Drought is a prominent abiotic stress factor that poses a significant global threat to food security by adversely affecting plant growth and productivity (Seleiman et al., 2021). Its primary association with osmotic stress disrupts cell division and proliferation, hinders normal growth and development, and triggers the overproduction of reactive oxygen species (ROS), collectively leading to diminished yield both quantitatively and qualitatively (Bashir et al., 2021). In recent decades, numerous research studies have proposed that the induction of anthocyanins under drought conditions could serve as a mechanism for osmoregulation and the scavenging of excess ROS (Kaku et al., 1992; Chalker-Scott, 2002). Another formidable abiotic stressor, salinity, imposes simultaneous osmotic and ionic stresses on plants. Perturbed Na⁺ and Cl⁻ homeostasis restricts water and mineral uptake, damages cellular membranes, and diminishes photosynthetic efficiency, consequently reducing crop yield (Isayenkov and Maathuis, 2019). In a manner analogous to their role in responding to drought stress, anthocyanins induced by salt stress have been suggested to alleviate ionic and osmotic damage while scavenging ROS (Eryılmaz, 2006). However, the elucidation of the precise molecular mechanisms underlying the beneficial effects of anthocyanins on plant drought and salt tolerance/resistance has only become possible with the recent application of advanced omics technologies and gene editing tools. These innovative approaches have enabled a more in-depth exploration of the intricate pathways through which anthocyanins contribute to enhancing plant resilience against drought and salt stresses.

Anthocyanins are water-soluble pigments that display hues of blue, purple, or red. They are primarily located in fruits, tubers, and flowers and are produced through the flavonoid metabolic pathway (Khoo et al., 2017). Anthocyanins typically serve multifaceted ecological roles, including the attraction of pollinators and facilitation of seed dispersal. Additionally, they function in photoprotection by shielding against intense light and absorbing ultraviolet radiation. Furthermore, anthocyanins exhibit antioxidative properties by scavenging ROS, contributing to cellular redox homeostasis. Moreover, these pigments play a role in maintaining osmotic balance within plant tissues (Dabravolski and Isayenkov, 2023). Anthocyanins undergo synthesis in the cytosol, initiated by the phenylpropanoid pathway from phenylalanine, forming naringenin chalcone. Enzymatic steps involve early biosynthesis genes such as; Chalcone synthase (CHS), Chalcone isomerase (CHI), Flavanone 3-hydroxylase (F3H), Flavonol synthase (FLS) and late biosynthesis genes Dihydroflavonol 4-reductase (DFR), UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT), Anthocyanidin synthase (ANS), Glutathione S-transferase (GST) for modification (Dabravolski and Isayenkov, 2023). DFR plays a crucial role in converting precursor molecules into leucoanthocyanidins, further modified by ANS through glycosylation, acylation, and methylation (Saigo et al., 2020). Transport to vacuoles involves GST and the MATE (Multidrug and Toxic Compound Extrusion) system. Despite gaps in late modification pathways, transcriptional regulation is driven by Myeloblastosis Protein (MYB), Basic Helix-Loop-Helix (bHLH), and Tryptophan-Aspartic acid 40 (WD40) transcription factors, orchestrating the expression of structural genes and downstream regulators (Cui et al., 2021; Khusnutdinov et al., 2021).

