Breeding Drought-Tolerant Maize ( Zea mays ) Using Molecular Breeding Tools: Recent Advancements and Future Prospective

: As a most signiﬁcant cereal crop, maize provides vital nutritional components to humans and livestock. Drought stress curtails maize growth and yield by impairing several morphological, physiological, and biochemical functions. The rising threats of drought stress signiﬁcantly affect global food security and increase the ratio of hunger and starvation. The use of molecular breeding techniques has enabled maize researchers to deeply examine the genetic control of drought tolerance and the genetic differences between genotypes to drought stress. Despite the signiﬁcant progress in molecular genetics, the drought tolerance mechanism is still not fully understood. With the advancements in molecular research, researchers have identiﬁed several molecular factors associated with maize tolerance to drought stress. Quantitative trait loci (QTL) mapping and genome-wide association study (GWAS) analysis have led to identifying QTL, and genes linked to drought tolerance in maize that can be further exploited for their possible breeding applications. Transcriptome and transcription factors (TFs) analysis has revealed the documentation of potential genes and protein groups that might be linked to drought tolerance and accelerate the drought breeding program. Genetic engineering has been used to develop transgenic maize cultivars that are resistant to drought stress. Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) is a new ray of hope to edit the gene of interest to enhance drought tolerance in maize and save both time and cost in cultivar development. In the current review article, we have tried to present an updated picture of the advancements of drought tolerance in maize and its future prospects. These organized pieces of information can assist future researchers in understanding the basis of drought tolerance to adopt a potential breeding tool for breeding drought-tolerant maize cultivars.


Introduction
Crop production faces unpredictable encounters due to global climate variations [1,2]. Abiotic stresses such as drought, salinity, and heat stress constantly threaten the yield of essential crops and increase the risk of danger to global food security [3][4][5][6]. Improving food security is critical, given that over 800 million people remain affected by insufficient food supplies [7]. Conflicts and wars have pushed this situation to a dead end [8,9]. Rising  Maize needs more water for its growth; therefore, improving its water usage efficiency (WUE) must improve its production under changing environmental conditions [27]. Understanding responses to drought stress is a prerequisite to understanding the molecular Agronomy 2023, 13, 1459 3 of 21 mechanism, and, therefore, several potential genes have been identified at the transcriptional level [28,29]. Considering the importance of maize, increasing its yield under adverse environments has been the main objective of maize breeders [30]. Improving drought tolerance in maize is significant in ensuring food security [31]. Classical breeding methods have shown limited progress in improving maize tolerance to drought stress; hence, identifying how the plant responds to drought stress will provide new insights into how genetic modification affects crop yield under drought conditions [32]. Many molecular breeding tools have been employed to enhance the drought tolerance of maize, and hundreds of drought-tolerant maize cultivars have been developed.
QTL mapping is the most powerful way to identify novel genomic regions for drought tolerance. Earlier studies have identified many novel QTL governing the drought tolerance of maize. Hao et al. [33] conducted a meta-analysis of QTL for drought tolerance and studied 239 QTL under well-watered conditions and 160 under stressed conditions. These QTL were detected using 12 experiments and 22 populations [33]. GWAS has identified several genes, QTL, and single nucleotide polymorphisms (SNP S ) linked to drought tolerance in maize, and it is considered an effective molecular tool for developing tolerant maize cultivars. The use of GWAS has increased the speed of plant breeding to increase crop tolerance to abiotic stresses [27]. Transcriptome and TFs analyses provide a deep insight into the molecular mechanism of drought tolerance in maize by identifying gene/TF families under drought stress conditions. Hence, it is crucial to locate the drought-tolerant regulatory genes for the genetic improvement of maize [34]. An earlier study by Min et al. [35] detected several genes regarding drought tolerance in maize, which can be potential candidates for drought-tolerant breeding [35]. TF analysis identified several TF families that govern maize's drought tolerance [36]. Hence, these techniques have enormous potential to accelerate drought breeding [35]. Genetic engineering and CRISPR/Cas9 are potential molecular tools that play a crucial role in the genetic improvement of crops for desired traits [37][38][39][40]. Transgenic maize plants developed by Wei et al. [37] showed significant tolerance to drought stress [37]. This review summarizes the applications of potential molecular tools for developing drought-tolerant maize genotypes. There are sufficient studies on this aspect, but a detailed overview is missing. Hence, this comprehensive review will help future breeders to adopt more realistic molecular breeding methods to accelerate drought-tolerant breeding in maize. This information will expedite further research progress in the future.

