	Flowering	-	[83]
	Leaf number	-	[84]
	Boll size	-	[85]
	Premature boll opening	-	[85]
Water logging	Nutrient deficiency	+	[86]
	Leaf chlorosis	+	[87]
	Shoot length	-	[88]
	Leaf area	-	[89]
	Fiber quality	-	[90]
	Root length	-	[91]
	Stomatal density	-	[92]
	Trichome density	+	[92]
	Flowering	-	[89]
	Boll size	-	[92,93]
	Premature boll opening	+	[94]

Table 1. Impact of abiotic stress on cotton physiological changes. Growth impacts are indicated based on the severity of the effect observed, ranging from negative (-) to positive (+).

Stress factor	Impact on Development and Traits	Impact	Citations
Heat stress	Leaf wilting	+	[44]
	Photosynthesis efficiency	-	[45]
	Fiber quality	-	[46]
	Leaf number	-	[47]
	Stomatal density	-	[48]
	Trichome density	+	[49]
	Flowering	-	[50]
	Boll size	-	[51]
	Root length	-	[45]
	Shoot length	-	[52]
	Premature boll opening	+	[53]
Drought	Leaf rolling	+	[54]
	Leaf growth	-	[55]
	Leaf area	-	[56]
	Fiber quality	-	[57]
	Root length	-	[58]
	Shoot length	-	[59]
	Stomatal density	+	[60,61]
	Trichome density	+	[62]
	Flowering	--	[63]
	Leaf number	--	[64]
	Boll size	-	[65]
	Premature boll opening	+	[63]
Salinity	Root length	-	[66,67]
	Leaf necrosis	+	[68]
	Nutrient absorption	-	[25]
	Fiber quality	-	[69]
	Stomatal density	+	[70]
	Boll size	-	[71]
	Premature boll opening	+	[72]
	Leaf rolling	+	[73]
	Leaf area	-	[74]
	Shoot length	-	[75]
	Trichome density	-	[76]
	Flowering delay	+	[77]
	Leaf number	-	[78]
Heavy metal toxicity	Shoot length	-	[79]
	Leaf chlorosis, necrosis	+	[80]
	Leaf rolling	+	[81]
	Leaf area	-	[80]
	Fiber quality	-	[82]
	Root length	-	[79]
	Stomatal density	+	[80]
	Flowering	-	[83]
	Leaf number	-	[84]
	Boll size	-	[85]
	Premature boll opening	-	[85]
Water logging	Nutrient deficiency	+	[86]
	Leaf chlorosis	+	[87]
	Shoot length	-	[88]
	Leaf area	-	[89]
	Fiber quality	-	[90]
	Root length	-	[91]
	Stomatal density	-	[92]
	Trichome density	+	[92]
	Flowering	-	[89]
	Boll size	-	[92,93]
	Premature boll opening	+	[94]

2.2. Impact of Heat Stress on Cotton

Heat stress is a significant threat to cotton agriculture globally, affecting this key fiber crop’s growth, development, and productivity [95]. Heat stress adversely affects membrane stability, photosynthesis, root development, and stomatal conductance, and these physiological impairments are linked to heat-induced damage to the lipid membranes of cellular organelles [96,97]. Elevated temperatures can raise respiration rates in cotton plants, potentially surpassing photosynthesis, leading to energy deficits and reduced biomass accumulation [98]. This decline in photosynthesis may be attributed to stomatal closure and decreased PSII efficiency [99]. Heat stress exacerbates water loss through transpiration, resulting in water deficit circumstances and impairing the plant’s capacity to maintain adequate hydration [100]. Cotton varieties with efficient antioxidant systems, sustained photosynthesis rates (Pn), and stable stomatal conductance (Gs) exhibit enhanced resilience to heat stress. At the same time, osmoregulation-related genes and transcription regulatory factors further support regulating the plant’s internal environment under stress conditions [101,102]. Yousaf et al. (2023) demonstrated that heat stress negatively impacts cotton yield by reducing germination rate, net photosynthetic efficiency, relative cell stability, sympodial branches, and boll weight while increasing the abscission of flowers, squares, and bolls [103].

Moreover, heat stress also compromises lint quality, resulting in shorter fibers and decreased fiber fineness and uniformity [104]. In addition to these effects, heat stress induces wilting, reduces photosynthesis, and leads to flower and boll shedding, whereas cold stress causes chilling injury, stunted growth, and lower yields. Key factors influencing cotton yield include shortened boll development periods, increased bud and boll abscission, and extreme temperature fluctuations during flowering and boll formation. Heat stress, particularly during critical stages such as blooming and boll development, can degrade fiber quality by reducing fiber length and strength, altering micronaire values, and ultimately lowering the market value of cotton [105,106].

Cotton’s resilience to heat stress relies on a range of physiological and biochemical responses, including water regulation and cellular defense mechanisms such as heat shock proteins (HSPs) and antioxidants, essential for preserving cellular integrity and overall plant health under high temperatures. The genes GhHSP70, GhHSP90, GhMYB, GhbZIP, GhCAT, and GhPOD were upregulated in response to heat stress in cotton. Breeding programs should prioritize the development of heat-tolerant cotton cultivars by targeting key traits like increased HSP expression, enhanced photosynthetic efficiency, and improved tolerance to elevated temperatures. A comprehensive understanding of the impacts of heat stress and implementing proactive strategies are vital for maintaining cotton production, fiber quality, and yield stability amid rising global temperatures due to climate change [100]. Further exploration of stress-responsive genes can facilitate the creation of genetically engineered cotton varieties with superior tolerance to harsh environmental conditions [107].

2.3. Impact of Salinity Stress on Cotton

Salinity stress has been the primary limiting factor for agricultural productivity across the biosphere. Around 20% of the world’s cultivated area is impacted by salinity (salt) stress [108,109]. The increasing soil salinity in arable lands worldwide is driven by improper irrigation practices, particularly the use of saline water, along with faulty agronomic practices such as improper fertilization, excessive water usage, and pesticide applications. The issue of deteriorating soils is further exacerbated by limited precipitation, excessive surface evaporation, weathering of local rocks, and the impacts of climate change, all contributing to the growing salinity problem in agricultural soils [110]. Causes of the rising salinity problem include little precipitation, excessive surface evaporation, weathering local rocks, irrigation with saline water, and unfavorable cultural practices such as excessive water use, fertilizers, and pesticides [110]. Specifically, secondary salinization causes formerly productive agricultural lands to lose their suitability for agriculture because of substandard irrigation water.

