Improving Crop Tolerance to Abiotic Stress for Sustainable Agriculture: Progress in Manipulating Ascorbic Acid Metabolism via Genome Editing

Abstract

Plants often encounter challenging environmental factors, including intense sunlight, drought, extreme heat, cold temperatures, salinity, excessive metals, and nutrient shortages, which can heavily affect their growth and survival. In this regard, L-ascorbic acid (AsA) is not only an essential nutrient for human health but also plays a significant role in plant responses to environmental stresses, regulating various functions during growth and development, redox signaling, and phytohormone biosynthesis. The growing need to cope with climate change, together with the advancement of CRISPR/Cas9-editing technologies, stimulated new opportunities to enhance AsA biosynthesis to improve crop stress tolerance. In this review, we discuss the biosynthesis and regulation of AsA in abiotic stress response mechanisms. We also explore the latest advancements of CRISPR/Cas9 technologies, their applications, and their challenges as tools for modifying genes associated with AsA metabolism, aiming to develop crops more tolerant and resilient to environmental changes.

1. Ascorbic Acid to Counter Oxidative Damage

Plants are continuously exposed to environmental fluctuations, but unlike animals, they cannot relocate in response to these challenges. Thus, as sessile organisms, they must depend on their internal regulatory mechanisms to adapt to external changes. Under usual fluctuations, plants can adapt to regular environmental changes, maintaining their growth and development. However, when environmental conditions become extreme or persist for long periods, plants may experience what is known as abiotic stress. This form of stress occurs when environmental non-biological factors such as temperature, water deficit, nutrient imbalances (toxicity or deficiency), or salinity significantly challenge the plant’s ability to adapt. In the context of ongoing climate change, abiotic stress has emerged as a critical threat to agriculture, causing yield losses and thus affecting the supply of global food needs [1,2].

A common consequence of many abiotic stresses is the disturbance of redox balance, leading to the accumulation of reactive oxygen species (ROS) [3]. The generation of ROS includes free radicals like superoxide (O₂⁻) and hydroxyl radicals (OH⁻), along with non-radical molecules such as hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂) (Figure 1). In excessive quantities, these reactive molecules cause oxidative damage to cellular structures like proteins, lipids, and DNA, ultimately compromising the cell’s integrity (Figure 1). ROS also contribute to cell death when the stress is too intense and surpasses the cell’s antioxidant defenses and repair mechanisms [4]. Whether ROS causes severe cellular damage or helps plants acclimatization depends on where and how much ROS are produced, which are tightly regulated by antioxidant systems. To cope with abiotic stresses, plants implement a wide array of adaptive responses that enable them to maintain their homeodynamic equilibrium and regulate, among other things, the production of secondary metabolites [5,6]. In particular, plants have developed L-ascorbic acid (AsA) accumulation as a non-enzymatic antioxidant mechanism to detoxify ROS [7,8].

AsA is an effective antioxidant that protects organelles and, more generally, the cell from oxidative damage by ROS induced by environmental stress [9,10,11]. Furthermore, AsA plays a role in numerous processes, including regulating cell division and expansion, photosynthesis, and hormone biosynthesis, and it acts as a cofactor in many enzymatic activities [12,13]. AsA is present in various plant tissues, and its concentrations are high in meristems, fruits, and especially in the fully developed chloroplasts of mature leaves [14,15].

In most plant species, AsA concentrations are insufficient to effectively counteract abiotic stress effects [16]. Increasing AsA in plants has become pivotal and can be achieved through conventional breeding programs, such as marker-assisted selection using DNA level polymorphism [17]. However, these programs are often slow and labor-intensive [18,19,20]. Another method to enhance AsA biosynthesis involves the mutation breeding, using physical or chemical mutagens [21]. Unfortunately, mutagenesis results are unpredictable due to the simultaneous induction of off-target mutations, the phenomena of chimerism, and the difficulty to detect rare genetic variants. Apart from optimizing agronomic practices, such as irrigation and nutrient management [22], another way to increase AsA content involves the application of exogenous AsA [23,24,25]. Excellent reviews help to identify what research has been conducted on the exogenous application of AsA [10,16]. These studies demonstrate that external AsA treatments by foliar sprays, seed priming, and its incorporation into the rooting medium improved physiological and productive traits in crops [23]. Progress has been observed in plant yield and growth by regulating various physio-biochemical pathways in stressful conditions such as salinity, drought, and extreme temperatures. However, the exogenous application of AsA has notable limitations, such as its transient effect, which necessitates repeated treatments to maintain stress resistance, increasing labor costs. Furthermore, excessive AsA application can disrupt the plant’s natural redox balance, potentially reducing its overall effectiveness.

Advanced targeted genome editing on single or multiple target sites through the CRISPR/Cas9 system can address these limitations [26]. These tools enable precise interventions in plant metabolic pathways [27], and in many cases, these technologies have enabled the development of plant varieties with increased tolerance to abiotic stresses [28,29]. However, the potential for modulating the AsA pathway using the latest CRISPR/Cas9 techniques remains largely unexplored. In this review, we examine the biosynthesis and the important role of AsA in the mechanisms of plant responses to abiotic stress. We also explore the latest advancements in CRISPR/Cas9 technology, highlighting its applications in gene editing associated with AsA metabolism to develop crop varieties more tolerant to abiotic stresses for more environmentally sustainable agriculture.

2. Ascorbic Acid Biosynthesis and Modulation in Response to Abiotic Stresses

AsA biosynthesis in plants can occur via four proposed pathways: L-galactose (L-Gal), L-gulose, myo-inositol, and D-galacturonate (Figure 2). The L-Gal pathway, which shares an aldonolactone as the immediate precursor to AsA with the other pathways, is called “Smirnoff–Wheeler” [30,31]. Substantial evidence supports the L-Gal pathway as the primary route for plants ascorbate biosynthesis. In contrast, the roles of the L-gulose, myo-inositol, and D-galacturonate pathways remain debated [32].

The L-Gal pathway begins with glucose and involves several enzymatic steps (Figure 2). Prior to guanosine diphosphate (GDP)-D-mannose formation, the initial steps intersect with pathways related to the synthesis of cell wall polysaccharides and glycoproteins. D-fructose-6-phosphate derived from D-glucose is converted into D-mannose-6-phosphate via the enzyme phosphomannose isomerase (PMI) [33], which is followed by conversion to D-mannose-1-phosphate through phosphomannomutase (PMM). PMM is then transformed into GDP-D-mannose by the enzyme GDP-D-mannose pyrophosphorylase (GMP). From this point on, the reactions are dedicated exclusively to AsA biosynthesis.

GDP-D-mannose conversion to GDP-L-galactose is catalyzed by GDP-D-mannose-3,5-epimerase (GME), which belongs to the short-chain dehydratase/reductase protein family. The following steps in the pathway involve the transformation of GDP-L-galactose into L-galactose-1-phosphate, L-Gal, and finally L-galactono-1,4-lactone, which is catalyzed by three enzymes: GDP-L-galactose phosphorylase (encoded by VITAMIN C2 (VTC2) and VTC5 in Arabidopsis thaliana) [34], L-galactose-1-phosphate phosphatase (encoded by VTC4), and NAD-dependent L-galactose dehydrogenase (GalDH) [35]. Among these steps, the reaction catalyzed by GDP-L-galactose phosphorylase is considered the first committed step for AsA biosynthesis with regulation of the pathway occurring predominantly at the level of this gene [8,14].