Screening of Drought Tolerance in Maize
Improving drought tolerance is one of the main objectives of maize breeders [41] and drought tolerance is a complex process both at the physiological and molecular levels [41][42][43]. Drought tolerance is a multigenic trait with low heritability and is categorized by genotypeenvironment interaction (GEI) [44]. Screening for drought tolerance (Figure 2) requires the existence of considerable genetic variability in available germplasms. Many researchers have assessed the drought tolerance of maize by studying photosynthesis, hormone regulation, gene expression, and osmotic regulation [45,46]. Crop resilience refers to the adaptive ability to drought stress at a certain level. If the degree of stress does not exceed the resilience range of the crop, then the crop can recover after drought stress [47]. Plant genetic resources (PGRs) are valuable materials for present and future research studies. They have been categorized as an indispensable source of genetic variation for breeding new crop varieties [48]. Extensive efforts have been made to organize, store, and analyze the data collected during collection missions [49]. The global plan for preserving and utilizing PGRs was laid down by the Food and Agricultural Organization (FAO) in 2010. However, in the available germplasm stored in hundreds of gene banks, little information is known about the extent of genetic variability in traits of juvenile plants such as germination rate and seedling morphology traits in response to biotic and abiotic stresses. One of the largest gene banks in the world is the Federal Ex Situ Gene Bank of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), which is in Gatersleben [50][51][52]. Screening the large germplasm under drought stress conditions is essential to identify the tolerant genotypes. Badr et al. [53] evaluated the 40 accessions of maize under drought stress conditions for 27 responsive traits of germination percentage (GP) and seedling growth. The high heritability of shoot and root seedling traits provided a strong reason for further genetic analysis. Shoot and root traits showed a significant positive correlation under drought conditions [53]. The degree of drought tolerance in maize lines can be identified at the seedling stage by the measurement of dry shoot weight and root length of plants. These traits can be potentially used for drought-tolerant screening among maize lines [54]. Another study by Ali et al. [55] revealed that hybrid Sh-139×EV-340 showed significant variation for root and shoot development under water stress conditions at the seedling stage. This hybrid can be further evaluated to identify unknown loci for root and shoot development [55].
Agronomy 2023, 13, x FOR PEER REVIEW 4 of 21 germination rate and seedling morphology traits in response to biotic and abiotic stresses. One of the largest gene banks in the world is the Federal Ex Situ Gene Bank of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), which is in Gatersleben [50][51][52]. Screening the large germplasm under drought stress conditions is essential to identify the tolerant genotypes. Badr et al. [53] evaluated the 40 accessions of maize under drought stress conditions for 27 responsive traits of germination percentage (GP) and seedling growth. The high heritability of shoot and root seedling traits provided a strong reason for further genetic analysis. Shoot and root traits showed a significant positive correlation under drought conditions [53]. The degree of drought tolerance in maize lines can be identified at the seedling stage by the measurement of dry shoot weight and root length of plants. These traits can be potentially used for drought-tolerant screening among maize lines [54]. Another study by Ali et al. [55] revealed that hybrid Sh-139×EV-340 showed significant variation for root and shoot development under water stress conditions at the seedling stage. This hybrid can be further evaluated to identify unknown loci for root and shoot development [55]. The new indices such as PCI (the production capacity index) and YPSI (the yield production score index) are essential selection methods for identifying and categorizing maize hybrids based on their degree of drought tolerance. These methods were used by Bonea [56], and he selected HS 1156-14 with high resilience and productivity under drought stress [56]. In another experiment, seedlings of 55 inbred and five hybrids were shifted to the hydroponic solution. Seedlings were exposed to drought stress for five days. The new indices such as PCI (the production capacity index) and YPSI (the yield production score index) are essential selection methods for identifying and categorizing maize hybrids based on their degree of drought tolerance. These methods were used by Bonea [56], and he selected HS 1156-14 with high resilience and productivity under drought stress [56]. In another experiment, seedlings of 55 inbred and five hybrids were shifted to the hydroponic solution. Seedlings were exposed to drought stress for five days. The inbred lines, CM140, CML422, and hybrid DMRH1306 were identified as drought tolerant. Results confirmed that this phenotypic screening method is very effective, short, and simple, and can be used to screen large germplasm of maize for drought tolerance [57]. Another method of screening is based on photosynthetic pigments and antioxidant enzymes. Different maize genotypes and hybrids were evaluated under drought stress conditions for 50 days. The Yousafwala hybrid and FH-1137 exhibited a smaller decrease in photosynthetic and antioxidant parameters, indicating their tolerant nature [58]. Hence, the selection of genotypes and hybrids based on these traits is recommended for maize growth under water-deficit conditions [58]. Raj et al. [59] assessed the drought tolerance of maize using PEG-6000, which initiated the drought stress conditions. Single-sequence repeats (SSR) were used for drought tolerance screening at the molecular level. The hybrids AUK-30 and AUMH-8855 were tolerant to drought stress, which can be genetically analyzed for further studies [59].
It has been confirmed from research studies that screening for drought tolerance at the early growth stage can lead to a better understanding of the drought-tolerant nature of maize genotypes or lines. The maize inbred lines FLD 12, FLD 23, and FLD 24 were identified as drought tolerant in an earlier study conducted by Adhikari et al. [60]. This assessment was carried out based on morphological and physiological traits [60]. In another study, 24 super-sweet maize inbred lines were classified based on drought tolerance and yield potential. The inbred lines MPA90010/51-1 and MSH90011/82-1 were drought tolerant, as indicated by mean productivity (MP) values and the stress tolerance index (ST). These inbred lines could be used as source material for developing drought-tolerant maize genotypes [61]. The four inbred lines, Nub60 (1.56), Nub32, Nub66, and GZ603 (1.44) were the highest drought-tolerant inbred lines, as studied by Balbaa et al. [62]. Previous studies have extensively studied maize genotypes with phenotypic and genotypic variation for morphological, physiological, and biochemical traits. Emmanuel et al. [63] screened 12 Indian maize landraces for drought stress based on relative water content (RWC). RJ-2020, BPCH-6, and EC-3161 had high RWC and were selected as drought-tolerant landraces for further study [63]. Maize genotypes grown under drought stress conditions showed different degrees of drought tolerance. Masood et al. [64] revealed that genotypes 545, AES204, and WM13RA were selected based on superior performance under drought stress conditions. These genotypes can be used as parental material for developing droughttolerant maize hybrids [64]. In an earlier study, maize accessions 14927, 15155, 19191, and 15233 were identified as drought tolerant based on tolerance indices such as root length, shoot length, and wall thermostability linked to drought stress [65]. These results showed that landraces and inbred lines are potential sources of genetic variability for drought tolerance in maize. There is an urgent need to evaluate the wild maize relatives to screen for drought-tolerant traits that can be used to improve commercial cultivars grown in water-deficit soils. Field screening is relatively tough and costly; hence, screening maize genotypes in lab conditions would save time and accelerate plant breeding.