Soil salinity is a major abiotic stressor that hinders plant growth by disrupting water uptake and ion balance, leading to ion toxicity, osmotic stress, oxidative damage, and reduced nutrient uptake, leading to stunted growth, leaf burn, and reduced yield [111]. Salt stress induces morphological, physiological, biochemical, and metabolic changes that ultimately impair plant growth, development, and productivity. Elevated soil salinity significantly impacts cotton growth and overall productivity, reducing boll weight and the number of bolls, delaying flowering, and increasing fruit shedding, thus adversely affecting seed cotton yield [108,109,112]. Excessive salt concentration in the soil interferes with water uptake and ion balance in cotton plants, causing ion toxicity, reduced growth, leaf burn, and diminished yield. Excess soil salinity has a detrimental effect on cotton growth and productivity [109,112].

To resolve salinity stress with notable genotype variations, induced salt stress adversely affects germination and vegetative growth. This stress reduces seed germination, decreases water uptake by developing seeds and plant roots, and impairs photosynthesis and protein synthesis [111,113,114]. Soil salinity stress causes an excessive build-up and production of ROS in mitochondria and chloroplasts, such as superoxide anion (O⁻²), hydroxyl radicals (OH), and hydrogen peroxide (H₂O₂) [115,116,117] (Table 2). ROS are highly reactive, and without any protective mechanism, they can seriously disrupt normal metabolism through oxidative damage to lipids, proteins, and nucleic acids. However, plants develop defense strategies against salt stress by activating antioxidant enzymes [118,119].

The cotton plant has shown resilience against salt stress compared with other crops. Understanding cotton’s resilience to salt, its response, and management tactics can help develop solutions to enhance cotton performance in saline environments [120]. Proline and glycine betaine are effective ROS scavengers and protective agents acting as osmolytes for macromolecules under salt stress. Many plant species build up large amounts of proline in response to salinity and drought stress; this protein is involved in stress adaptation [77,121,122]. Several antioxidant enzymes, including SOD, glutathione reductase (GR), and ascorbate peroxidase (APX), are found in plants and are used to strengthen defenses against ROS, such as superoxide anion (O⁻²) [123]. The genes GhSOS1, GhNHX1, GhNAC, GhWRKY, GhHKT1, and GhNHX2 were upregulated in response to salinity stress.

Furthermore, transgenic plants overexpressing antioxidant enzymes can withstand salinity stress [124]. The amalgamation of these findings offers a comprehensive perspective on how cotton plants effectively manage physiological disruptions triggered by salinity stress. A better understanding of cotton’s resilience to salt, its response, and management tactics can help develop ways to enhance cotton performance in saline environments [47,125].

Table 2. Summary of the effects of various stress conditions on biochemical traits in cotton genotypes. Explant source refers to the tissue used for analysis, and the screening method indicates the technique employed for measurement.

Abiotic Stress	Biochemical Traits	Effects on Biochemical Traits	Explant Source	Screening Method	References
Drought stress	Proline	Increased levels under drought stress are indicative of osmotic adjustment.	Leaf	High-performance liquid chromatography (HPLC)	[126,127]
	Chlorophyll	Decreased levels under heat stress indicate photoinhibition.	Leaf	HPLC	[128,129]
	Antioxidant enzyme activity	Enhanced activity under oxidative stress protects against damage.	Leaf	Enzyme assays	[130,131]
	Ion homeostasis	Alterations occur in nutrient uptake, essential for plant growth.	Root	Ion analysis	[132]
Heat stress	Heat shock protein expression	Induced expression under elevated temperatures leads to aiding protein stability.	Leaf	Protein analysis	[133,134]
	Peroxidase (POD) activity	Changes in metabolic pathways impact plant growth and development.	Root	Enzyme assays	[135,136]
	Lipid peroxidation	Increased levels indicate membrane damage under stress conditions.	Leaf	Thiobarbituric acid assay	[137,138]
	Soluble sugar content	Accumulation acts as an osmoprotectant, maintaining cellular integrity.	Leaf	Spectrophotometry	[137,139]
	Total phenolic content	Elevated levels contribute to antioxidant defense against stress.	Leaf	Spectrophotometry	[140,141]
	Malondialdehyde (MDA) content	Elevated levels indicate lipid peroxidation and cellular damage.	Leaf	Spectrophotometry	[142,143]
	Superoxide dismutase (SOD) activity	Increased activity under oxidative stress leads to scavenging superoxide radicals.	Leaf	Enzyme assays	[144,145]
	Catalase (CAT) activity	Enhanced activity under oxidative stress leads to decomposing hydrogen peroxide.	Leaf	Enzyme assays	[146]
	Carotenoid content	Decreased levels impact photosynthetic efficiency under stress.	Leaf	HPLC	[147]
	Flavonoid content	Increased synthesis contributes to stress tolerance mechanisms.	Leaf	Spectrophotometry	[148]
	Ascorbic acid content	Decreased levels affect antioxidant capacity and stress tolerance.	Leaf	Titration method	[149]
	Glutathione content	Altered levels impact oxidative stress response and redox regulation.	Leaf	Enzymatic assay	[150]
	Polyphenol oxidase activity	Enhanced activity in response to stress leads to tissue browning.	Leaf tissue	Enzyme assay	[4]
Salinity	Proline	Increased levels under saline conditions are indicative of osmotic adjustment.	Leaf	HPLC	[112,115,116,117]
	Chlorophyll	Decreased levels under salinity stress indicate photoinhibition.	Leaf	HPLC	[122,123,124]
	Antioxidant enzyme activity	Enhanced activity under saline stress protects against oxidative damage.	Leaf	Enzyme assays	[120,130]
	Ion homeostasis	Altered nutrient uptake due to saline conditions.	Root	Ion analysis	[69,132]
Heavy metal toxicity	Antioxidant enzyme activity	Enhanced enzyme activity reflects an upregulated defense response to mitigate oxidative damage caused by metal toxicity.	Leaf	Enzyme assays	[79,84]
	Chlorophyll content	Significant reduction in chlorophyll levels leads to chlorosis and tissue necrosis because of metal accumulation.	Leaf	HPLC	[80,83]
	Proline accumulation	Increased proline levels serve as a protective osmolyte to counteract the osmotic stress induced by heavy metals.	Leaf	HPLC	[82,85]
Water-logging stress	Antioxidant enzyme activity	Elevated enzyme activity indicates a defensive response to the oxidative stress resulting from excess water.	Leaf	Enzyme assays	[86,87]
	Chlorophyll content	Reduction in chlorophyll concentration leads to chlorosis due to impaired photosynthesis under prolonged waterlogged conditions.	Leaf	HPLC	[92,93]
	Lipid peroxidation	Increased MDA levels suggest oxidative deterioration of cellular membranes under saturated conditions.	Leaf	Thiobarbituric acid assay	[89,94]

Table 2. Summary of the effects of various stress conditions on biochemical traits in cotton genotypes. Explant source refers to the tissue used for analysis, and the screening method indicates the technique employed for measurement.