The third pathway (Figure 2) originates from GDP-D-mannose, where GDP-D-mannose 3′,5′-epimerase converts it into GDP-L-gulose, which is then processed into L-gulose-1-phosphate by GDP-L-gulose-1-phosphate phosphatase [36,37,38]. The L-gulose-1-phosphate is further converted into L-gulose, and eventually, L-gulono-1,4-lactone is formed through L-gulose dehydrogenase. This compound is then used in the final step of AsA synthesis, which is catalyzed by L-gulono-1,4-lactone dehydrogenase.

Another significant pathway involves cell wall pectins (Figure 2), where methyl-galacturonate is converted into L-galactonate by methyl esterase and D-galacturonate reductase [39,40]. L-galactonate is then transformed into L-galactono-1,4-lactone through a reaction catalyzed by aldono lactonase, and this precursor is used in the final synthesis of AsA.

The fourth pathway involves myo-inositol (Figure 2), which is converted into L-gulono-1,4-lactone through three reactions catalyzed by myo-inositol oxygenase, glucuronate reductase, and aldono lactonase [41,42]. Finally, L-gulono-1,4-lactone is converted into AsA through a reaction catalyzed by a dehydrogenase.

In addition to scavenging ROS to reduce oxidative damage, AsA also regulates ROS homeostasis, affecting plant stress response mechanisms [15,43]. In A. thaliana, the vtc1-1 mutant showed a mutation in the gene encoding GMP, reducing GMP activity and significantly lowering AsA production [44]. VTC1, besides its role in AsA synthesis, has emerged as a pivotal player in plant growth, stress responses, and other physiological processes [45]. Specifically, VTC1 interacts with CSN5B, a subunit of CONSTITUTIVE PHOTOMORPHOGENESIS 9 (COP9) signalosome (CSN), to influence plant development and responses to environmental stress [46]. Interestingly, mutations in CSN components, such as csn5b and csn8, negatively regulate AsA levels, further highlighting the role of CSN in modulating AsA biosynthesis during oxidative stress or high salt concentrations [47,48].

Further investigation into AsA regulation has revealed other important genes [32]. For example, VTC2 plays a critical role in AsA production, and vtc2 mutants exhibit a reduction in AsA levels [35]. Despite its low expression, VTC2 works alongside VTC5 to support AsA biosynthesis [34,49]. The vtc2/vtc5 double mutant suffers from severe developmental defects, which can be reversed by supplementing the plants with AsA or its precursor L-Gal. Another critical regulatory mechanism of AsA biosynthesis involves a non-canonical upstream open reading frame (uORF) in the VTC2 gene, which modulates translation based on the cellular concentration of AsA [50]. When AsA levels are high, the uORF inhibits further AsA production by blocking the translation of GDP-L-galactose phosphorylase (GGP). At the same time, under low AsA conditions, GGP synthesis is promoted [51].

The enzymes involved in AsA metabolism are essential for both the synthesis and regeneration of AsA and for the control of its turnover within the plant system especially during abiotic stresses [reviewed by Xiao et al. (2021)] [13]. Therefore, AsA can exist in two oxidized forms: monodehydroascorbate (MDHA) and dehydroascorbate (DHA). For this purpose, AsA is oxidized to MDHA by ascorbate oxidase (AO) or ascorbate peroxidase (APX), and MDHA can spontaneously convert to DHA (Figure 3). Both forms can be reduced back to ascorbate through the activity of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) with NAD(P)H and glutathione serving as electron donors. In particular, APXs are involved in sustaining cellular redox homeostasis by using AsA to detoxify H₂O₂ [52]. Deficiency in APX6 activity leads to a reduction in seed AsA levels and the accumulation of DHA, which disrupts cellular redox balance and affects signaling pathways associated with ROS, abscisic acid (ABA), and auxin [53]. On the other hand, AOs, which oxidize AsA to MDHA, are primarily located in the cell wall and participate in cell signaling transduction. Unlike APX, AO uses molecular oxygen (O₂) to oxidize AsA [54].

In the apoplast, MDHA produced by the oxidation of AsA through AO can be converted to DHA and transported into the cytoplasm, where DHA is recycled back into AsA by DHAR. This process facilitates an efficient antioxidant exchange between the apoplast and the cytoplasm, which is critical for maintaining redox homeostasis, especially under abiotic stress conditions [55,56]. During stress, the rapid increase in DHA flux from the apoplast alters the redox balance in both compartments, signaling adaptive responses. In contrast, alterations in DHAR activity can significantly influence the AsA/DHA ratio, affecting plant responses to stress [13,57]. A low AsA/DHA ratio can impair auxin signaling pathways compromising plant responses to environmental challenges, while a high ratio is often associated with increased tolerance to abiotic stresses [58,59,60].

AsA is also important in hormonal signaling networks orchestrating plant stress responses [13]. The influence of AsA on gene expression is particularly relevant in the context of ABA signaling. During early stress responses, ABA induces and activates ROS production and stress signaling pathways [61,62]. For example, in A. thaliana, the vtc1 mutant, deficient in AsA, exhibits lower stress tolerance and accelerated flowering due to the modulation of ABA synthesis [63,64]. Furthermore, AsA can improve drought tolerance in maize by increasing ABA synthesis, reducing water loss and improving resistance to osmotic stress [65]. In other cases, high amounts of reduced AsA stimulate the synthesis of ethylene, which regulates fruit ripening and responses to environmental stress [66]. In maize, under heat stress conditions, AsA can increase heat tolerance by decreasing ABA and indole acetic acid (IAA) levels while promoting the accumulation of salicylic acid (SA) [67]. In wheat, AsA enhances salt tolerance by promoting the synthesis of gibberellins (GAs), IAA, zeatin, and brassinosteroids (BRs), helping to improve plant response to salt stress [68].

3. Genome Editing to Improve Abiotic Stress Resistance

3.1. CRISPR/Cas System

One of the most efficient strategies to enhance plants’ resistance to abiotic stress is to modify the crucial pathways involved in antioxidant activity. Genome editing (GE) approaches can be applied to perform these modifications. Among the GE techniques, the “domestication” of the natural system called CRISPR/Cas allows a low-cost tool of manipulating specific DNA/RNA sequences which is also efficient and adaptable [27]. The acronym CRISPR/Cas derived from “Clustered Regularly Interspaced Short Palindromic Repeat-Associated Protein Cas” and is an adaptive immune system developed by bacteria and archaea to defend themselves against virus attacks [69]. The system is mainly composed of two components: the CRISPR-associated protein 9 (Cas9) endonuclease and variants (e.g., Cas12 and Cas13) [70] responsible for cutting DNA/RNA, and a double-guide RNA (crRNA and tracrRNA) required to deliver the endonuclease to a specific target DNA sequence to be cut. When a virus injects its DNA into bacteria, the CRISPR/Cas system cuts the exogenous DNA/RNA in a double strand break (DSB), inactivating it [71]. It has recently been found in viruses [72] and eukaryotic organisms [73], highlighting how much remains to be discovered about this system.