QTL Mapping Analysis for Drought Tolerance in Maize
QTL mapping has been effectively used for the genetic dissection of drought-tolerant genomic regions in maize [66]. Earlier and recent studies have identified the QTL linked with drought tolerance in maize, and some have been cloned successfully. Sheoran et al. [67] summarized the 542 QTL reported in 33 published papers for abiotic stress tolerance to conduct a meta-analysis. They have detected 32 meta-QTL possessing 1907 genes for abiotic stresses (waterlogging and drought). These QTL could facilitate drought-tolerant breeding in maize [67]. A double haploid (DH) population of 217 lines was subjected to drought stress to identify the genomic regions associated with drought tolerance in maize. A total of 9 QTL were detected for water stress conditions. QTL, qWS-GY3-1, and qWS-ESP3-1 (Table 1) contributed to drought tolerance, and these QTL were in edQTL regions (environment-dependent QTL) [68]. Another study reported 54 QTL affecting different photosynthetic traits in maize under well-watered and drought stress conditions. A total of 43 QTL identified under drought stress indicated that tolerance to photoinhibition is a key factor influencing drought tolerance in maize. These QTL could support MASbased breeding of drought tolerance in maize [69]. QTL sequence analysis is one of the reliable ways to identify the potential QTL/genes underlying drought tolerance in maize. Zhang et al. [70] used leaf relative water content (LRWC) as drought tolerance indices for maize drought tolerance. QTL sequence analysis identified four QTL, qLRWC2, qLRWC10a, qLRWC10b, and qLRWC10c for LRWC under water-deficit conditions. These QTL may help carry out additional research studies in the future [70]. In two QTL, qDTA3-3 and qDTA10, days to anthesis (DTA) were identified under drought stress conditions, which may be promising candidates for drought-tolerant breeding in maize [71].
Identifying QTL linked with yield-related traits can play a key role in developing high-yielding maize cultivars that sustain yield under water-deficit conditions. A total of 9 QTL were identified under water stress conditions in maize which linked to weight, kernel weight, and 100-kernel weight. The QTL qEL4s and qKW4s could be used for fine mapping and acceleration of MAS breeding as they were found in the same region in water stress conditions [72]. In the same way, Almeida et al. [73] identified 83 and 63 QTL for grain yield (GY) and anthesis-silking interval (ASI) through the individual environmental analysis of these traits. QTL, qWS-GY-10, and qWS-GY-7 were identified for GY under water stress conditions [73]. Another study identified 45 QTL (5 for yield and 40 for eight yield components) for yield and yield components. These QTL were distributed on all maize chromosomes except chromosome 8 [74]. A total of 203 QTL were identified for the traits ear per plant (EPP), stay green (SG), plant-to-ear height ratio (PEH), and anthesis-silking interval (ASI) linked with drought tolerance in maize. The QTL linked with SG and EPP were distributed in chromosomes 1 and 2. These QTL should be further analyzed for their use in MAS selection [75]. The haplotype QTL qPH-HP383-10 was detected on chromosome 10 and linked with drought tolerance, as reported by Lu et al. [76]. Another study identified many QTL for yield and yield-related traits under drought stress conditions. The QTL qES3 for ear setting (ES) was located in chromosome 3, which can contribute to drought tolerance in maize [77]. This comprehensive analysis of drought-tolerant QTL showed that these QTL could be used for QTL pyramiding and MAS breeding to develop drought tolerance in maize genotypes. However, additional studies are required to improve the efficiency of QTL mapping and the development of novel mapping populations, which can increase the chances of identifying novel regions for drought tolerance.

Overview of Association Mapping Studies for Drought Tolerance
Genome-wide association study (GWAS) is one of the most powerful and influential ways to dissect the genetic makeup of drought tolerance in crops. GWAS gives a reliable estimate of traits-marker association by screening many germplasms. A detailed overview of GWAS-based QTL/genes identification is critical to search for the most potential candidate genomic regions which can be exploited in drought-tolerant breeding. Wang et al. [79] reported the natural variation for drought tolerance in the maize seedling stage using GWAS. They have reported that natural variation in ZmVPP1 contributed significantly to the trait. ZmVPP1 overexpression in maize transgenic lines increased drought tolerance by increasing photosynthesis and root development [79]. GWAS identified 365 SNPs in 354 genes, and 52 of these genes exhibited significant differential expression in inbred line B73 under well-watered and water-stressed conditions. These alleles could improve drought tolerance in maize [27]. In another study, Wang et al. [80] evaluated 201 maize ILs and identified 206 significant SNPs in 115 candidate genes for drought tolerance and linked traits, including GY, plant height, etc. Besides this, GWAS also identified nine QTL, and these genomic regions might contribute to drought tolerance in maize [80]. SNPs are a common form of genetic variability in the maize genome. A pair linkage disequilibrium and association mapping analysis with phenotypic traits performed under well-watered and water stress conditions revealed a decline within 100-500 kb along with the physical distance of each chromosome. Results showed a strong association of 29 SNPs with two phenotypic traits in one or more environments. SNPs can be converted to functional markers and used for MAS programs [81].