Abiotic Stress	Biochemical Traits	Effects on Biochemical Traits	Explant Source	Screening Method	References
Drought stress	Proline	Increased levels under drought stress are indicative of osmotic adjustment.	Leaf	High-performance liquid chromatography (HPLC)	[126,127]
	Chlorophyll	Decreased levels under heat stress indicate photoinhibition.	Leaf	HPLC	[128,129]
	Antioxidant enzyme activity	Enhanced activity under oxidative stress protects against damage.	Leaf	Enzyme assays	[130,131]
	Ion homeostasis	Alterations occur in nutrient uptake, essential for plant growth.	Root	Ion analysis	[132]
Heat stress	Heat shock protein expression	Induced expression under elevated temperatures leads to aiding protein stability.	Leaf	Protein analysis	[133,134]
	Peroxidase (POD) activity	Changes in metabolic pathways impact plant growth and development.	Root	Enzyme assays	[135,136]
	Lipid peroxidation	Increased levels indicate membrane damage under stress conditions.	Leaf	Thiobarbituric acid assay	[137,138]
	Soluble sugar content	Accumulation acts as an osmoprotectant, maintaining cellular integrity.	Leaf	Spectrophotometry	[137,139]
	Total phenolic content	Elevated levels contribute to antioxidant defense against stress.	Leaf	Spectrophotometry	[140,141]
	Malondialdehyde (MDA) content	Elevated levels indicate lipid peroxidation and cellular damage.	Leaf	Spectrophotometry	[142,143]
	Superoxide dismutase (SOD) activity	Increased activity under oxidative stress leads to scavenging superoxide radicals.	Leaf	Enzyme assays	[144,145]
	Catalase (CAT) activity	Enhanced activity under oxidative stress leads to decomposing hydrogen peroxide.	Leaf	Enzyme assays	[146]
	Carotenoid content	Decreased levels impact photosynthetic efficiency under stress.	Leaf	HPLC	[147]
	Flavonoid content	Increased synthesis contributes to stress tolerance mechanisms.	Leaf	Spectrophotometry	[148]
	Ascorbic acid content	Decreased levels affect antioxidant capacity and stress tolerance.	Leaf	Titration method	[149]
	Glutathione content	Altered levels impact oxidative stress response and redox regulation.	Leaf	Enzymatic assay	[150]
	Polyphenol oxidase activity	Enhanced activity in response to stress leads to tissue browning.	Leaf tissue	Enzyme assay	[4]
Salinity	Proline	Increased levels under saline conditions are indicative of osmotic adjustment.	Leaf	HPLC	[112,115,116,117]
	Chlorophyll	Decreased levels under salinity stress indicate photoinhibition.	Leaf	HPLC	[122,123,124]
	Antioxidant enzyme activity	Enhanced activity under saline stress protects against oxidative damage.	Leaf	Enzyme assays	[120,130]
	Ion homeostasis	Altered nutrient uptake due to saline conditions.	Root	Ion analysis	[69,132]
Heavy metal toxicity	Antioxidant enzyme activity	Enhanced enzyme activity reflects an upregulated defense response to mitigate oxidative damage caused by metal toxicity.	Leaf	Enzyme assays	[79,84]
	Chlorophyll content	Significant reduction in chlorophyll levels leads to chlorosis and tissue necrosis because of metal accumulation.	Leaf	HPLC	[80,83]
	Proline accumulation	Increased proline levels serve as a protective osmolyte to counteract the osmotic stress induced by heavy metals.	Leaf	HPLC	[82,85]
Water-logging stress	Antioxidant enzyme activity	Elevated enzyme activity indicates a defensive response to the oxidative stress resulting from excess water.	Leaf	Enzyme assays	[86,87]
	Chlorophyll content	Reduction in chlorophyll concentration leads to chlorosis due to impaired photosynthesis under prolonged waterlogged conditions.	Leaf	HPLC	[92,93]
	Lipid peroxidation	Increased MDA levels suggest oxidative deterioration of cellular membranes under saturated conditions.	Leaf	Thiobarbituric acid assay	[89,94]

2.4. The Impact of Heavy Metals and Waterlogging on Cotton Growth and Yield

Various abiotic factors, such as heavy metal toxicity and waterlogging, can adversely affect cotton plants, reducing growth and output. Exposure to heavy metals like chromium, cadmium, manganese, and zinc has been found to increase antioxidant enzyme activity [79,84]. This heightened activity serves as a defensive response, indicating the activation of the plant’s defense mechanisms to counteract the oxidative damage caused by these harmful metals. However, heavy metal stress also significantly decreases chlorophyll content, resulting in necrosis and chlorosis in the leaves, thus directly hampering the plant’s ability to conduct photosynthesis. Reduced leaf area and overall growth are the consequences of the impaired photosynthetic system [80,83].

Furthermore, exposure to heavy metals increases lipid peroxidation, indicating oxidative damage to cell membranes. Various ROS species accumulation causes oxidative stress, which increases cellular damage and may even induce cell death [81,85]. Proline is an osmoprotectant that helps the cells maintain osmotic balance in these unfavorable conditions, and cotton plants exposed to heavy metal stress also generate more of it [82,85]. The genes GhMT1 and GhPCS were upregulated in response to heavy metal toxicity Heavy metal stress also impacts other elements of cotton physiology, such as increased stomatal density, which changes transpiration and gas exchange, reduces flowering and fiber quality, and lowers yield [80,83].