Anthocyanins, natural compounds, are produced in response to environmental stresses like low temperatures, salt, drought, nutritional deficiency, pathogen attacks, and heavy metals. They contribute diverse colors (purple, red, blue) to various plant tissues and organs (Kaur et al., 2023). A study on Brassica napus (L.) investigated the functional role of anthocyanins in drought defense, focusing on the purple-stem genotype (Chen et al., 2022). The purple-stem genotype exhibited superior antioxidant capability and drought stress tolerance compared to the green-stem genotype. This was evident through heightened expression and activity of antioxidant enzymes, elevated levels of proline and soluble sugar in the purple-stem genotype. These biochemical features enable more effective mitigation of drought-induced ROS and malondialdehyde (MDA) accumulation, demonstrating the protective role of anthocyanins in drought stress response. Additionally, a study on millet (Panicum miliaceum L.) investigated the transcriptomic and metabolomic responses to drought stress in the drought-resistant and drought-sensitive varieties (Cao et al., 2022). After 6 hours of drought stress, 97 structural genes associated with anthocyanin biosynthesis were identified. Notably, both varieties exhibited up-regulation of 25 GST, 94 CL, 8 F3H, 4 PAL, 2 CHS, 2 CHI, 1 DFR, and 1 OMT genes. Additionally, the drought-resistant variety specifically up-regulated 8 GST, 3 F3H, 2 OMT, and 14 CL genes. These findings suggest that while both varieties activated anthocyanin biosynthesis metabolically under drought stress, the specific set of up-regulated anthocyanin-metabolism-related genes in drought-resistant plays a crucial role in its drought-resistant phenotype (Cao et al., 2022). Another study shows that, anthocyanin-rich tomato exhibited superior salinity and drought tolerance compared to lower anthocyanin content genotypes, displaying effective regulation of Na⁺ (sodium ion), K⁺(potassium ion), and Ca²⁺ (calcium) levels, higher antioxidant enzyme activity (Superoxide Dismutase (SOD), Peroxidase (POD), and Catalase (CAT)), reduced MDA and ROS production, and less impact on seed germination and root elongation under stress conditions (Napar et al., 2022). Through transcriptomic and metabolomic analysis, it was observed that drought stress induces an increase in anthocyanin content in A. thaliana and heightened anthocyanin accumulation was found to mitigate ROS levels in response to drought stress (Nakabayashi et al., 2014). Studies have shown that, elevated anthocyanin levels in transgenic plants have consistently demonstrated heightened resistance to drought, establishing a positive correlation between anthocyanin accumulation and drought tolerance in Arabidopsis, tobacco, and tomato. Overexpression of the snapdragon bHLH transcription factor Delila (Del) in tobacco further validated this correlation, showcasing increased anthocyanin accumulation that enhanced antioxidant capacity, preserved relative water content, and reduced lipid peroxidation in leaves, collectively improving drought stress tolerance (Waseem et al., 2019; Naing and Kim, 2021; Xiang et al., 2021; Li and Ahammed, 2023).

ABA and SA function as key regulators in augmenting abiotic stress tolerance by initiating antioxidant responses, leading to the mitigation of reactive oxygen species such as MDA, hydrogen peroxide (H₂O₂), and superoxide ions (O₂ ^•−). Recent research demonstrated that ABA plays a crucial role in governing physiological and biochemical adaptations for drought stress tolerance, with a potential impact on the regulation of plant phenolic compound biosynthesis, particularly anthocyanins that accumulate during drought stress (González-Villagra et al., 2019). Nagira et al. (2006) found that osmotic stress in Torenia fournieri plants increased ABA levels before anthocyanin biosynthesis, indicating ABA’s crucial role in modulating anthocyanin induction during drought stress. Meanwhile, González-Villagra et al. (2017) proposed a model suggesting ABA involvement in anthocyanin biosynthesis regulation through microRNA156, enhancing the expression of anthocyanin biosynthesis genes. Despite limited reports connecting changes in endogenous ABA levels to anthocyanin biosynthesis induction, the precise role of ABA in regulating anthocyanin concentrations under drought stress conditions remains unresolved. SA serves as a key plant hormone and elicitor, crucially enhancing plant defense mechanisms against various biotic and abiotic stresses throughout the plant’s life cycle, from seed germination to flowering and fruit ripening (Tiwari et al., 2017; Koo et al., 2020). A study shows that application of SA increased anthocyanins accumulation in grapes until 3 fold (Obinata et al., 2003). Moreover, proline, a significant osmoprotectant, assumes a crucial role in abiotic stress responses. Elevated levels of proline, soluble sugars, and antioxidant enzyme activity were observed in the anthocyanin-rich plant, contributing to a more effective mitigation of the heightened accumulation of ROS and MDA induced by drought stress (Dabravolski and Isayenkov, 2023). Comparable outcomes were observed in lucerne plants overexpressing miRNA156, linking enhanced drought tolerance to elevated levels of ABA, antioxidants, and augmented proline accumulation (Arshad et al., 2017). This suggests that increased proline content in response to drought stress actively contributes to the amelioration of its adverse effects.