Xue et al. [82] dissected the genetic complexity of drought tolerance using 350 maize ILs genotyped by using 1365 SNPs. GWAS has identified 42 linked SNPs in 33 genes for 126 traits × environment × treatment combination. Gene GRMZM2G12577 was linked with hundred kernel weight and relative ear position [82]. The morphological or yield traits and secondary metabolites can also contribute to drought tolerance in maize. In an earlier study, 123 significant SNPs linked with 63 loci were identified for phenotypic and metabolic traits under two water conditions. Of 63, 23 loci exhibited a significant effect between QTL and water status, indicating that these loci linked with metabolites were associated with drought tolerance [83]. The flowering stage is the most critical because any abiotic stress can cause a decline in the function of seed development. Hence, identifying loci linked with drought tolerance at the flowering stage should be a key priority for maize breeders. Recently, Khan et al. [84] evaluated 279 maize ILs under drought stress and well-watered conditions for three consecutive years. Plant height (PH), anthesis-to-silk interval (ASI), and ear biomass at silking stage (EBM) were studied for drought tolerance. GWAS identified 71, 159, and 21 SNPs (Table 2) linked with three traits, ASI, EBM, and PH. Only a few genes were linked with drought-tolerant traits. For example, ARABIDILLO 1 was linked with ASI and SF16 protein for PH [84]. Drought mainly affects the root traits of crops; hence, an improvement of root-based drought tolerance is critical to sustaining maize growth under drought stress. GWAS-based analysis of seminal root length (SRL) has identified 62 loci under two water treatments. This strongly supports the further analysis and use of GWAS for a better understanding of drought tolerance in maize [85]. Grain yield (GY) and flowering time are critical traits affected by drought stress. GWAS and genomic prediction (GP) analyses of 300 maize ILs identified 688 candidate genes under drought stress and well-watered (WW) conditions. These genes were enriched into 15 ontology terms, and 46 genes showed significant differential expression under WW and water stress conditions. The genetic information and loci of flowering time and grain yield (GY) identified in this study can be used to rapidly develop stress-tolerant maize germplasm [86].
Genomic selection (GS) has gained importance because of the hindrances in traditional breeding methods. Two hundred forty maize subtropical lines were phenotyped for drought tolerance using 29,619 SNPs. A total of 77 SNPs were significantly associated with 10 TFs responsible for drought tolerance. TFs were involved in the drought tolerance of morphological and physiological traits [87]. The comprehensive analysis of 240 maize lines was performed using high-density markers for the genetic mechanism of drought tolerance. Out of 61 SNPs, 48% were critically linked with drought-tolerant genes. These SNPs were associated with stomatal closure, flowering, root development, detoxification, and reduced water potential. Interaction of SNPs via functional traits can improve drought tolerance in maize. SNPs linked with ABA signaling improved drought tolerance by regulating flowering, auxin, root development, etc. [88]. Earlier researchers, Osuman et al. [89], conducted GWAS analysis for yield and yield-related traits under terminal drought conditions. One hundred sixty-two maize tropical lines were evaluated under drought stress and genetically analyzed for potential SNPs/genes. In total 66, 27, and 24 SNPs were associated with traits evaluated under combined heat and drought (CHD), terminal drought (TD), and their combined effects. Four SNPs had pleiotropic effects on days to anthesis (AD), and silking (SD) under CHD. A total of 12 genes were implicated in regulating plant response to multiple abiotic stresses [89]. A collection of 210 maize ILs was analyzed for genomic regions governing drought tolerance. GWAS identified 696 traits-associated SNPs under water-stressed (WS) and 413 SNPs under well-watered (WW) conditions. These results showed the importance of genomic selection (GS) for drought breeding [90].
Maize landraces have been a potential source of genetic diversity for drought tolerance. Identifying genes in landraces and their transfer can eventually improve drought tolerance in maize; however, it demands the large-scale GWAS-based genetic analysis of landraces. A GWAS technique was employed on 1326 maize landraces developed by the CIMMYT Genetic Resource Program. GWAS was employed to identify the marker-trait and grain yield (GY) associations for two irrigation treatments. Two genes were linked to drought stress response and showed that landraces had a significant genetic diversity and could be used to improve drought tolerance in maize [91]. As mentioned in earlier analyses, GWAS showed that it is a powerful technique to unfold the genetic background of drought tolerance and can be widely exploited for other drought breeding programs. More studies may be conducted to analyze the biochemical-based drought tolerance in maize using GWAS analysis. Candidate genes identified in GWAS studies must be transformed using the latest molecular breeding techniques.