The possible use of the CRISPR/Cas mechanism as a GE tool became evident when the double-guided Cas9 system was simplified by the designing of a single-guided chimeric RNA (sgRNA). Due to the complementary between sgRNA and the target region, the endonuclease Cas9 can be delivered anywhere in the genome, producing a DSB [27,74]. DSBs are the most deleterious type of DNA/RNA damage, resulting in the loss of large chromosomal regions in many cases [75]. The CRISPR/Cas9 mechanism takes advantage of the natural DNA repair system, consisting mainly of NHEJ (non-homologous end joining) and HDR (homology-directed repair) to implement genome modification. In fact, when the CRISPR/Cas9 system induces a DSB in a genome, the cell activates these repair mechanisms to reunite the ends of the double helix [76]. The NHEJ system, being faster but less precise, can introduce errors such as insertions, deletions, and substitutions. In contrast, HDR uses a homologous sequence, such as the sister chromatid or the homologous chromosome, as a template to repair the damage precisely [77]. NHEJ has been exploited to induce short frameshift insertions–deletions in the sequence, changing the reading frame of mRNA with the formation of premature stop codon (knock-out) and HDR to introduce specific mutation (knock-in). Although the knock-out approach is the most widely used, the CRISPR/Cas system is continuously updated by fusing the Cas proteins with domains encoding effectors for epigenome editing [78], base editing [79], activation/repression genes [80], and prime editing [81]. Recently, Rogo et al. (2024) [27] reviewed these noteworthy topics in more depth.

3.2. CRISPR/Cas for Plant Resilience Against Abiotic Stresses

Plants have evolved diverse mechanisms to cope with abiotic stresses such as salt, drought, and heat. These responses, regulated by a complex interplay of genes, require basic studies to delve into the function of specific genes. These studies are crucial to identifying candidate genes that could be modified for breeding programs, thereby developing crop varieties resistant to abiotic stresses. Biotechnological advances in plant biology based on CRISPR/Cas systems promise a new generation of crop plants [82].

3.2.1. Improvement of Salt Tolerance by CRISPR/Cas-Dependent Genome Editing

Saline soils reduce osmosis in the primary stress phase, when the soil solution is rich in soluble salts and stomatal opening is reduced; as a result, water stress occurs that hinders plant growth [83]. In rice, a large deletion (366 bp) of the Drought and Salt Tolerance (DST) gene, obtained using two different gRNAs, resulted in moderate osmotic stress tolerance and a high level of salt stress tolerance, suggesting that DST is a negative regulator of salt stress tolerance [84]. Another negative regulator of salt tolerance in rice is Decreased grain size1 (OsDGS1), which belongs to a family of RING genes that are important for their role in the ubiquitination process. CRISPR/Cas9 mutant lines for this gene showed improved salt tolerance compared with wild-type (WT) plants at both germination and seedling stages, as showed by increases in plant height, root length, and total fresh weight, as well as total chlorophyll and relative water content under salt stress conditions [85]. Members of the chloride channel (CLC) family located in intracellular organelles are required for anion accumulation, pH adjustment, and salt tolerance. In maize, the voltage-gated chloride channel ZmCLC gene was mutated through the CRISPR/Cas9 system. Three guides were designed to target the three gene exons in different plants. The mutant plants were self-pollinated to obtain homozygous mutant lines. All three Zmclc mutants showed a more significant reduction in root length, root fresh weight, shoot length, and shoot fresh weight than WT under 100 mM NaCl treatment [86]. Plant hybrid proline-rich proteins (HyPRPs) are putative proline-enriched cell wall proteins composed of an N-terminal repetitive proline-rich domain and a conserved C-terminal eight-cysteine-motif domain [87]. In some plant species, the HyPRP genes play various functional roles in responses to biotic and abiotic stresses [88]. In tomato, targeted deletion of the SlHyPRP1 gene resulted in enhanced salinity tolerance during the germinative and vegetative stages under experimental conditions. CRISPR/Cas9-based domain editing can be an effective tool for engineering multi-domain proteins in key food crops to address global climate change, promote sustainable agriculture, and ensure future food security [89].

3.2.2. Improvement of Drought Tolerance by CRISPR/Cas-Dependent Genome Editing

In recent years, drought has become one of the most impactful problems for global agriculture. Plants in this condition experience damage by drought and display stunted growth, withering foliage, and disruption in blossoms and buds throughout their developmental stages. Plants adapt their water balance dynamically under drought conditions to reduce water loss or improve water uptake. Several studies highlight that through the CRISPR/Cas9 system, it is possible to create tolerant plants without unduly compromising productivity. Transcription factors (TFs) from diverse families, for example, DREB, BZIP, WHY, AP2/ERF, HD-Zip, bHLH, AREB/ABF, MYB, NAC, and WDR TFs, play multiple roles in drought stress tolerance and represent a crucial opportunity for the development of drought-tolerant plant varieties [90]. In rice, drought tolerance was enhanced by using CRISPR/Cas9 to knock-out Semi-rolled leaf 1, 2 (SRL1, SRL2), and ENHANCED RESPONSE TO ABA1 (ERA1) genes [91]. SRL1 is allelic to Curled leaf and dwarf 1 (Cld1), which encodes a putative glycosylphosphatidylinositol-anchored protein and regulates leaf rolling on the adaxial side by increasing the number of bulliform cells [92]. ERA1 encodes the β-subunit of farnesyltransferase and regulates ABA signaling and the dehydration response [93]. Moreover, rice grew better in drought conditions when the expression of regulatory genes, such as DERF1, PHOTOPERIOD-SENSITIVE GENIC MALE STERILE 3 (PMS3), MutS-Homolog1 (MSH1), MYB5, and SPP, was down–regulated [94]. Similarly, maize plants in which the ARGOS8 gene, a negative regulator of ethylene response that modulates hormone signal transduction, has been mutated show a slight decrease in yield during normal growth and a significant increase in grain yield under dry conditions. These results were further evaluated and confirmed in the fields even during the dry season [95]. CRISPR/Cas9 has also been used in oilseed crops. For example, in Brassica napus, knock-out of the REPRESSOR OF GIBBERELLIC ACID (BnaA6.RGA) gene results in a notable improvement in drought resistance [96].