The primary objective of our research is to comprehensively investigate the impact of anthocyanins on mitigating drought stress in rice plants. Given the scarcity of published data on this subject, our study aims to provide valuable insights into the influence of anthocyanins on enhancing drought stress tolerance in rice. Additionally, we seek to elucidate the defense mechanisms employed by exogenous anthocyanins to enable rice plants to effectively counteract the adverse effects of drought stress. Through a systematic and thorough assessment, our research will not only contribute to the understanding of the physiological and biochemical changes induced by anthocyanins but also shed light on the intricate pathways through which these compounds bolster the plant’s resilience to drought stress. By elucidating the specific mechanisms involved, we aspire to provide a foundation for the development of sustainable strategies to enhance drought tolerance in rice crops, ultimately contributing to agricultural resilience in the face of changing environmental conditions.

The data presented herein provides insight into a potential mechanism whereby anthocyanin orchestrates the modulation of plant tolerance in response to drought stress. Our investigation reveals that anthocyanin exerts regulatory influence across multiple stress tolerance pathways. Specifically, it impacts antioxidant enzyme activity, initiates the phenylalanine ammonia-lyase (PAL) pathway, influences the accumulation of phytohormones, facilitates melatonin accumulation, modulates drought-related gene expression, and governs the regulation of essential macronutrients. These findings underscore the multifaceted role of anthocyanin in enhancing plant resilience under conditions of drought-induced stress.

Our study demonstrates that the application of exogenous anthocyanin positively influences various key indicators of plant growth under drought conditions. Notable improvements were observed in parameters such as shoot and root length, leaf area, chlorophyll content, relative water content, panicle length and number, as well as seed number and weight ( Figure 1 ). These findings suggest that anthocyanin supplementation effectively mitigates the adverse effects of drought stress, fostering normal plant growth and development. Furthermore, our investigation reveals a significant reduction in H₂O₂, O₂ ^•− and MDA contents in drought-stressed plants treated with exogenous anthocyanin, as depicted in Figure 2 . This reduction underscores the role of anthocyanin in ameliorating oxidative stress associated with drought conditions. The observed decline in ROS and MDA levels is indicative of anthocyanin’s capacity to serve as a reactive oxygen species scavenger, thereby shielding plants from oxidative damage and augmenting their resilience. This aligns with existing literature highlighting the antioxidative properties of anthocyanins, emphasizing their potential to enhance plant sustainability in challenging environmental conditions (Dabravolski and Isayenkov, 2023).

We conducted a comprehensive analysis, monitoring the accumulation of naringenin, cyanidin, delphinidin, total flavonoids, and key pathway genes (PAL, CHS, CHI, F3H, and ANS) in both anthocyanin-treated and non-treated plants subjected to drought stress. Our findings reveal a consistent upregulation of all examined metabolites and their associated biosynthesis genes in anthocyanin-treated plants compared to their non-treated counterparts under drought stress conditions ( Figure 3 ). These results provide evidence that the exogenous application of anthocyanin positively influences the PAL pathway genes, thereby augmenting the endogenous accumulation of anthocyanins. A recent study have reported that, under drought conditions, plants activate survival mechanisms, particularly biosynthesis of secondary metabolites such as phenolics and flavonoids, influencing the accumulation of polyphenolic substances, including anthocyanins and other flavonoids (Park et al., 2023). Our investigation aligns with prior studies demonstrating a significant elevation of anthocyanin levels in pea and grape berry plants under drought stress conditions (Nogués et al., 1998; Castellarin et al., 2007). The stress-induced anthocyanins function as potent free radical scavengers, thereby mitigating oxidative stress associated with drought by facilitating their transport to the vacuole (Zahedi et al., 2021). The proposed role of anthocyanins as osmoregulators in stressed plants involves the regulation of water homeostasis, contributing to enhanced resistance to drought stress. Cirillo et al. (2021) suggested that plant species with higher anthocyanin levels tend to exhibit greater resistance to drought conditions (Cirillo et al., 2021).