TFs Analysis and Their Role in Drought Tolerance
TFs analysis is one of the most powerful techniques to recognize the genomic regions involved in tolerance to abiotic stresses. TFs regulate gene expression for several abiotic stresses [93]. Various TFs families such as WRKY, NAC, and bZIP have a key role in plant response to abiotic stress [94][95][96]. A detailed and updated picture of TF iden-Agronomy 2023, 13, 1459 9 of 21 tification and application in drought tolerance in maize is critical. WRKY is one of the largest families of TFs, which regulates several critical responses under drought stress. Zhao et al. [97] screened a new WRKY IIa TF, a substrate protein of ZmMPK6. Overexpression of ZmMPK6 enhanced drought tolerance in maize. ZmWRKY104 phosphorylated by ZmMPK6 was key in the ABA-induced antioxidant defense response (Table 3) [97]. Earlier, Wang et al. [98] identified a WRKY TF, ZmWRKY40, which is in the nuclei of mesophyll protoplasts. ZmWRKY40 was induced by drought stress and ABA. This study has provided a candidate gene and genetic mechanism of ZmWRKY40 to improve drought tolerance in maize [98]. The function of another maize WRKY TF, ZmWRKY79 was elucidated by drought stress and ABA-biosynthesis. ZmWRKY79 boosted the ROS scavenging, reduced the H 2 O 2 content, and increased the antioxidant defense system. ZmWRKY79 also targeted the gene ZmAAO3 in maize protoplast via acting on the W-boxes of corresponding gene promoters [99].
The basic helix-loop-helix (bHLH) TFs family contributes to maize's root and low phosphate development. ZmPTF1 of the bHLH family regulated ABA synthesis, signaling pathways, and drought tolerance. The ZmPTF1-overexpressed maize lines had improved root systems and enhanced ABA content. Hence, ZmPTF1 was found to be a useful gene for transgenic breeding in maize [100]. The bHLP TF can be a potential breeding target for drought tolerance in maize. CgbHLH001 is a bHLP TF from Chenopodium glaucum that increased drought tolerance in maize. CgbHLH001-overexpressed maize lines showed improved drought tolerance by accumulating soluble sugars and increased activities of antioxidants such as superoxide dismutase (SOD), and catalase (CAT) [101]. The BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1(BZR1) TFs regulate drought tolerance in maize via different mechanisms. Two genes, ZmBES1/BZR1-3 and ZmBES1/BZR1-9, were cloned from a maize inbred line, B73, and they were functionally characterized by investigating their pattern of expression and transcriptional activation activity. These genes were linked to responses to oxidative stress and amino acid metabolic processes. Further analysis is required for the detailed mechanism of drought tolerance in maize and Arabidopsis [102]. The Apetala2/ethylene response factor (AP2/ERF) family of TFs plays a major role in abiotic stress tolerance in maize. Unfortunately, few members of this family have been characterized in maize and demand more studies. ZmEREBP60, a member of TFs, was functionally characterized in maize, and its expression was induced by drought stress in roots and leaves. ZmEREBP60 enhanced drought tolerance in maize by reducing the content of H 2 O 2 . Hence, ZmEREBP60 is a positive regulator of drought tolerance in maize and can be used for drought breeding [103]. NAC TFs play a key role in response to abiotic stresses. However, information about stress-induced NAC genes in maize is insufficient. Wang et al. [104] identified 87 NAC TFs in maize under drought stress conditions that showed differential expression [104]. Mao et al. [105] cloned a NAC TF, ZmNAC55, and studied its role in drought tolerance, and expression of this TF was induced by drought stress. ZmNAC55 enhanced drought tolerance in wild Arabidopsis and was a positive regulator of drought tolerance [105]. The NAC TF, ZmNAC49, improved drought tolerance in maize by reducing stomatal density. ZmNAC49 overexpression decreases the stomatal density, rate of transpiration, and stomatal conductance. The expressions of genes ZmTMM and ZmSDD1 were also induced by ZmNAC49 [36]. Many studies have reported the identification and cloning of genes that showed the differential mechanism for drought tolerance. HD-ZIP is one of the largest families of TFs, characterized by its role in drought tolerance in maize. Qiu et al. [106] identified the 42 ZmHDZ genes using maize transcriptome data. Results showed that the expression of several ZmHDZ genes was induced by drought stress, and maize lines with overexpression of ZmHDZ9 showed enhanced activity of SOD and peroxidase (POD) and enhanced soluble protein content. Hence, this study reported the evolutionary HD-ZIP TFs homologs in maize [106]. Another HD-ZIP TF, ATHB-6, reduced the content of malondialdehyde in lines overexpressing ATHB-6 compared to the wild type. Hence, further studies will help to deeply understand the functions of HD-ZIP TFs in maize [107]. MYB TFs family is mainly found in eukaryotes and contributes to crop drought tolerance. The research study showed that the mechanism of ZmMYB-CC in maize is unknown. ZmMYB-CC10 enhanced drought tolerance in maize by reducing oxidative damage and decreasing the content of H 2 O 2 . The activation of the ZmAPX4 function by ZmMYB-CC10 (Table 3) alleviated drought stress in maize [108]. Nuclear factors Y (NF-Ys) are significant TFs; however, their function in the underlying tolerance mechanism in crops is largely unknown. Two nuclear factors Y (NF-Ys) members were functionally characterized for their role in drought tolerance. ZmNF-YB16and ZmNF-YA1 improved drought tolerance in maize by similar biological functions under drought stress; however, they had different roles in other biological processes. The TF, ZmNF-YA1, promoted root development in maize [34]. The ethylene response factor (ERF) also plays a key role in improving drought tolerance in maize. An ERF TF, ZmERF21, is mainly expressed in roots and leaves, and its expression was induced by drought stress. The overexpression of ZmERF21 increased chlorophyll content and antioxidant activities under drought stress. ZmERF21 may regulate the genes linked to hormones and calcium signaling [109]. The detailed functional analysis of MYB and NF-Ys needs further studies because their genetic mechanism for drought tolerance is largely unknown. Identifying more TFs of MYB and NF-Ys would be useful.