3.2.3. Improvement of Heat Tolerance by CRISPR/Cas-Dependent Genome Editing

The crop varieties with higher yields under heat stress have high photosynthetic rates, modified membrane structure, appropriate ROS levels, and water use efficiency (WUE). Characterizing key stress-responsive genes and related physiological mechanisms is always a first step toward the development of stress-tolerant cultivars [97]. For example, the role of the mitogen-activated protein kinase (SlMPAK3) gene was investigated in tomato. Knock-out of this gene highlighted a negative regulation of stress response, as mutant plants were more tolerant to higher temperatures than WT plants [98]. Further, 9-cis-epoxy carotenoid dioxygenase 4 (LsNCED4) knockdown improved lettuce seed germination under heat stress [99]. In addition, maize plants mutated for the ZmTMS5 gene, a member of the thermo-sensitive genic male sterility (TGMS) gene family, showed an increase in male fertility temperature from 24 to 28 °C [100].

Table 1. Genes targeted by CRISPR/Cas system to improve tolerance against salt, drought, and heat stresses.

Stress	Species	Target Genes	References
Salt	Oryza sativa	OsDST	[84]
	Oryza sativa	OsDSG1	[85]
	Zea mays	ZmCLC	[86]
	Solanum lycopersicum	SlHyPRP1	[89]
Drought	Oryza sativa	OsSRL1, OsSRL2, and OsERA1	[91]
	Oryza sativa	OsDERF1, OsPMS3, OsMSH1, OsMYB5, and OsSPP	[94]
	Zea mays	ZmARGOS8	[95]
	Brassica napus	BnaA6.RGA	[96]
Heat	Solanum lycopersicum	SlMPAK3	[98]
	Lactuca sativa	LsNCED4	[99]
	Zea mays	ZmTMS5	[100]

reports a list of target genes mutagenized by genome editing.

3.2.4. CRISPR/Cas Strategy to Increase Ascorbic Acid Content and to Investigate Putative Role of Specific Proteins in the Vitamin C Pathway

To date, some interesting results have been obtained through genome editing programs with the aim of modifying the level of AsA and evaluating the role of specific TFs. For example, in tomato, the expression of the ASCORBATE PEROXIDASE 4 (SlAPX4) gene during fruit ripening was analyzed. Mutation of SlAPX4 using the CRISPR/Cas9 system increased the ascorbate content in ripened tomato fruits, while the AsA content in leaves was not significantly changed. However, phenotype analysis revealed that the SlAPX4 mutation did not adversely affect the growth of tomato plants [101]. In the same crop, edited CRISPR/Cas9 lines for the SlSGR1 gene, which encodes a STAY-GREEN protein that plays a critical role in regulating chlorophyll degradation in leaves and fruits, showed a higher content of AsA equivalent (AAE) compared to the WT [102]. Therefore, genomic editing in tomato has sometimes made it possible to ascertain that some proteins not directly involved in biosynthesis may still have specific roles at the endogenous level. In line with this consideration, the analysis of the cpk28 mutant generated via CRISPR/Cas9 clarified that calcium-dependent protein kinases (CPK28) have a direct role in APX2 protein phosphorylation, and these edited genotypes are more sensitive to heat stress [103].

In kiwifruit, the response to cold stress is influenced by the TF AcePosF21, which interacts with a MYB factor (AceMYB102); this molecular step is followed by direct transcriptional control on the AceGGP3 gene with a modification of AsA levels. Plants edited in the AcePosF21 gene respond to stress with low AsA levels and increased ROS [104]. Undoubtedly, the GGP gene plays a key role in AsA biosynthesis, and in fact, ggp3 mutant of Actinidia eriantha obtained by genome editing are characterized by low AsA levels compared with WT plants [105]; in contrast, it was established in A. thaliana that the knock-out of the uORF of GGP by CRISPR/Cas9 increased the concentration of ascorbate content [32]. Following uORF editing of GGP1-2 in lettuce, Zhang et al. (2018) [106] observed a 1.4- and 2.6-fold increase in oxidative stress tolerance and ascorbate content, respectively. In tomato, editing the uORF of GGP2 resulted in a 1-4-fold increase in ascorbate in leaves [107]. More recently, Deslous et al. (2021) [108] showed that SluORF-GGP1-edited tomato plants, although having increased AsA content than WT plants, showed negative effects related to a dwarf and bushy phenotype and also pollen sterility [108].

Interestingly, using targeted deletions in the PAS/LOV gene sequence by genome editing in tomato has provided relevant data to define the molecular mechanism underlying blue light-dependent in AsA synthesis [109].

In A. thaliana, genome editing techniques were used to assess the role of the Myo-inositol pathway in AsA biosynthesis. In particular, Ivanov Kavkova et al. (2019) [110] demonstrated that mutants with impaired protein glucurokinase1 produced only glucuronic acid, and the AsA content in leaves was not altered compared to the control. The collected data support the conclusion that the Myo-inositol pathway is not important in controlling AsA levels [110].

Table 2. Edited genes through CRISPR/Cas9 involved in AsA level regulation.

Species	Target Genes	Effect on AsA	References
Solanum lycopersicum	SlAPX4	Increased	[101]
	SlSGR1	Increased	[102]
	SlCPK28	Unaltered	[103]
	SluORF-GGP1	Increased	[108]
	SluORF-GGP2	Increased	[107]
	PAS/LOV	Increased	[109]
Actinidia eriantha	AcePosF21	Decreased	[104]
	AceGGP3	Decreased	[105]
Arabidopsis thaliana	AtuORF-GGP1	Increased	[32]
	AtGLCAK	Unaltered	[110]
Lactuca sativa	LsORF-GGP1/2	Increased	[106]

contains a list of representative genes mutagenized by CRISPR/Cas9.

4. Genes Coding for Transcription Factors as Putative Targets for Genome Editing to Modify AsA Content Under Abiotic Stress Conditions

4.1. Transcription Factors

In recent years, many genes encoding for TFs involved in environmental stress responses have been discovered, and some of them also regulate genes involved in AsA biosynthesis and metabolism [32,111,112]. Therefore, we have reported below some of the most interesting genes studied for their influence on AsA content. These genes could be potential targets for future GE programs across various species using CRISPR/Cas technology. In general, TFs recognize and bind specific cis-acting elements in promoters of target genes to control their expression. Certain TFs enhance the AsA pool size by promoting the transcription of biosynthetic genes, while others could negatively affect AsA concentrations.

4.1.1. Salt Stress

In Arabidopsis, the over–expression of the Ethylene response factor (ERF)98, a member of the AP2/ERF TF superfamily with a conserved DNA-binding domain, led to increased AsA levels and improved plant tolerance to salt stress [113].