Comparably, cotton under stress from water logging likewise experiences several physiological alterations. Waterlogged cotton plants have higher activity of antioxidant enzymes as a defense against oxidative stress brought on by extended exposure to too much water. Increased enzyme activity mitigates oxidative stress-related damage [86,87]. Waterlogging reduces chlorophyll, leading to chlorosis and damage to the photosynthetic machinery. This negatively impacts plant growth and photosynthetic efficiency [92,93]. Waterlogged soil increases lipid peroxidation levels in cotton, similar to heavy metal stress. This suggests that cellular membranes suffer oxidative damage, significantly contributing to cellular injury and reduced plant functionality [92,93]. In response to waterlogging, cotton increases its proline levels as a defense mechanism to maintain osmotic balance within the plant cells. Proline is crucial to helping plants adapt to and survive extreme water stress by acting as an osmoprotective agent [88,91]. GhLEA and GhRAB show increased expression under waterlogging stress. Ultimately, the combined physiological reactions to waterlogging, including shortened shoot and root lengths, reduced leaf area, and hampered blooming and boll size, impact cotton fiber quality and yield [89,92,93].

3. Mechanisms of Cotton Plants in Response to Abiotic Stress-Signaling Pathways

Cotton has developed various morphophysiological approaches to drought tolerance, such as photosynthetic response, osmotic adjustment, stomatal regulation, low leaf water loss, high relative water content (RWC), and enlarged tap roots [151]. Plants adopt drought recovery mechanisms following successful stress signal transduction. Plant cell membranes act as sensors for stress signals, triggering both self-activated and hormone-dependent signaling mechanisms. These processes often involve mitogen-activated protein kinase (MAPK) networks. Calcium ions (Ca²⁺) frequently serve as common secondary messengers in stress signaling pathways, influenced by drought stress as well as various hormones like abscisic acid (ABA), jasmonic acid (JA), and ethylene. ABA interacts with SnRK2 proteins, initiating molecular and physiological responses to drought stress [152,153,154,155,156,157,158]. Improvements in physio-morphological characteristics are crucial for mitigating the effects of drought [159].

Crosstalk between these hormones fine-tunes plant responses to complex stress scenarios [41,160]. Transcription factors like DREB, ERF, HSF, and MYB act as critical regulators of gene expression in response to abiotic stress, promoting or repressing the transcription of target genes [161]. Transcriptional changes lead to modulating genes cascade, leading to physiological and biochemical adaptations, such as synthesizing osmolytes, antioxidant enzymes, and chaperones, enabling plants to maintain cellular homeostasis, protect cellular components, and survive under adverse conditions. Drought and salinity tolerance are specific to the upstream activation of the genes [162]. The impact of various abiotic stresses and the intrinsic relationship of genes and hormones for the development of stress tolerance are depicted in Figure 1.

3.1. Roles of ABA Signaling Pathway Genes in Cotton’s Abiotic Stress Responses

The abscisic acid (ABA) signaling pathway is vital for cotton’s response to abiotic stresses. It primarily regulates stomatal closure to minimize water loss and activates genes associated with stress tolerance (Figure 1). Several genes involved in ABA-dependent pathways have been identified as crucial for enhancing cotton’s drought tolerance and stress resilience. GhANN1 has boosted cotton salinity tolerance by modulating ABA biosynthesis, ion homeostasis, and the phenylpropanoid pathway. WRKY transcription factors govern stress responses and detoxifying processes in cotton. The relevance of WRKY genes in stress responses in cotton stresses their significance in plant adaptability to changing environmental conditions [163]. The complex interaction of WRKY transcription factors with abscisic acid and ethylene adds to the intricacies of stress signaling in cotton [164,165]. The interaction of ABA signaling pathways and WRKY proteins is critical for plant response to diverse stresses [166]. Overexpression of GhANN1 results in increased ABA levels and upregulation of stress-responsive genes and shows GhANN1 interaction with GhWRKY40-like, which collectively enhance salt stress responses [167]. The transcription factor CaHB12 improves drought tolerance in cotton by enhancing photosynthetic yield and water use efficiency. It achieves this by modulating ABA-dependent pathways and reducing indole-3-acetic acid (IAA) levels to prevent leaf abscission under stress. The upregulation of critical ABA signaling genes in transgenic cotton corroborates CaHB12’s role in bolstering water deficit resilience [168].

GhPYL9-5D and GhPYR1-3A, ABA receptors in cotton, exhibit high expression levels following PEG or NaCl treatment. These receptors are co-expressed with redox signaling components, transcription factors, and auxin signals, indicating their central role in cotton’s adaptation to salt and osmotic stress by integrating various signaling pathways [169]. GhMYB102 contributes to drought resistance by regulating genes responsive to drought and influencing ABA biosynthesis, enhancing cotton’s ability to cope with water scarcity [170]. The GhMAP3K62-GhMKK16-GhMPK32 MAPK cascade is essential to cotton’s drought response. This cascade regulates ABA-dependent stomatal movement and promotes ABA synthesis through a feedback mechanism, thus playing a critical role in the plant’s adaptation to drought stress [171]. Additionally, mepiquat chloride priming has been found to enhance cottonseed salt tolerance by promoting ABA-regulated GABA signaling, which controls the ascorbate–glutathione cycle [172]. These findings underscore the importance of ABA signaling pathway genes in regulating cotton’s stress responses, offering potential targets for genetic improvements in stress tolerance.

3.2. Enhancing Cotton Stress Tolerance through Reactive Oxygen Species Signaling

Reactive oxygen species play a dual role in plant biology: they are crucial signaling molecules for growth and stress adaptation at low concentrations. Still, ROS can harm cellular metabolism at elevated levels under stress conditions [20,173]. Reactive oxygen species signaling is crucial in cotton plant response to stress, activating defense mechanisms and stress-responsive genes. To maintain ROS homeostasis, plants utilize a sophisticated antioxidant defense system that includes both enzymatic and nonenzymatic components, which work together to regulate ROS levels and keep them at low and stable concentrations to mitigate oxidative damage [174]. Tolerant cotton genotypes have demonstrated lower levels of H₂O₂ and MDA accumulation and higher activities of antioxidant enzymes like SOD, peroxidase (POD), and catalase (CAT) under stress [175]. These genotypes have also shown increased expression of ROS-scavenging enzymes, contributing to their better performance than susceptible genotypes, which have more significant oxidative damage and lower enzyme activities [175].

This pathway is vital for plant health and stress tolerance. Transgenic cotton expressing AtHDG11 demonstrated enhanced stress tolerance, improved root development, increased proline and soluble sugar content, and elevated activities of ROS-scavenging enzymes. These plants also exhibited reduced leaf stomatal density and more giant stomatal and epidermal cells, leading to enhanced drought tolerance and superior agronomic performance, including higher yields under normal and drought conditions [176]. Similarly, cotton plants overexpressing SbHKT1 showed improved germination rates, biomass, and root development compared with wild-type plants. Overexpression of SbHKT1 enhanced salt tolerance by improving potassium uptake, maintaining K⁺/Na⁺ balance, and boosting antioxidant enzyme activity, increasing ROS scavenging efficiency [177].