Transcriptome Analysis for Drought Tolerance
Transcriptome analysis gives an understanding of the molecular mechanisms of drought tolerance in maize and has been regarded as the best research tool. Hence, analysis of the gene expression profile of maize genotypes under drought stress conditions will enhance our understanding of genetic control of drought tolerance [111]. Dozens of studies have been published on this topic, but an updated picture of recent studies is critical for future researchers Zheng et al. [111]. analyzed the gene expression profile of two maize inbred lines with different levels of drought tolerance. They detected 24, 220, and 4551 differentially expressed genes (DEGs) in inbred line 287M (drought tolerant) after 24, 48, and 72 h of drought stress. Likewise, 16, 29, and 2641 genes were identified in 753F (drought sensitive) after 24, 48, and 72 h of drought stress. The difference between the two inbred lines was linked with the scavenging of ROS, signal interaction network, etc. In an earlier study, 957 DEGs in maize genotypes subjected to melatonin and non-melatonin treatment were identified. MT-based regulated genes were linked with calcium signaling transduction and the biosynthesis of jasmonic acid (JA). MT interacted with other hormones to enhance drought tolerance. This drought tolerance mechanism would be highly useful for drought breeding [112].
One of the best ways is to conduct the comparative transcriptome analysis of droughttolerant and sensitive genotypes. A transcriptome study has identified 4552 DEGs in C7-2t (drought-tolerant mutant) and C7-2 wild-type maize. Results showed that drought tolerance of inbred line C7-2t (drought-tolerant mutant) was due to its water-holding capacity and improved photosynthesis rate [113]. Two constating maize ILs, CML69 (drought-tolerant) and LX9801 (susceptible), were treated with drought stress at the seedling stage for five days. RNA sequence analysis has identified 10,084 DEGs. Drought-tolerant genes were divided into two sets; 4687 were genotype-specific, and 2219 were common drought-responsive genes. Tolerant genes are linked with the scavenging of ROS, drought avoidance, osmotic regulation, etc. [114]. Along with introgression lines (ILs), different hybrids were evaluated to study the transcriptome analysis under drought stress conditions in maize. Two hybrids, ND476 (drought tolerant) and ZX978 (drought sensitive), were evaluated under drought stress and control conditions. Transcriptome analysis identified 3451 and 4088 DEGs in ND476 and ZX978. Genes in ND476 were associated with starch and sucrose metabolism, while genes in ZX978 were linked to the ribosome and pentose [115]. Another hybrid, Zhengdan538, showed an enhanced response to drought stress in maize. A total of 2994-4692 DEGs were identified under WW and drought stress conditions, which were recognized by comparison with their parents. The potential genes were involved in energy biosynthesis and photosynthesis. This expression level dominance (ELD) was key in hybrid tolerance to water-deficit conditions [116]. Recently, Gillani et al. [117] evaluated two contrasting maize ILs, 478 (drought tolerant) and H21 (susceptible), to conduct the transcriptome analysis for drought tolerance under water stress conditions. They identified 68%, 48%, and 32% of drought-responsive genes (DRGs) in 478, and genes involved in drought tolerance were linked with starch and sucrose metabolism, plant hormonal signal transduction, etc. [117].
627 RNA sequence analyses were performed for 224 maize accessions under three water regimes. Results showed that 73,573 eQTL were identified for 30,000 genes. Ninetyseven genes were prioritized as related to drought tolerance because of their different expressions. These prioritized drought-tolerant genes may be a direct target for alleles mining [118]. These results showed the significance of RNA sequence analysis in identifying the DEGs in maize under drought stress conditions. Dong et al. [119] identified 666, 2417, and 7375 DGEs at the flare, tasseling, and grain-filling stages. Gene, DnaJ, and a putative WAK family receptor-like protein kinase were linked with molecular chaperon activities and cell signal transduction. DnaJ gene expression can be the best tolerance index for drought tolerance screening [119].
Identifying genes at the tasseling stage may help mitigate the harmful effects of drought stress in maize and avoid yield loss. Ten maize ILs were evaluated under drought stress to perform the RNA sequence analysis of early-developing tassels under control and water stress conditions. A total of 19,001 DEGs were identified in 10 ILs [120]. The application of growth hormones and osmolytes such as glycine betaine (GB) also enhanced drought tolerance in maize, as shown by transcriptome analyses. Two drought-tolerant and susceptible inbred lines, Chang 7-2 and TS141, were subjected to drought stress and GB treatment, and 562 up-regulated and 824 downregulated DEGs were found in ILs. The exogenous application of GB induced the upregulation of 1061 and 424 DEGs in ILs. The expression of 9 DEGs was constant with their transcriptome expression profile [1]. Pollen development is one of the most sensitive stages of maize reproduction. Hence, transcriptome analysis at the pollen-setting stage helps identify the potential genes involved in maize reproduction. A maize hybrid was evaluated under drought stress. It was observed by transcriptome analysis that the expression profile of 6424 genes and 1302 (Table 4) transcripts were changed in pollen grains after seven days of drought stress. Different genes were involved in pollen development and responses to drought stress. The identification of tolerant genes during pollen development and change in the expression profile of transcripts can help to prevent the loss caused by water shortage during the pollen development stage [121]. Transcriptome analysis to identify the potential genes in wild relatives of maize would accelerate drought-tolerant breeding. These ideas should be implemented in future research studies. Table 4. A List of genes expressed under drought stress conditions in maize.