The Dof (DNA-binding with one finger) proteins are TFs characterized by a highly conserved DNA-binding region known as the Dof domain [114]. In tomato, SlDof22, a member of the Dof family, plays a negative role in regulating AsA accumulation [115]. In transgenic lines, RNA interference of SlDof22 enhanced AsA content in both fruits and leaves and influenced the expression of genes linked to the D-mannose/L-galactose and recycling AsA pathways. Furthermore, SALT OVERLAY 1 (SOS1), a gene crucial for maintaining Na⁺ and K⁺ homeostasis [116], was significantly down–regulated in SlDof22 RNAi plants, causing a decrease in salt stress tolerance [115]. According to Li et al. (2018) [48], salt stress in Arabidopsis induces the zinc-finger protein SIZF3, which interferes with the interaction between CSN5B and VTC1. This mechanism promotes AsA accumulation and improves salt tolerance. On the other hand, the Lipid transfer protein-1 (LTP1) gene plays a role in regulating genes linked to the ascorbate–glutathione cycle and stress response, including APX, catalase (CAT), Superoxide dismutase (SOD), and HSP. Its over–expression increased AsA levels and improved tolerance to salt, heat, and water stress [117]. In maize, the ZmbHLH55 plays a positive function in salt tolerance through the modulation of AsA biosynthesis. In fact, gene expression analysis shows that ZmbHLH55 activates the expression of ZmPGI2, ZmGME1, and ZmGLDH. Surprisingly, it negatively regulates the expression levels of ZmGMP1 and ZmGGP genes [118]. The ectopic expression of MrWRKY30 from Muscadinia rotundifolia in Arabidopsis enhanced its resistance to downy mildew pathogen Peronospora parasitica [119]. In transgenic plants, pathogenesis-related (PR) genes, such as AtPR1, AtPR4, AtPR5, and AtPDF1.2, were up-regulated after P. parasitica inoculation. In particular, transgenic seedlings exhibited lower salt tolerance, and antioxidant enzyme genes AtAPX, AtCAT, and AtGST were suppressed in transgenic plants, which might result to ROS-mediated sensitivity to salt stress [119]. ABA INSENSITIVE 4 (ABI4) is a key TF involved in the ABA signaling pathway. In Arabidopsis, ABI4 negatively regulates salt tolerance. In particular, ABI4 binds directly to the VTC2 promoter, inhibiting VTC2 transcript expression and reducing AsA biosynthesis [120]. ABI4 is also required for the ascorbate-dependent control of growth, which is a process that involves enhancing SA signaling and inhibiting JA signaling pathways. Low redox buffering capacity reinforces both SA and JA interactions through the mediation of ABA and ABI4 to fine-tune plant growth and defense in relation to metabolic cues and environmental challenges [121]. Notably, ABI4 directly combines key ROS production and scavenging genes to modulate ROS metabolism during seed germination under salinity stress [122]. Moreover, Kakan et al. (2021) [123] found that AsA partially recovers the salt stress sensitivity of plants over–expressing ABI4, which exhibits lower AsA content and higher ROS accumulation. In addition, ABI4 is transcriptionally repressed by ETHYLENE-INSENSITIVE3 (EIN3) in ethylene-regulated AsA biosynthesis [124]. Therefore, ethylene and ABA antagonistically control AsA biosynthesis and ROS accumulation in response to environmental stimuli through the EIN3-ABI4-VTC2 transcriptional cascade [124].

4.1.2. Drought Stress

The WAX1 gene, which encodes a MYB TF of sea cress (Eutrema salsugineum), was associated with the induction of genes related to AsA biosynthesis, such as GGP1, L-GalDH, and Myo-inositol oxygenase (MIOX)4 [125]. The expression of WAX1 under the constitutive promoter increased AsA levels but also caused alterations in plant growth and development. However, when expressed under a stress-inducible promoter (RD29A), WAX1 increased AsA levels under drought stress conditions, significantly improving tolerance to such conditions [125]. Wang et al. (2017) [126] identified drought-responsive genes through Illumina sequencing in Pugionium cornutum (L.) Gaertn., which is a xerophytic plant. The differentially expressed genes (DEGs) were primarily associated with photosynthesis, nitrogen metabolism, and plant hormone signal transduction pathways with a notable focus on ascorbate metabolism. This may contribute as an alternative mechanism pathway to increase the antioxidant capacity of P. cornutum in response to drought stress. The authors showed that the transcription of most genes involved in AsA metabolism was affected by drought conditions [126]. Interestingly, 93 genes encoding drought-inducible TFs were identified in DEGs, including DREB, AP2/EREBP, B-2a, ERF2, MYB, and the Zinc finger family. These genes represent promising candidates for improving drought stress tolerance in other species through genetic engineering. The double transgenic tomato developed by introducing heterologous genes, AtDREB1A and BcZAT12, showed significant drought tolerance, reducing oxidative stress and increasing yield [127]. In particular, transgenic plants showed increased activity of antioxidant enzymes, like CAT, SOD, GR, APX, DHAR, MDHAR, and guaiacol peroxidase (POD). Additionally, they accumulated a greater amount of non-enzymatic antioxidants like AsA and glutathione compared to single transgenic or WT plants [127].

4.1.3. Light Stress

Recently, in cabbage (Brassica rapa ssp. chinensis), it has been shown that the ethylene-responsive BcERF070 TF (homologous to AtERF98 in Arabidopsis) binds the dehydration responsive elements (DREs) of seven target gene promoters with a consequent modification of the AsA content [128]. In particular, ERF98 increased the transcript levels of the VTC1, VTC2, GDH, and GLDH genes. In addition, rapid light response and ROS signaling were analyzed using the glutathione- and ascorbate-deficient pad2 and vtc1 mutants [129]. The transcriptional response of two TFs, ERF6 and ERF105, which respond rapidly to light, was deregulated in the pad2 mutant but not in the vtc1 background. Further analyses, combining low-to-high light transfer with methyl viologen pretreatment, highlighted the importance of glutathione in strongly modulating the retrograde signal from the chloroplast to nuclear gene expression [129].

4.1.4. Methyl Viologen Stress

Another TF, HZ24, belonging to the HD-Zip I family, was identified in tomato by a yeast hybrid assay, targeting the promoter region of the GMP3 gene [130]. HZ24 positively regulates several genes of the L-galactose pathway, including PMM, GMP4, GME1, GME2, GGP, GPP1, GPP2, and L-GalDH. The over–expression of HZ24 in tomato increased AsA levels and improved resistance to methyl viologen-induced oxidative stress [130]. Another important TF is NL33, a member of the subfamily of nucleotide-binding site (NBS) and leucine-rich repeat (LRR) proteins (NL), which negatively regulates the transcription of genes related to AsA biosynthesis and recycling. Knocking down NL33 expression by RNA interference (RNAi) in tomato increased AsA levels and improved resistance to methyl viologen-induced oxidative stress [131].

4.1.5. Heavy Metal Stress

In Populus yunnanensis, the over–expression of PyWRKY75, achieved by genetic transformation technology, increased AsA and other metabolites such as GSH and phenolic compounds, enhancing poplar plants to cadmium stress to remediate Cd-contaminated soils [132].

4.1.6. Cold Stress

In birch leaf pear, TF MYB5 can interact with the promoter region of the DHAR2 gene, which is involved in the ascorbate recycling cycle [133]. MYB5 expression is induced by various types of stress, such as drought, salinity, and cold. It positively regulates genes associated with AsA recycling, the ascorbate–glutathione cycle, and genes activated during stress, including MDHAR, DHAR2, APX, C-repeat/DRE binding factor 1 (CBF1), CBF2, and CBF3. The over–expression of MYB5 in tobacco increased AsA levels, improving cold tolerance [133]. The tomato SlICE1 gene encodes a TF that belongs to the basic helix–loop–helix DNA-binding protein (bHLH) superfamily. The over–expression of SlICE1 induced AsA accumulation by improving their cold tolerance. However, activated target genes that might be involved in AsA metabolism have not yet been identified [134,135].