The overexpression of GhEXLB2 in cotton has been shown to improve water use efficiency, increase levels of soluble sugar, and enhance chlorophyll content, leading to enhanced drought tolerance at different growth stages [178]. Similarly, overexpression of GhMYB4 in Arabidopsis increased flavonoid levels, enhanced tolerance to salt and drought stresses, and upregulated genes involved in flavonoid and proline biosynthesis and ROS scavenging [179]. Additionally, overexpression of SOD3 in cotton boosted antioxidant enzyme activity, reduced cell membrane damage, and improved agronomic traits under normal and drought conditions [180]. The cytokinin oxidase/dehydrogenase gene GhCKX6b-Dt alleviated salt stress in cotton by enhancing the antioxidant system. Silencing GhCKX6b-Dt led to increased proline and MDA levels, decreased SOD activity, and reduced ROS scavenging capacity, resulting in more significant oxidative damage under salt stress. This underscores the role of GhCKX6b-Dt in mitigating salt stress through ROS management [181]. ROS production and antioxidant defense genes also exhibited strong responses to waterlogging stress in cotton [182]. Understanding the molecular mechanisms of ROS generation and scavenging is vital to exploring the effects of co-expressing multiple antioxidant genes and studying the interactions between plant hormones and external signaling molecules to understand plant responses to concurrent stressors [183]. This comprehensive analysis highlights the significant role of ROS signaling and targeted genetic modifications in enhancing cotton’s tolerance to various abiotic stresses, offering promising strategies for improving stress resilience in crops.

3.3. Role of Heat Shock Proteins in Cotton Stress Tolerance

Heat shock proteins (HSPs) help cotton plants withstand heat, drought, and salinity by stabilizing cellular structures, ensuring proper protein folding, and preventing aggregation under stress. This aspect is crucial for survival in adverse environments. The 70-kDa heat shock protein GhHSP70-26 has been shown to enhance drought tolerance in cotton. It was evidenced by increased expression levels in response to stress and improved stress tolerance in overexpressed tobacco plants while silencing GhHSP70-26 reduced stress tolerance in cotton [134,184]. Additionally, Unraveling the genetic and molecular basis of heat stress in cotton [133,185]. A natural variation in the promoter region of the GhHSP70-26 gene, including a 360 bp insertion, has been identified as a critical factor in enhancing drought stress tolerance by upregulating gene expression during drought conditions. The selection of this allele has improved drought resistance in cotton [186]. Heat-responsive protein (GhHRP) is vital to cotton’s heat tolerance. It regulates hormone signaling and protects chloroplasts. GhHRP and the GhPIF4/GhEIN3 complex enhance cotton’s heat tolerance [187]. In conclusion, the critical role of HSPs, particularly GhHSP70-26 and GhHRP, in enhancing cotton’s stress tolerance underscores their potential as crucial targets for developing more resilient cotton varieties in the face of increasing environmental challenges.

3.4. Calcium Signaling in Cotton Stress Responses

Calcium signaling is essential to cotton’s adaptation to stress by acting as a secondary messenger. Elevated cytosolic calcium levels in response to stress conditions such as drought and salinity initiate various downstream effects, including the activation of stress-responsive genes and modulation of cellular processes to enhance stress resilience. The calcium-binding protein GhCBL3, which shows increased expression under stress, plays a critical role in this signaling pathway. Knockdown of GhCBL3 has been shown to alter ROS levels, impacting stress tolerance [188]. GhMPK3 contributes to cold, drought, and salt stress tolerance by stabilizing cell membranes, reducing water loss, increasing antioxidant enzyme activity, and upregulating stress-responsive genes, and silencing GhMPK3 decreases drought tolerance in cotton [189].

Silencing GhMAPKK5 in cotton results in increased susceptibility to drought and salt stress, while its overexpression in Arabidopsis enhances stress tolerance, likely through a MAPK signaling cascade involving GhMEKK-GhMAPKK5-GhMAPK and regulation of vital stress-related genes [190]. This aspect highlights GhMAPKK5 as a crucial regulator of stress responses in cotton, offering potential targets for genetic improvement. Melatonin enhances salt tolerance in cotton by modulating ROS scavenging and calcium signaling, highlighting its potential in stress management [128,191]. Additionally, overexpression of GhCIPK6a improves resilience to abiotic stresses through involvement in ROS scavenging and MAPK signaling pathways [192]. Calcium signaling plays a pivotal role in cotton’s stress responses, with essential proteins like GhCBL3, GhMPK3, and GhMAPKK5 emerging as vital regulators of stress tolerance, offering promising targets for enhancing resilience in cotton through genetic improvement.

3.5. Stress-Responsive Genes and Proteins

Abiotic stresses, such as cold, drought, and salinity, challenge crop growth. Plants have evolved stress-responsive mechanisms and pathways. ABA plays a crucial role in stress signaling, with crosstalk between ABA-dependent and independent pathways. Identifying the molecular mechanisms that drive stress responses in cotton is critical for increasing its resilience to varied environmental challenges—Table 3 summarizes differential gene expression in the stress-responsive genes in response to abiotic stress. The draft genome sequencing of diploid cotton G. raimondii and allotetraploid cotton G. hirsutum L. TM–1 has significantly advanced cotton genomics, providing valuable resources for investigating stress-responsive genes [193,194,195,196,197]. Analysis of stress-related pathways using this genomic data offers insights into cotton’s molecular mechanisms governing stress responses. This knowledge facilitates the identification of stress-tolerance genes, informing targeted genetic improvement strategies for enhancing cotton resilience and productivity under various environmental stressors [198].

Investigating drought coping strategies, such as improving crop water use efficiency, has been a focal point in cotton research. The involvement of WRKY transcription factors in abiotic stress responses is well established. WRKY transcription factors are signaling and regulatory components in plant stress responses [199,200,201]. The WRKY gene family, extensively studied in model plants and crops, plays a crucial role in stress signaling. The intricate network of transcription factors in cotton and other plants responds to abiotic and biotic stresses [202,203,204,205]. The involvement of GhWRKY15 in disease resistance and plant development underscores the multifaceted roles of these transcription factors [206]. WRKY transcription factors govern stress responses and detoxifying processes in cotton. The relevance of WRKY genes in stress responses in cotton stresses their significance in plant adaptability to changing environmental conditions [163]. The complex interaction of WRKY transcription factors with abscisic acid and ethylene adds to the intricacies of stress signaling in cotton [164,165]. The interaction of ABA signaling pathways and WRKY proteins is critical for plant response to diverse stresses [166]. Here, we discussed the generation of ROS and the activation of transcription factors that initiate the stress response.