Transgenic Breeding and CRISPR/Cas9
Genetically modified (GM) crops were first introduced in the United States (US) in the mid-1990s, and they were extensively adopted by growers around the world [125]. A total of 189.8 million hectares of land were used to plant GM crops alone in 2017 [126]. To save time and money, it has become essential to employ GM tools to combat the rising threats of drought stress in maize. A transgenic drought-tolerant maize line, SbSNAC1-382, with overexpression of the gene, SbSNAC1, showed enhanced tolerance to drought stress, as studied by Zeng et al. [127]. Earlier, two transgenes, BetA, and TsVP, were expressed in maize plants produced by cross-pollination. Pyramided transgenic maize plants showed higher contents of GB and H + -PPase activity compared to parental lines. Pyramid plants had vigorous growth and higher yields than their wild type and parents. This study proved that several transgenes could improve drought tolerance in transgenic maize plants [37]. To obtain effective transgenic plants, a suitable promoter that permits transgene expression at desired levels is very significant [128]. Therefore, scientists must isolate various promoters and identify their characteristics. The isolation and functional characterization of abscisic acid (ABA) promoters are of great importance for the genetic improvement of drought tolerance in maize. A ZmSOPro gene was isolated from the maize genome and analyzed for its role in drought tolerance. The minimal ZmSOPro was induced by drought stress in transgenic maize plants. This gene and promoter, the 119-bp promoter, could be an ideal candidate for the genetic engineering of drought tolerance in maize [129].
Earlier, TsVP was transformed from a Thellungiella halophila into maize. Transgenic maize plants exhibited a higher percentage of seed germination and less membrane damage than wild-type plants. V-H + -PPase action of transgenic maize enhanced drought tolerance in maize plants [130]. Further characterization and analysis of transgenes can improve the speed of drought-tolerant breeding in maize. The identification and transformation of genes from other crops can also increase maize's tolerance to drought stress. ERECTA (ER) is a leucine-rich repeat-receptor-like kinase gene (LRR-RLK) encoding a protein identified in Arabidopsis. Two ERECTA (ER) genes, SbER1-1 and SbER2-1, were isolated from droughttolerant sorghum (Sorghum bicolor). The SbER2-1 gene increased drought tolerance in maize by increasing the net photosynthetic rate. Transgenic maize plants have a higher lignin content and upgraded metabolism under drought stress. SbER2-1 can be used to breed genetically modified cultivars [131].
In the same way, HVA1 from barley was transferred to develop transgenic maize plants to cope with drought stress conditions. The T3 plants with transgene overexpression showed higher root and leaf biomass and RWC, indicating GE's promising role in drought tolerance [132]. Plant hormones play an important role in abiotic stress response. AtG2ox1, a member of the GA2ox family, is used to develop maize plants with gibberellins (GA) deficiency. AtG2ox1 enhanced the chlorophyll content and growth of transgenic plants compared to wild-type plants. AtG2ox1 can be used as a candidate gene for breeding drought-tolerant maize cultivars [133]. It has been concluded that studies on genetically modified drought-tolerant maize are limited. This issue must be taken seriously to map the potential genes from local genotypes or wild type and other crop genomes to breed the transgenic maize plants.
CRISPR/Cas9, a novel gene editing tool [134], is valuable in accelerating molecular breeding for abiotic stress tolerance in crops ( Figure 3). The genome editing tools, transcription activator-like effector nucleases (TALENs), meganucleases, zinc-finger nucleases (ZFN), and CRISPR/Cas9 have ensured the targeted gene editing in plants [135][136][137]. CRISPR/Cas9 is highly efficient and easiest to implement. Cas9 edits the targeted DNA sequence in the genome. CRISPR/Cas9 gene editing is accomplished by introducing a DNA double break (DSB) in the targeted genes through Cas9, followed by DNA repair using non-homologous end joining (NHEJ) and homology direct repair (HDR). This tool has been used in maize [138]. ARGOS8 is a negative regulator of ethylene responses. To study the targeted use of ARGOS8 native expression variation in drought-tolerant breeding, a set of 400 inbred maize was examined for ARGOS8 mRNA expression. The CRISPR-Cas9 tool was used to generate new variants of the ARGOS8 gene. Compared to the wild type, a field study showed that ARGOS8 variants enhanced grain yield by five bushels per acre under flowering stress conditions and had no loss in yield under well-watered conditions. These outcomes showed the greater ability of CRISPR/Cas9 to generate novel drought-tolerant mutants [38]. Recently, Pan et al. [139] generated two allelic mutants of ZmSRL5 using CRISPR/Cas9. The wax-related genes were not changed in the srl5 mutant. Hence, ZmSRL5 is obligatory for the structure formation of cuticular wax and can enhance drought tolerance [139]. In another study, the zmcpk37 mutant was generated using CRISPR/Ca9. The transgenic lines of ZmCPK35 and Zmcpk37 were obtained using Agrobacterium-mediated transformation in maize IL. Maize hybrid and IL with overexpression of ZmCPK35 and Zmcpk37 showed higher yields under drought stress [140]. Despite all of the progress, the use of CRISPR/Cas9 in maize is limited regarding breeding for drought tolerance. Targeted genes can be edited to generate desired mutants in maize to expand the use and efficiency of CRISPR/Cas9. New editing systems (base editing and prime editing) would increase the precision of genome editing in maize.