4.1.7. Ozone Stress

AsA Mannose Pathway Regulator 1 (AMR1), an F-box protein, acts as a negative regulator of AsA synthesis in Arabidopsis thaliana. Loss of function in AMR1 leads to a significant increase in AsA levels, suggesting that it inhibits critical enzymes in the biosynthetic pathway, including GMP, GME, GGP1, L-galactose-1-phosphate phosphatase (GPP), L-GalDH, and L-GalLDH [111]. When AMR1 loses its function due to a mutation, AsA levels increase, improving the plant’s ability to resist ozone stress.

4.1.8. Heat Stress

Heat stress TF A2s (HsfA2s) are key regulators in plant response to high temperatures. The ectopic expression of CtHsfA2b from Cynodon transvaalensis improved heat tolerance in Arabidopsis and restored heat-sensitive defects of the Arabidopsis hsfa2 mutant [136]. Notably, CtHsfA2b was shown to bind to the heat shock element (HSE) on the promoter of AtAPX2, thereby boosting the transcriptional activity of AtAPX2 [136].

Interesting examples of TFs that could be subject to mutagenesis through genome editing are summarized in

Table 3. Examples of TFs that could be chosen as putative molecular targets for genome editing programs based on their involvement in plant response to abiotic stresses in association with AsA level modification.

Stress	Species	Target TFs	References
Salt	Arabidopsis thaliana	AtERF98	[113]
		MrWRKY30 (from Muscadinia rotundifolia)	[119]
		AtABI4	[120]
	Solanum lycopersicum	SlDof22	[115]
	Solanum tuberosum	StLTP1	[117]
	Zea mays	ZmbHLH55	[118]
Drought	Arabidopsis thaliana	EsWAX1 (from Eutrema salsugineum)	[125]
	Pugionium cornutum	PcDREB, PcAP2/EREBP, PcB-2a, PcERF2, PcMYB, and PcZinc finger	[126]
	Solanum lycopersicum	AtDREB1A and BcZAT12 (from Arabidopsis thaliana and Brassica carinata, respectively)	[127]
Light	Arabidopsis thaliana	AtERF98	[129]
Methyl Viologen	Solanum lycopersicum	SlHZ24	[130]
		SlNL33	[131]
Heavy Metal	Populus yunnanensis	PyWRKY75	[132]
Cold	Nicotiana tabacum	NtMYB5	[133]
	Solanum lycopersicum	SlICE1	[134,135]
Ozone	Arabidopsis thaliana	AtAMR1	[111]
Heat	Arabidopsis thaliana	CtHsfA2b (from Cynodon transvaalensis)	[136]

.

4.2. Further Transcription Factors Involved in the Ascorbic Acid Regulation and Putative Targets for Genome Editing Programs

In addition to the above examples, further evidence has been obtained for other TFs that may alter AsA levels when associated genes are deregulated in transgenic plants. These additional TFs, especially those with a role under abiotic stress conditions, could represent further potential targets in genome editing programs. The following are some significant examples.

Recently, the CCAAT-binding factor NFYA10 has been characterized as a negative regulator of AsA biosynthesis in tomato. Transgenic lines over–expressing NFTA10, regulating the expression of genes SlGME1 and SlGGP1, encoding for the GDP-Man-3′,5′-epimerase and GDP-L-galactose phosphorylase enzymes, respectively, showed decreased levels of gene expression and, consequently, decreased AsA concentration in leaves and fruits, which was accompanied by enhanced sensitivity to oxidative stress [137]. Ye et al. (2019) [138] conducted a genome-wide association study on 302 accessions of tomato in two different environments, leading to the identification of an ascorbate quantitative trait locus, TFA9 (TOMATO FRUIT ASCORBATE ON CHROMOSOME 9). This locus, which co-localizes with the TF SlbHLH59, is crucial in promoting high ascorbate accumulation by directly binding to the promoter of structural genes implicated in the D-mannose/L-galactose pathway. In tomato, BRI1-EMSUPPRESSOR1 (BES1), a gene encoding a brassinosteroid (BR) response TF Brassinazole resistant 1 (BZR1), has been identified as a promoter of AsA accumulation [139]. This TF is a master regulator of multiple gene expressions and thus has pleiotropic functions. When plants are affected by biotic and abiotic agents, the campesterol precursor is converted to BR, which activates BZR1.

In Ziziphus jujuba, several genes (ZjERF17, ZjbZIP9, and ZjGBF4) have been identified that encode TFs controlling the AsA level in the fruit. The high correlation between the expression levels of these TFs and some AsA biosynthesis-related genes indicates their key role in regulating AsA biosynthesis [140]. However, we do not know whether certain types of oxidative stress are reduced in these plants. In rice, microRNA528 (OsmiR528) increased the cell viability, growth rate, antioxidant content, and activity of both APOX and SOD. It also reduced ion leakage and the accumulation of thiobarbituric acid reactive substances (TBARSs) under low-temperature stress conditions in Arabidopsis, Pinus elliottii, and rice [141]. Specifically, OsmiR528 decreased the expression of TF OsMYB30 by targeting a gene containing an F-box domain protein, which functions as a positive regulator of OsMYB30. In rice plants transgenic for OsmiR528, reducing the expression of OsMYB30 resulted in increased expression of the β-amylase (BMY) genes OsBMY2, OsBMY6, and OsBMY10. Transcript levels of OsBMY2, OsBMY6, and OsBMY10 increased with OsMYB30 deletion. However, the over–expression of OsMYB30 in OsmiR528 transgenic cell lines decreased the expression of these BMY genes. This suggested that OsmiR528 may increase low-temperature tolerance by modulating the expression of TF related to stress response [141]. More recently, in Rosa roxburghii, through transcriptomic and metabolomics analyses, two significant gene networks/modules and 168 TFs putatively involved in AsA synthesis were identified [142]. The promoter analysis of two genes involved in AsA synthesis, RrGGP and RrGalUR, revealed a retroviral long terminal repeat (LTR) insert in the RrGalUR promoter. Additionally, Su et al. (2024) [142] demonstrated that the TFs RrHY5H and RrZIP9 bind to the promoter of RrGGP to promote its expression. In particular, RrZIP6 and RrWRKY4 bind to the LTR region in the RrGalUR promoter to promote its expression. Other factors, including nucleotide sugar pyrophosphorylase-like proteins (KONJAC1 and 2) [143], and a calmodulin-like protein (CML10) [144], have been identified as stimulators of the enzyme activity of AsA-related genes, revealing novel mechanisms of how the AsA pool is regulated. In particular, KONJAC1 and KONJAC2 can stimulate GMP activity, while CML10 can stimulate PMM activity, resulting in increased cellular levels of mannose-1-phosphate and, consequently, increased reactions in the AsA biosynthetic pathway. The genetic under–expression of KONJACs and CML10 significantly reduced AsA concentrations [145]. When plants are subjected to oxidative stress, CML10 gene expression is induced in parallel with the increase in intracellular Ca²⁺. Subsequently, the synthesized CML10 protein binds calcium ions, undergoes a conformational change and complexes with the PMM enzyme. In this way, AsA biosynthesis increases, enabling plants to overcome oxidative stress [144]. AIR12 (Auxin Induced in Root culture) encodes for a mono-heme cytochrome b [146]. The over–expression of MfAIR12 from Medicago falcata resulted in the accumulation of H₂O₂ in apoplast and improved cold tolerance. This enhanced tolerance was blocked by H₂O₂ scavengers, indicating that the increased cold tolerance depended on the accumulated H₂O₂. It has been hypothesized that AIR12 confers cold tolerance due to H₂O₂ alteration in the apoplast, signaling CBF regulation in the cold response pathway and ascorbate homeostasis [146]. In Actinidia chinensis, Chen et al. (2021) [147] identified a candidate TF named AcERF91, which potentially regulates the expression of a GDP-galactose phosphorylase gene (AcGGP3). Over–expressed lines through recombinant plasmid 35S-AcERF91 and silenced lines through pTRV2-AcERF91 were obtained, highlighting a higher and lower AsA content than the controller. In addition, given the high gene expression correlation between AcERF91 and AcGME1, AcGME2, AcGMP2, AcGMP4, AcGGP1, AcGLDH, and AcGDH, along with the presence of ERF binding sites in the promoter regions, AcERF91 may also regulate their expression [147].