Table 3. Overview of stress-responsive differential gene expression in response to abiotic stress. The table provides information on specific genes, the corresponding abiotic stress, the plant part affected, the impact on gene expression regulation, the analysis method, and references for further reading.

Sr. No	Genes	Abiotic Stress	Plant Part	Impact on Gene Expression	Regulation	Method	Ref.
1.	GhRD29A, GhDREB1A	Drought	Roots	Activation of genes related to osmotic regulation	Up	RNA-Seq	[207,208]
2.	GhP5CS, GhBADH	Drought	Leaves	Activation of genes involved in proline biosynthesis	Up	qPCR	[209]
3.	GhPIP, GhTIP	Drought	Leaves	Regulation of aquaporin genes involved in water transport	Up	RNA-Seq	[210,211,212]
4.	GhHSP70, GhHSP90	Heat	Leaves	Upregulation	Up	qRT-PCR	[186,213,214]
5.	GhMYB, GhbZIP	Heat	Leaves	Activation of transcription factor genes	Up	qPCR	[215]
6.	GhCAT, GhPOD	Heat	Leaves	Induction of antioxidant enzyme genes	Up	RNA-Seq	[216,217]
7.	GhSOS1, GhNHX1	Salinity	Roots	Altered expression of ion transport genes	Up	Microarray	[218,219]
8.	GhNAC, GhWRKY	Salinity	Roots	Modulation of stress-responsive transcription factor genes	Up	RNA-Seq	[220,221,222,223]
9.	GhHKT1, GhNHX2	Salinity	Roots	Alteration in ion homeostasis-related gene expression	Up	RNA-Seq	[224,225]
10.	GhLEA, GhRAB	Water-logging	Roots	Induction of genes related to water logging tolerance	Up	Microarray	[226,227]
11.	GhAPX, GhSOD	Oxidative stress	Leaves	Upregulation of antioxidant enzyme genes	Up	qRT-PCR	[228,229]
12.	GhDHN, GhERF	Cold stress	Leaves	Modulation of genes related to cold response	Up	qRT-PCR	[230,231]
13.	GhMT1, GhPCS	Heavy metal toxicity	Roots	Induction of metal detoxification genes	Up	qPCR	[232,233,234]
14.	GhUVR8, GhCOP1	UV-B radiation	Leaves	Activation of genes involved in UV protection	Up	RNA-Seq	[235,236]
15.	GhPAL, GhCHS	UV-B radiation	Leaves	Regulation of genes involved in phenylpropanoid biosynthesis	Up	qRT-PCR	[237,238,239,240]

Table 3. Overview of stress-responsive differential gene expression in response to abiotic stress. The table provides information on specific genes, the corresponding abiotic stress, the plant part affected, the impact on gene expression regulation, the analysis method, and references for further reading.

Sr. No	Genes	Abiotic Stress	Plant Part	Impact on Gene Expression	Regulation	Method	Ref.
1.	GhRD29A, GhDREB1A	Drought	Roots	Activation of genes related to osmotic regulation	Up	RNA-Seq	[207,208]
2.	GhP5CS, GhBADH	Drought	Leaves	Activation of genes involved in proline biosynthesis	Up	qPCR	[209]
3.	GhPIP, GhTIP	Drought	Leaves	Regulation of aquaporin genes involved in water transport	Up	RNA-Seq	[210,211,212]
4.	GhHSP70, GhHSP90	Heat	Leaves	Upregulation	Up	qRT-PCR	[186,213,214]
5.	GhMYB, GhbZIP	Heat	Leaves	Activation of transcription factor genes	Up	qPCR	[215]
6.	GhCAT, GhPOD	Heat	Leaves	Induction of antioxidant enzyme genes	Up	RNA-Seq	[216,217]
7.	GhSOS1, GhNHX1	Salinity	Roots	Altered expression of ion transport genes	Up	Microarray	[218,219]
8.	GhNAC, GhWRKY	Salinity	Roots	Modulation of stress-responsive transcription factor genes	Up	RNA-Seq	[220,221,222,223]
9.	GhHKT1, GhNHX2	Salinity	Roots	Alteration in ion homeostasis-related gene expression	Up	RNA-Seq	[224,225]
10.	GhLEA, GhRAB	Water-logging	Roots	Induction of genes related to water logging tolerance	Up	Microarray	[226,227]
11.	GhAPX, GhSOD	Oxidative stress	Leaves	Upregulation of antioxidant enzyme genes	Up	qRT-PCR	[228,229]
12.	GhDHN, GhERF	Cold stress	Leaves	Modulation of genes related to cold response	Up	qRT-PCR	[230,231]
13.	GhMT1, GhPCS	Heavy metal toxicity	Roots	Induction of metal detoxification genes	Up	qPCR	[232,233,234]
14.	GhUVR8, GhCOP1	UV-B radiation	Leaves	Activation of genes involved in UV protection	Up	RNA-Seq	[235,236]
15.	GhPAL, GhCHS	UV-B radiation	Leaves	Regulation of genes involved in phenylpropanoid biosynthesis	Up	qRT-PCR	[237,238,239,240]

4. Breeding and Biotechnological Approaches to Improving Abiotic Stress Tolerance in Cotton

4.1. Breeding for Stress Tolerance

Breeding has traditionally been used to produce resilient cotton cultivars. Because of breeding efforts, cotton’s abiotic stress tolerance has significantly improved recently. This review summarizes the findings of significant studies focusing on genetic analyses, molecular markers, and physiological responses related to abiotic stress tolerance in cotton. A genetic investigation of drought tolerance in Egyptian cotton (G. barbadense L.) yielded important insights into the heritability of drought tolerance features. A thorough exploration of the genetic foundations underlying tolerance to drought and salinity has been undertaken [241]. Integrating these diverse stress resistances is imperative in developing robust cotton varieties [242]. Marker-assisted selection (MAS) has helped in creating gene maps and identifying quantitative trait loci (QTL) associated with improved stress tolerance in cotton [243]. Genome-wide association studies covering the entire genome have been crucial in detecting major QTLs that enhance abiotic stress tolerance in cotton [244]. Studies have revealed distinct genomic regions linked to osmotic stress tolerance, dehydration tolerance, and drought and salt tolerance in upland cotton, providing potential targets for marker-assisted selection in breeding programs [245,246,247]. Genome-wide association studies have identified markers linked to stress tolerance and have explored tolerance to biotic and abiotic stresses in a population of upland cotton [248]. Numerous studies have focused on identifying drought and salt-tolerant cotton germplasm to conserve and utilize genetic resources with stress tolerance [249]. Mapping studies of QTLs offer valuable insights into the genetic foundations of stress tolerance, providing potential avenues for marker-assisted breeding strategies. Identifying QTLs for salt tolerance through the interspecific cross of G. tomentosum with G. hirsutum illustrates the potential of incorporating wild cotton species into breeding programs [250,251,252].