Furthermore, our investigation delves into the gene expression of the phenylalanine ammonia lyase (PAL) pathway under drought stress. Our results indicate a significant upregulation of PAL, CHS, CHI, F3H, and ANS genes in response to exogenous anthocyanin during drought stress. Notably, these genes also exhibited significant enhancement under drought stress in normal conditions (non-treated plants) compared to control plants. This finding is consistent with previous studies, such as the observed increase in flavonoids and relevant genes, including PAL, CHS, CHI, F3H, FLS, DFR, and ANS, in Achillea pachycephala Rech.f during drought stress exposure (Gharibi et al., 2019). Similarly, significant induction of flavonoid biosynthesis genes was reported in buckwheat under drought stress (Hou et al., 2019). In accordance with our findings and previous research, we conclude that the heightened levels of anthocyanins and total flavonoids under drought stress are closely associated with the upregulation of flavonoid biosynthesis genes, including PAL, CHS, CHI, F3H, FLS, DFR, and ANS (Ma et al., 2014). This suggests a coordinated regulatory mechanism governing the enhanced synthesis of these secondary metabolites in response to drought-induced stress. Further, upon closer examination, it was observed that the expression of drought-responsive genes, namely DHN and DREB, was notably upregulated with the application of anthocyanin under both normal and drought conditions ( Figures 5C, D ). These findings suggest a positive correlation between DHN and DREB genes and anthocyanin, indicating a potential involvement of anthocyanin in the regulation of these genes. Even though, regulation of DREB on anthocyanin biosynthesis is completely unknown, however a recent study predicted that DREB gene expression in Ammopiptanthus mongolicus enhanced the accumulation of anthocyanin under drought stress.

The production of phytohormones, notably ABA and SA, is a well-established defense mechanism against drought stress. In our investigation of the relationship between anthocyanin and ABA and SA biosynthesis during drought stress, we observed that the exogenous application of anthocyanin significantly heightened the accumulation of ABA and SA compared to both control and drought-exposed plants ( Figures 4C, D ). Numerous studies have suggested that the drought stress-induced increase in ABA, proline, and sugars may play pivotal roles in anthocyanin biosynthesis (Das et al., 2012; Sperdouli and Moustakas, 2012; González-Villagra et al., 2019). Previous research, such as that on Torenia fournieri, has proposed that ABA precedes anthocyanin biosynthesis during drought stress, with ABA induction occurring before the regulation of anthocyanin (González-Villagra et al., 2019). These findings elucidate that ABA induction occurs during periods of drought stress, subsequently triggering the biosynthesis of anthocyanin. However, in our study, the exogenous application of anthocyanin resulted in an enhanced accumulation of ABA in drought-stressed plants, indicating a synergistic association between ABA and anthocyanin during drought stress. While SA is traditionally known for its role in biotic stress, recent studies have implicated its active participation in plant tolerance to drought stress (Brito et al., 2018; Khan et al., 2022). Consistent with these findings, we observed an increase in SA levels in drought-stressed plants compared to control plants. Moreover, anthocyanin-treated plants exhibited even higher SA accumulation under drought stress, suggesting that anthocyanin application enhances SA accumulation in rice during drought stress. In support with previous study, Our study posits that SA may mitigate drought stress by triggering an antioxidant system that alleviates oxidative stress, as reported in Aristotelia chilensis plants (González-Villagra et al., 2022).

Interestingly, our results revealed a substantial induction of proline in anthocyanin-treated plants compared to non-treated plants under drought stress ( Figure 4B ). Proline, an amino acid with a well-established response to osmotic stress in plants, acts as a crucial component in stress reduction (Cirillo et al., 2021). This includes functioning as a buffer for cellular redox potential, serving as a ROS scavenger, stabilizing proteins and membranes, and inducing genes related to drought and salt stress (Satoh et al., 2002; Carillo, 2018). Additionally, proline can be rapidly metabolized when no longer needed, providing essential resources for the recovery and repair of stress-induced damages (Carillo et al., 2008). Our findings, aligned with previous reports, underscore the association between increased anthocyanin accumulation and a notable metabolic shift involving proline in response to drought stress.