4.3. Gene Involved in the Recycling Pathway and Putative Targets in Mutagenesis

Other attempts have also been made to understand the role and effects of genes encoding enzymes involved in the AsA turnover pathway. For example, over–expression of the wheat DHAR gene increased foliar and kernel AsA levels 2- to 4-fold. In transgenic tobacco and maize plants, both the AsA amount and the ascorbate redox state increased significantly [148]. Yin et al. (2009) [149] showed that the over–expression of Arabidopsis DHAR induced better root growth than WT plants under aluminum stress in tobacco plants. In Triticum aestivum, 15 TaMDHAR genes were identified [150]. The presence of multiple cis-regulatory elements in the promoter region and their interaction with numerous TFs indicate their involvement in specific functions of growth and development and responses to light, phytohormones, and stress. In particular, their differential gene expression and increased enzyme activity during drought, heat and salt treatments highlighted their key role in the response to abiotic stresses. The interaction of MDHARs with various antioxidant and biochemical enzymes related to the ascorbate–glutathione cycle also suggested their synchronized functioning. In addition, interaction with auxin indicated the likelihood of cross-talk between antioxidants and hormone signaling. The interactions of miR168a, miR169, and miR172 with various TaMDHARs genes supported the hypothesis of their association with developmental processes and stress responses [150]. Eltayeb et al. (2007) [151] showed that tobacco plants over–expressing the cytosolic MDHAR gene exhibited 2.2-fold higher levels of reduced AsA compared to non-transformed control plants. In addition, the transgenic plants also showed enhanced stress tolerance to ozone, salt, and polyethylene glycol stresses. However, in tomato plants, the over–expression of the AtMDHAR3 gene induces a reduction in AsA levels in mature green fruits by 0.7-fold [152]. Ascorbate levels in over–expressed MDHAR and silenced MDHAR lines have been evaluated in tomato leaves [153]. All the over–expressed lines showed a significant decrease in reduced ascorbate, whereas all the silenced lines exhibited a significant rise. The same decrease in AsA levels response has been shown when the MDHAR3 of Actinidia eriantha was over–expressed in tomato transgenic lines [154]. The unexpected effect of the MDHAR gene with a negative impact on ascorbate levels cannot be explained by changes in the expression of the “Smirnoff–Wheeler” pathway genes or by the activity of enzymes involved in the degradation or recycling of ascorbate, suggesting an unrelated mechanism that regulates ascorbate levels [155]. Furthermore, the suppression of the AO gene in tomato showed the highest AsA levels and increased salinity tolerance [156]. Thylakoid membrane-bound ascorbate peroxidase (tAPX) plays a role in regulating H₂O₂ levels. In Arabidopsis, microarray analysis showed that tAPX silencing affected the expression of a wide range of genes, some involved in cold and pathogen response [157]. In response to tAPX silencing, CBF1 transcript levels were suppressed, resulting in a high cold sensitivity of tAPX-silenced plants. In addition, the silencing of tAPX increased SA levels and response to SA. Notably, genes responsive to tAPX silencing were up– or down–regulated by bright light, and tAPX silencing had a negative effect on the expression of ROS-responsive genes under high light (HL) conditions, suggesting a synergistic and antagonistic role of chloroplastic-H₂O₂ in the response to HL [157].

5. Epigenetic Regulation of AsA Content

The recruitment of epigenome editing effector domains by CRISPR/Cas systems allows the site-specific control of modifications to DNA, histones, and chromatin architecture [158,159]. The cytosine methylation of DNA is one of the main epigenetic factors affecting gene activities. It is a chemical signal related to genome stability, gene imprinting, plant development, and environmental response without changing the DNA sequence. In plants, DNA methylation also contributes to stress response by affecting gene expression and genome stability [159,160,161]. Cytosine methylation occurs at the 5′ position in symmetric (CG and CHG) and asymmetric (CHH) sequence contexts (H represents A, T, or C) [162].

Notably, AsA is a cofactor for the ten-eleven translocation of dioxygenases responsible for DNA methylation. Also, AsA is required for histone demethylation, since it operates as a cofactor of Jumonji C-domain-containing histone demethylases [163]. AsA may be a mediator between the environment and the genome. In the seminal work by Xue and colleagues (2019) [164], Chlamydomonas reinhardtii showed that AsA provides glycerol to C4–C6 for a unique demethylation modification, C5-glyceryl-methylcytosine (5gmC) catalyzed by the 5mC modifying enzyme (CMD1). Compared with WT, C. reinhardtii shows a 60% decrease in 5gmC levels and a doubling of 5mC with CMD1 knock-out. Meanwhile, 5gmC levels decrease by 80% and 5mC doubles in the vtc2 mutant of C. reinhardtii with AsA deficiency [164].

Wang et al. (2022) [165] showed that 5-azacytidine, a methyltransferase inhibitor, promotes tomato ripening. At the same time, they found a significant increase in AsA content in treated leaves and fruits. The results suggested a new unexpected function of DNA demethylation in fruit ripening. Even more interesting was the observation that a tomato AsA biosynthetic gene, SlGalUR5, which codes for D-galacturonic acid reductase, showed reduced DNA methylation levels and higher transcription levels in Slmet1 mutant (knock-out for METHYLTRANSFERASE 1 gene), while it showed a converse pattern in Sldml2 mutant (knock-out for DEMETHYLASE 2 gene) [165]. Collectively, these findings suggest a promising approach to enhancing AsA levels in crops.

