*3.2. Heat/Temperature Stress*

Plants have a preferred temperature, any rise or fall in that temperature can significantly impede their development and productivity. The third most important abiotic factor is heating stress, which may decrease crop production considerably. For instance, every 1 ◦C augmentation in atmospheric temperature diminishes wheat yield by 6%, rice yield by 10–20%, and corn yield by 21–31% [164–166]. Significant yield losses were caused by high heat stress, which is now recognized as a severe problem that will simply become worse in the future. All phases of plant growth, from germination to harvest, are severely harmed by heat stress [167,168]. Heat stress not only increases plant mortality rates but also reduces plant quality [169,170].

In severe cases, a bad alteration in temperature results in plant mortality because plants are more susceptible to temperature changes. The ideal temperature would normally be better for crop growth and development; conditions below and above the optimum temperature have a harmful effect on productivity. For every 10 ◦C rise, followed by 20 ◦C and 30 ◦C, mostbiochemical and enzymatic procedures double in speed [171]. Abiotic stressors, predominantly high and low heat, have a harmful effect on the premature stage of the male gametophyte in a range of agricultural crops, including maize, rice, barley, wheat, sorghum, and chickpea [172]. Due to temperature stress, the functions of tapetal cells are diminishedduring the reproductive growth period, and the anther is dysplastic. Pollen discharge is insufficient and indehiscence happens as a result of increased heat preventing pollen grains from escalating. Plants have developed precise physiological and chemical reactions to manage temperature stress [173].

The presence of genes that are responsive to heat stress, signal transduction, and the synthesis of metabolites are only a few of the complex molecular systems that plants activate in response to heat stress. Different temperature-stress-related genes have been identified and characterized to improve plants' ability to withstand heat as a result of developments in structural and functional genomics technologies in plants. The heat stress reaction, which is connected to the accumulation of ROS, is mediated by the heat shock transcription factors (HSFs) and the heat shock proteins (HSPs) [174].Therefore, by enhancing plants' ability to resist ROS components, temperature stress tolerance can be improved [175]. This indicated that higher tolerance might increase the antioxidant properties of crops. Plant temperature tolerance was significantly increased via metabolite production and temperature-induced gene expression. To explore the molecular processes associated with temperature stress and improve plant heat tolerance, CRISPR-Cas9 is a cutting-edge technology among all genome-editing techniques [176] (Table 3).

A cultivable HS-inducible rice mutant was created using CRISPR/Cas9 technology [177]. The orthologs of mitogen-activated protein kinase 3 and agamous-like 6 were modified using CRISPR to increase tomato sensitivity to heat stress, whereas ADP-ribosylation factor 4 enhanced tomato sensitivity to salinity shocks. According to Bouzroud et al. [178], these CRISPR-edited mutant plants had improved agronomic characteristics and were resilient to abiotic stresses. As a component for heat tolerance, BRZ1 positively regulates the formation of ROS in the tomato apoplastic area. This was confirmed by the CRISPR-Cas9-based bzr1 mutants, which showed reduced temperature tolerance and respiratory burst oxidase homolog 1 (RBOH1) with diminished hydrogen peroxide generation in the apoplast [179]. In comparison to wild-type crops, the development of CRISPR/Cas-mediated heat-stress-sensitive albino 1 (HSA1) mutants of tomato showed greater sensitivity to temperature stress [180].

The thermosensitive genic male sterile gene was altered by CRISPR in maize to promote thermo susceptible male-sterile plants [181]. In lettuce, knockouts of NCED4, a crucial regulating enzyme in abscisic acid production, allowed the seeds to germinate at a higher temperature. As a result, LsNCED4 mutants may have commercial significance in manufacturing environments with high temperatures [182]. In order to make a plant more resistant to heat, the hsps gene, which increases osmolyte levels and prevents cell protein damage, can be overexpressed [183]. The protein kinase SAPK6 and the transcription factor OsbZIP46CA1 in rice also increase the capacity for responding to heat stress [184].

#### *3.3. Cold Stress*

Cold stress, which includes chilling (20 ◦C) and freezing (0 ◦C) temperatures, hinders plant growth and development and severely limits plant geographic expansion and agricultural productivity [185]. Plants are directly inhibited from responding metabolically to low temperatures, which results in osmotic stress, oxidative stress, and other types of stress. Due to mechanical damage and metabolic dysfunction caused by extreme cold temperatures, plant growth and development are halted [186]. The physiological, biochemical, and molecular behavior of plants during their growth and expansion is adversely affected by cold stressors. The photosynthetic capacity and crop anatomy are brutally impacted by cold exposure, especially throughout the winter [187,188].Cold stress during the seedling stage may cause impaired germination and emergence. Long-term exposure impairs source–sink relationships, growth, nutrient localization, and leaf chlorosis [189]. Membrane formation, which amplifies other cold-stress-related downstream processes, is the main consequence of cold stress on crops [190]. In-generic or inter-specific hybridization has been successful in boosting the cold tolerance of significant crops using conventional breeding methods. For creating non-transgenic genome-edited crops to combat climate change and ensure future food security, CRISPR/Cas9 is a clever and practical approach [191,192] (Table 4).

To increase the plant's resistance to cold, genome editing is employed to target a few of the depressant regulator transcription factors in rice. A transcription factor called OsMYB30 attaches to the amylase gene promoter and negatively affects cold tolerance. According to Lv et al. [193], under conditions of cold stress, OsMYB30 forms a compound with OsJAZ9 and slows down the expression of the amylase gene, which may contribute to increasing cold sensitivity by causing maltose buildup and starch breakdown. In order to determine the specific function of the TIFY1a, TIFY1b, and Ann3 genes in rice's ability to withstand cold stress, CRISPR/Cas9 technology has also been applied to these genes. The mutant outperformed the natural variation in terms of yield, temperature tolerance,

and amount of germination prior to harvest [194]. Using CRISPR base editing, suppression of photosynthetic genes in rice plants under cold stress has been shown to cause the white-striped leaves phenotype in the white stripe leaf 5 (wsl5) mutant line [195,196].

PRPs are proline-rich proteins that not only aid in dealing with low temperatures but also reduce nutrient loss, boost antioxidant activity, and aid in the production of chlorophyll. Rice capacity for cold tolerance was improved by the CRISPR/Cas9 deletion of OsPRP1, which encodes a proline-rich protein [197]. In a recent work using CRISPR/Cas9, three rice genes, viz., OsPIN5b, GS3, and OsMYB30were altered to increase spike length, grain size, and resilience to cold stress [198]. The CRISPR/Cas9 technology altered the G-complexrelated genes i.e., OsRGA1, OsGS3, OsDEP1, and OsPXLG4 to make rice more resistant to chilling stress [199].Because tomato plants are prone to chilling stress, their fruits are more vulnerable to damage from the cold. C-repeat binding factor 1 (CBF1) was shown using CRISPR-Cas9-based cbf1 mutants to protect the tomato plant next to it from cold/chilling damage and decrease electrolyte leakage [200]. These plants also demonstrated excellent addition of hydrogen peroxide and indole acetic acid, resulting in tomato plants tolerant of chilling stress.

**Table 3.** List of reported targeted gene(s) via CRISPR/Cas9 technology in different plant species for development of tolerant genotypes against drought and heat stresses.



**Table 4.** List of reported targeted gene(s) via CRISPR/Cas9 technology in different plant species for development of tolerant genotypes against cold stresses.