of ZmCPK35 and Zmcpk37 showed higher yields under drought stress [140]. Despite all of the progress, the use of CRISPR/Cas9 in maize is limited regarding breeding for drought tolerance. Targeted genes can be edited to generate desired mutants in maize to expand the use and efficiency of CRISPR/Cas9. New editing systems (base editing and prime editing) would increase the precision of genome editing in maize. Figure 3. The application of molecular tools such as CRISPR/Cas9 and genetic engineering have greatly accelerated the speed of drought breeding in maize. Expanding these tools would be highly useful in breeding tolerant genotypes that can combat the threats of drought stress in the future. This figure was created with Biorender.com.

Conclusions and Outlook
Maize is one of the most significant cereal crops that provides nutrition to human beings and livestock. Maize plays a key role in the gross domestic product (GDP) of many countries. Drought stress has curtailed the maize growth and yield in a large area and created an imbalance in the food supply chain. Human popul0ation growth and increasing consumption tend to increase the emissions of climate-changing greenhouse gases. Global warming has negative consequences on crop growth and yields in large areas. The threat of drought stress will be increased soon because of the continuous depletion of water resources and global warming. Maize breeders have used several conventional breeding tools to breed drought-tolerant maize genotypes and have achieved significant results. However, these breeding tools are time-consuming and costly, hindering the success of drought breeding. Maize tolerance to drought stress is polygenic, and, hence, the complex genetic mechanism of drought tolerance has hindered the large-scale use of conventional

Conclusions and Outlook
Maize is one of the most significant cereal crops that provides nutrition to human beings and livestock. Maize plays a key role in the gross domestic product (GDP) of many countries. Drought stress has curtailed the maize growth and yield in a large area and created an imbalance in the food supply chain. Human popul0ation growth and increasing consumption tend to increase the emissions of climate-changing greenhouse gases. Global warming has negative consequences on crop growth and yields in large areas. The threat of drought stress will be increased soon because of the continuous depletion of water resources and global warming. Maize breeders have used several conventional breeding tools to breed drought-tolerant maize genotypes and have achieved significant results. However, these breeding tools are time-consuming and costly, hindering the success of drought breeding. Maize tolerance to drought stress is polygenic, and, hence, the complex genetic mechanism of drought tolerance has hindered the large-scale use of conventional breeding methods. The maize plant uses several morphological, physiological, and biochemical processes to cope with drought stress; however, this is not a long-term solution to increase crop growth. In the past years, scientists have brought a significant revolution in molecular breeding to counter the threat of rising climatic changes. QTL mapping has identified the novel genomic regions controlling drought tolerance in maize and has shown marvelous success. It has also enhanced the use of MAS selection as well as the pyramiding of QTL, which enhanced the drought tolerance of several maize genotypes. Despite its large-scale use, QTL mapping has several limitations, such as the use of large sample sizes and recombination during the development of RIL. These challenges for QTL mapping must be addressed in future studies to enhance the efficiency of this technique. The above-reported QTL should be cloned and transferred to susceptible genotypes to increase their tolerance to rising episodes of drought stress. GWAS has effectively mapped the novel QTL and SNPs by analyzing a large sample size. Several QTL and SNPs have been identified that are involved in maize drought tolerance. GWAS cannot identify all genetic determinants of complex traits and can only identify the modest action of heritability.
Transcriptomes and TF analysis techniques have been used to identify the novel genomic regions for drought tolerance in maize. However, not all of them have been cloned and targeted by molecular breeding. It is necessary to screen the identified genes and examine their regulatory networks for their role in drought tolerance in maize. Some families of TFs are not completely understood for their possible role in drought tolerance. Genetic engineering techniques have shown tremendous success in many crops and maize to increase the resistance to certain abiotic stresses. The transgenic genotypes of many crops have significant resistance to drought stress; however, there is still insufficient knowledge about transgenic breeding applications in maize. Therefore, developing transgenic maize genotypes is urgently needed to cope with drought stress. The large-scale screening of maize germplasm is critical in identifying the tolerant genotypes as well as tolerant traits associated with drought tolerance. Abiotic stresses have threatened maize genetic diversity; hence, it is important to safeguard it using several molecular breeding tools. Comprehensive genome sequencing can give a better overview of the gene networks involved in drought tolerance in wild types.
We should expand the use of CRISPR/Cas9, a novel gene editing tool, to generate drought-tolerant mutants in maize. The use of CRISPR/Cas9 will accelerate droughttolerant breeding. Using CRISPR/Cas9 will increase the success rate of drought breeding and enhance the precision of genome editing. We suggest using novel gene editing systems such as prime editing (PE) and base editing (BE), which can edit large fragments of genetic material. Overall, the studies mentioned above showed that drought stress is still a burning issue and will continue to affect crops in large areas. Hence, improving existing molecular breeding tools and developing new methods is necessary to improve the genetic makeup of crops to deal with drought stress issues. This comprehensive and well-organized review paper will serve as a valuable source of information for future maize researchers to understand the basis of drought tolerance and adopt a more powerful method to breed drought-tolerant maize genotypes.