In higher plants, AsA has a considerable influence on the bifunctional demethylase/glycosylases Repressor of silencing 1 (ROS1) that negatively regulates the process known as the RNA-directed DNA methylation (RdDM) pathway [166,167]. The role of ROS1 and intracellular 5mC levels in stressed higher plants, along with the intricate relationship between the epigenome and AsA, will need to be further explored [168]. In addition, other epigenetic modifications, such as histone methylation and the post-translational modification of histones, including acetylation, deacetylation, ubiquitination, and non-coding RNAs, play essential roles in gene expression regulation [169]. These modifications can affect plant traits and responses to environmental stresses, enabling plants to maintain productivity despite variable climates or disease conditions. The relationships between these additional epigenetic mechanisms and AsA metabolism remain unclear. However, further research is expected to delve deeper into the connections between the epigenome, AsA metabolism, and plant adaptation to environmental challenges.

6. Ascorbic Acid in Plant–Biotic Interaction

The role of AsA in counteracting biotic stresses due to the attack of plant pathogens and pests has been investigated in some species, and interesting reviews about the topic can be suggested [57,170,171]. Studies on plant response in biotic interactions have been performed in mutants deficient in AsA biosynthesis and genotypes with wild-type vitamin C content both in the presence and absence of exogenous treatments. However, to date, the exact role that AsA plays toward biotic stresses appears to be poorly known. Nonetheless, it has been shown that the localization of ascorbate changes in the plant cell in response to biotic stress, and a complex biochemical network has been described in which the action of AsA is strongly correlated with other secondary metabolites and several phytohormones [57,171]. Enhanced ROS formation is a common response of plant cells to pathogen infection. Several studies have documented how AsA, interacting with other components of the redox system, activates several defense responses that reduce plant pathogen growth and promote plant growth and development under biotic stress conditions [57]. On the other hand, AsA can play specific roles in the host and aggressor contributing to the complication of biochemical interactions among the organisms involved.

7. Understanding Ascorbate Metabolism to Improve Plant Tolerance to Stresses

The dramatic increase in average temperature over the last century had an enormous influence on the salinization of arable land through increased evaporation rates, instability of soil water content due to floods or drought, and fluctuation in precipitation paradigms with severe impacts on global food security [172]. Relying on plants’ natural adaptation to stressors to sustain high-quality production would undoubtedly take an impractically long time. In addition, incorporating key resistance traits from wild species into cultivated crops through conventional breeding approaches also requires a lot of work and time [158]. Consequently, new breeding technologies (NBTs) should be considered powerful tools to revolutionize agriculture. Having so many candidate genes linked to AsA metabolism, the CRISPR/Cas technique can be a valuable tool to increase the concentration of this “anti-ROS” molecule in plants.

It is important to recognize that several critical challenges remain in achieving the goal of modulating AsA levels in crop plants [173,174,175,176,177,178,179]. In fact, a significant increase in AsA level is often difficult to achieve, as demonstrated in multiple studies of transgenic plants [112,180]. Moreover, as some author pointed out, it is not necessarily the case that an increased in AsA level is a positive trait for a specific crop for productivity purposes [108,181]. AsA is one of the most abundant antioxidant molecules in plants and is an important defense against ROS by protecting plant cells from many environmental factors that induce oxidative stress. However, it should be pointed out that stress tolerance is a complex evolutionary adaptation of which AsA is one of the players within a complex regulatory network (Figure 4).

AsA has vital roles not only in stress responses but also regulates multiple aspects of plant development, not to mention that there are a complex series of interactions between AsA and major plant hormones [169,182,183]. Thus, increasing the nutritional quality of a crop and/or enhancing its performance under biotic and abiotic stress conditions without compromising some important aspects of the phenotype may still be a daunting challenge. Based on these considerations, advancements in genomic editing technologies are expected to become increasingly efficient and versatile [184].

8. From Genomic Diversity to Precision Editing

The genetic diversity in many crops is often underexplored, and as climate variability intensifies, there is a deep need to quickly adapt plants to extreme conditions [185]. Understanding the complex interactions between plant genomes and environmental stressors is one of the biggest challenges in developing resilient crop varieties. In the last few years, approaches to predictive genomics and genetic vulnerability analyses have been applied to highlight key genetic factors contributing to balancing stress conditions and plant survival. These approaches include Genome-Wide Association Studies (GWASs), RNA-Seq analysis, QTL Mapping, Whole-Genome Sequencing (WGS), Machine Learning, etc. [186]. For example, the integration of genetic variations, derived from re-sequencing hundreds of individuals or population groups, with environmental modeling schemes are used to predict a highly spatiotemporal shift in this species in response to future climate change. Furthermore, the most tolerant populations provide candidate genes and variants that may be useful for breeding programs [187,188,189].

Unlike genome-wide approaches focused on locus variability, direct investigations in ascorbic acid regulation may play a critical role in plant adaptation against climate change. However, while the emergence of new technologies such as CRISPR/Cas9 has revolutionized plant genome editing, several challenges remain. In many species, the major bottleneck is the lack of efficient transformation protocols. It was estimated that fewer than 0.1% of 370,000 higher plants could be manipulated [190]. Therefore, although the CRISPR/Cas9 technique was born in 2012, it is still being updated and refined to increase the range of plants that can be genetically manipulated.

9. Conclusions

One of the most relevant effects of anthropogenic climate change is the high levels of atmospheric CO₂ that may allow plants to benefit from the carbon fertilization effect. However, we must consider that climate change affects factors critical to plant growth, such as nutrient deficiencies, temperature, atmospheric pollutants, and water. For example, the high amount of CO₂ in the atmosphere will allow plants to produce more carbohydrates but simultaneously result in nitrogen dilution in the leaves. If nitrogen is limited, the plant will likely fail to use the extra CO₂ in the atmosphere, and the increase in productivity may be short-lived. Increasing temperature has negative effects on nitrogen fixation. As temperatures rise above 25 °C, the rate of nitrogen fixation by bacteria would decrease, resulting in reduced plant productivity. Higher temperatures can make enzymes critical for photosynthesis less efficient. In particular, as temperatures rise, the ability of ribulose bisphosphate carboxylase/oxygenase (rubisco) to fix CO₂ becomes less efficient, and the enzyme begins to fix oxygen, reducing the efficiency of photosynthesis and altering the electron transport chain. Plant response to climate change suggests that abiotic and biotic stressors will be exacerbated, increasing ROS production and oxidative stress and reducing plant productive capacity. The role of L-ascorbic acid and the ascorbate recycling system enzymes in the response of plant cells to environmental changes is a fundamental part of a highly complex system involving the activation of TFs, genes directly involved in stress response, phytohormones, osmolytes, and epigenetic cell memory mechanisms, which should make plants able to tolerate stress conditions.

In conclusion, traditional agricultural methods fused with advanced genetic technologies present resilient prospects to accelerate plant evolution in response to abiotic stresses. It is crucial to develop climate-resilient crops capable of outperforming traditional crops under abiotic stress conditions, including drought, the high salt concentration of soils, heat, cold, and heavy metals. These crops will allow agricultural activities to be managed sustainably, preserve and restore critical habitats, contribute to better water use, and improve soil health.