Gene acquisition remains a significant barrier in plant genetic engineering. Advances in whole-genome sequencing and omics technologies, including genomics, proteomics, and metabolomics, hold promise for discovering novel genes expressed under stressful circumstances [253,254]. Newly discovered genes hold potential as options for improving plant stress tolerance. Identifying stress-related metabolites in crops is also critical to developing stress resilience. Since overexpressing single genes in crops provides limited stress tolerance, the gene pyramiding method, which involves simultaneous expression of numerous functionally related genes, appears to be a more sensible strategy [255,256,257]. Recent progress in enhancing plant tolerance to abiotic stresses via multi-gene assembly raises hopes for mitigating the adverse effects of abiotic stress. Sustainable agriculture requires using learned knowledge to cultivate crops that flourish and reproduce effectively in complex conditions.

4.2. Transgenic Approaches

Recent advancements in genetic engineering aimed at enhancing abiotic stress tolerances in cotton are being evaluated through critical studies to understand these strategies better. Researchers have utilized various genetic engineering approaches to overcoming abiotic stresses. This comprehensive review provides an in-depth analysis of genetic engineering approaches to breeding abiotic stress-tolerant cotton. The overexpression of genes associated with drought tolerance has improved drought resistance in transgenic cotton. Targeting critical genes involved in stress response pathways holds promise for developing cotton varieties resilient to water scarcity. Genetic engineering approaches to enhancing drought tolerance include expressing drought-responsive genes and transcription factors. A genome-wide analysis of the calcium-dependent protein kinase gene family in G. raimondii reveals a network involved in stress signaling.

Similarly, GhABF2, a bZIP transcription factor, is pivotal in conferring drought and salinity tolerance in cotton [258]. Enhancement of drought and salt tolerance in cotton on overexpression of rice NAC1 gene highlights the potential of cross-species gene transfer to improve stress resilience [259]. Overexpressing the Thellungiella halophila H⁺-PPase gene enhances salt tolerance and improves growth and photosynthetic performance [260].

Genetic factors such as the calcium sensor GhCaM7 and the GhCDPK1 gene are essential in promoting cotton fiber elongation and enhancing drought tolerance [261,262]. Additionally, overexpression of specific genes, such as AtDRE2A-CA and GhABF2, has shown promising results in improving cotton drought and salt stress tolerance [258,263,264]. Research on salt tolerance and understanding the balance between ROS and antioxidant defenses, as well as root traits and transcription factors like NAC and MYB, is crucial for developing stress-resistant cotton varieties [265,266,267,268]. These findings highlight the importance of genetic and molecular approaches in developing resilient cotton varieties and novel agricultural practices.

4.3. CRISPR/Cas in Cotton: Challenges and Solutions

The revolutionary clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) offers precise capabilities for genome editing (GE) [233,269,270,271]. Recent applications of CRISPR/Cas in cotton, specifically in whole genome-sequenced cotton species, have opened new possibilities for targeted genetic modifications related to stress response pathways [272,273]. In the post-genomic era, developing efficient biotechnological tools for studying gene functions, such as GE, reverse genetics, or omics-based approaches, has become necessary [274,275]. Since the early 2000s, gene silencing technologies have been widely used to uncover gene function, revealing regulatory mechanisms and metabolic pathways [276]. However, the limitations of existing gene silencing technologies, like RNA interference, have led to the exploration of CRISPR/Cas-based GE tools, which have diverse applications beyond the introduction of stable and heritable mutations [277,278].

Various approaches are available for target-specific GE, such as CRISPR/Cas, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) [279]. The CRISPR/Cas system has transformed the field of plant GE with its simple design and high efficiency, which uses short RNA molecules called single guide RNAs (sgRNAs). This system has overshadowed the complexity and limitations associated with ZFNs and TALENs in designing and cloning desired gene constructs [280,281]. Its cost-effectiveness and ease of use have made CRISPR/Cas the preferred choice for GE in plants, transforming the landscape of genetic research. Cotton, a vital fiber, biofuel, and oil crop, requires efficient GE tools for large-scale gene functional studies [193,282].

While CRISPR/Cas has been successful in GE for various crops and model systems, its applications in cotton have faced challenges, partly due to the time-consuming transformation process and polyploidy [283]. Several studies targeting genes, such as MYB25, GhVP, GhCLA1, or GhARG, have demonstrated the potential of CRISPR/Casin cotton GE [284,285,286,287]. In a recent study, the CRISPR/Cas system was efficiently used to generate knockout plants for the GhNAC3 gene [288]. The GhNAC3 gene encoded an NAC family transcription factor involved in drought stress tolerance through abscisic acid-related and independent pathways [289]. Mutant lines exhibited phenotypic variations, confirming the role of GhNAC3 in average growth and development, apart from drought stress tolerance. CRISPR/Cas-based base editing allows for precise nucleotide substitutions, enabling accurate base modifications and directed evolution of intended genetic loci [290]. Different research groups have already shown the potential use of base editing tools for cotton GE [291,292,293]. For example, Wang and colleagues [293] utilized the recent version of ABE (GhABE8e) to install A-to-G base changes in GhTFL1, an anti-florigen encoding gene. The evolved GhTFL1 led to the generation of new cotton germplasm with unique plant architectural features, such as compact size, shortened breeding cycle, and moderate height, which are beneficial for harvesting, and potentially suitable features to evade drought stress situations. This study demonstrates the potential of base editing technology for directed evolution studies in the cotton genome. It can be applied to produce weak or strong alleles in abiotic stress-related genes, which would have been considered an unattainable task in the past.

The transient gene expression is also essential for understanding abiotic stress research, which helps to comprehend stress tolerance mechanisms and develop strategies to mitigate abiotic stresses. A rapid method for validating sgRNAs for CRISPR/Cas-based GE was devised to tackle the challenges associated with time-consuming transformation methods in cotton [294]. This method involves transient expression in cotyledons, enabling experimental validation in cotton. Successfully applied for multiple purposes, including validation of sgRNAs for individual genes (GhPDS, GhCLA1, and GhEF1), simultaneous editing of homologous genes, and genomic fragment deletions, this new method showcased its versatility. CRISPR/Cas-induced mutations in stably transformed cotton plants targeting GhCLA1 also resulted in typical albino phenotypes [295,296,297,298,299,300]. By providing a swift validation method for sgRNAs, this approach overcomes the hurdles associated with prolonged transformation processes, making CRISPR/Cas-based methods more accessible for a wide range of applications in cotton [283,301].

The integration of reverse genetics methodologies and targeted GE technologies has propelled significant advancements in understanding gene function [302]. CRISPR/Cas-based techniques enable the production of transgene-free cotton plants through selfing or backcrossing, aligning with current guidelines for genetically modified organisms (GMOs) [303]. Prospective application of CRISPR/Cas-based methods across the entire cotton genome provides a novel avenue to bolster cotton productivity, enhance genetic traits, confer pathogen resistance, and optimize agronomic characteristics, as reported for other crops [304,305,306]. CRISPR/Cas, with its simplicity and efficiency, stands out as a powerful tool for large-scale gene functional studies in cotton [307]. As the GE field continues to evolve, advanced GE technologies like base editing and prime editing hold immense promise for unraveling gene function complexities, driving new breeding innovations in cotton crop improvement, and developing abiotic stress-tolerant varieties (Figure 2).

5. Future Prospects and Challenges

5.1. Advanced Biotechnological Interventions in Mitigating Abiotic Stress

Conventional breeding has indeed led to substantial improvements in crop yield, but it has fallen short of boosting crops’ resilience to abiotic stresses. This shortfall can be attributed, in part, to breeders’ inclination to assess genetic materials solely under optimal conditions. However, this approach overlooks the intricate nature of abiotic stresses and the fact that plants exhibit varying sensitivities to different stressors at different life cycle stages. As a result, conventional breeding faces the challenges of effectively selecting for increased stress tolerance. Therefore, there is a pressing need for an alternative approach to enhancing abiotic stress tolerance and improving both crop yield and quality.

In recent decades, scientists have successfully created transgenic plants by modifying the expression of specific genes using various transformation methods and tissue culture methods [177,180,308]. Manipulating individual genes in plants through transgenic approaches will help to understand a network of pathways related to stress response [309]. Modern genome-wide profiling and engineering technologies present unprecedented opportunities for understanding the dynamics of epigenetic modifications in response to stress [310]. Understanding epigenetic regulation in response to abiotic stress offers insights into enhancing crop resilience [311]. A transgenic approach focusing on osmolyte production, antioxidant defense against harmful ROS, adjustments to biochemical, physiological and cellular processes, signal perception, transduction cascades, activation of transcription factors, gene expression or modulation, and synthesis of functional proteins will lead to the development of stress-tolerant varieties [311,312,313,314,315,316,317]. Emerging technologies like CRISPR/Cas-based GE tools hold promise for the future of crop biotechnology [318]. CRISPR/Cas offers the advantage of editing a few nucleotides in the organism’s original DNA without introducing large foreign DNA fragments, potentially easing the acceptance of GMOs worldwide due to minimal changes in crop genomes.

5.2. Challenges of Mitigating Abiotic Stress in Cotton

The need for increased cotton output, driven by changing climates and a growing human population, has historically relied on conventional breeding methods to introduce genes into superior cultivars. Modern GE technologies have emerged as promising solutions to tackle these challenges more effectively. They encompass techniques such as ZFNs, TALENs, and CRISPR/Cas. The CRISPR/Cas system is notable for its simplicity, efficiency, and adaptability [317]. Leveraging CRISPR/Cas, researchers aim to enhance cotton’s tolerance to both biotic and abiotic stresses, modify gene expression, and stack genes for desired traits, all while addressing regulatory concerns associated with GMOs through the transgene clean strategy [318]. This approach underscores a range of research opportunities, including improving fiber quality, plant architecture, and flowering and addressing challenges related to epigenetic changes and gene stacking for economically significant traits in cotton cultivation.

Along with genetic techniques, biotic and abiotic factors provide considerable obstacles to cotton farming, jeopardizing growth, output, and fiber quality. Abiotic stresses such as high temperatures, drought, heavy metal toxicity, and salinity affect cotton’s antioxidant systems. We propose using modern agricultural practices and molecular tools in breeding strategies to help mitigate different abiotic stresses. Interdisciplinary methodologies integrating physiological, biochemical, and molecular investigations are essential for comprehending how cotton adapts to stress and fostering resilience in sustainable agriculture.

6. Conclusions

In conclusion, this review comprehensively explores cotton’s physiological, biochemical, and molecular responses to abiotic stressors, particularly heat, drought, and salinity. Through an in-depth analysis, we have gained significant insights into the responses of cotton genotypes to this complex challenge. The review highlights cotton plants’ impressive resilience and capacity to activate defense mechanisms to mitigate the adverse effects of various abiotic stresses. Advanced molecular genomic technologies, including marker-assisted selection, QTL mapping, high-throughput DNA sequencing, RNA sequencing, single-cell sequencing, chromatin immunoprecipitation sequencing, GWAS studies, genomic selection, proteomics, metabolomics, metagenomics, multi-omics techniques, and CRISPR-based genome engineering techniques, play a critical role in understanding the intricate mechanisms of abiotic stress in plants. It will significantly enhance our understanding of the robust signaling pathways, which, in turn, will aid in identifying candidate genes for functionally essential traits related to abiotic stress tolerance. Integrating physiological, biochemical, and molecular studies through interdisciplinary approaches is vital to comprehending cotton’s stress adaptation and fostering resilience for sustainable agriculture. While several stress-resistant genes and transcription factors have been identified and successfully transferred into cotton through genetic engineering, resulting in improved traits, these abiotic stress-resistant varieties are not yet widely cultivated. Still, the focus is on developing transgenic cotton lines with multiple resistance genes, potentially resulting in varieties with enhanced tolerance to various abiotic stresses. Genetic engineering techniques, especially CRISPR/Cas9, hold significant promise for creating stress-tolerant cotton varieties by incorporating multiple stacked resistance genes.