Melatonin, classified as an indole molecule, exhibits pronounced free radical scavenging capabilities, positioning it as a pivotal candidate for mitigating oxidative stress (Sun et al., 2023). Analogous to proline, melatonin functions as an inhibitor of oxidative stress by scavenging ROS induced by drought stress (Sharma and Zheng, 2019). The outcomes of our investigation reveal that the application of anthocyanin leads to an augmentation of melatonin levels in rice plants subjected to drought stress, as depicted in Figure 4A . Intriguingly, a recent study underscores the reciprocal relationship between melatonin and anthocyanin. It elucidates that melatonin not only induces the expression of anthocyanin biosynthesis genes, fostering the accumulation of anthocyanin, but also manifests stress-reducing properties through chelation mechanisms (Sun et al., 2023). The intricate interplay observed between melatonin and anthocyanin suggests a synergistic dynamic that may contribute significantly to enhancing overall stress tolerance in plants, particularly under drought conditions. Numerous researchers have posited diverse mechanisms through which melatonin imparts drought stress tolerance. These mechanisms encompass the preservation of water, maintenance of cell membrane integrity, reduction of cytotoxic biochemicals such as H₂O₂ and MDA, and augmentation of antioxidant enzyme activity (Yan et al., 2016; Wei et al., 2018; Khan et al., 2020). Collectively, these actions underscore melatonin’s multifaceted role as a protective agent during periods of drought-induced stress. In alignment with these findings, a contemporary study anticipates that the application of anthocyanin serves as a catalyst for drought stress tolerance by facilitating the accumulation of melatonin. This predictive insight highlights the potential synergistic relationship between anthocyanin and melatonin, offering a promising avenue for bolstering plant resilience in the face of challenging environmental conditions.

Plants actively accumulate essential macronutrients such as Na⁺, K⁺, and Ca⁺ within the cytosol to support fundamental physiological processes. While high levels of K⁺ initially facilitate increased water absorption during drought stress, a subsequent reduction in K⁺ levels is observed in the later stages (Fayyaz et al., 2013). Ca⁺ plays a pivotal role in plant adaptation to adverse conditions under water-deficit circumstances, serving as a regulatory element in plant cell metabolism (Bowler and Fluhr, 2000). The application of Ca⁺ has been shown to enhance drought resistance by inhibiting the synthesis of active oxides, preserving the integrity of the plasma membrane, sustaining normal photosynthesis, and modulating the metabolism of various plant hormones and other essential chemicals (Pathak et al., 2020). Some studies have indicated that elevated Na⁺concentrations can impede the accumulation of K⁺, elevate osmotic pressure, induce oxidative damage, and hinder productivity, growth, and metabolic activities (James et al., 2011; Jan et al., 2021b). Contrary to the typical response observed under drought stress, our findings reveal a simultaneous reduction in these macronutrients in untreated plants, whereas anthocyanin-treated plants exhibit a significant increase in Ca⁺, K⁺, and Na⁺ levels concurrently ( Figure 6 ). The supplementation of anthocyanins appears to mitigate the impact of drought stress by regulating the levels of these essential macronutrients in rice plants. However, a comprehensive understanding of the molecular mechanisms underlying this regulatory effect requires further investigation.

In conclusion, our study has shown that the application of anthocyanins significantly enhances the phenotypes of rice plants, mitigates the effects of drought-induced oxidative stress, and modulates crucial physiological and molecular responses under drought conditions. Anthocyanin-treated plants exhibit notable improvements in various morphological and biochemical traits, including increased shoot and root length, chlorophyll content, relative water content, panicle number, and seed weight. Additionally, anthocyanin application reduces oxidative damage by lowering levels of reactive oxygen species, lipid peroxidation, and enhancing antioxidant enzyme activities. The observed upregulation of anthocyanins, flavonoids, and associated biosynthetic genes points to their role in alleviating stress, while changes in melatonin, proline, ABA, and SA levels highlight their regulatory effects on stress-responsive pathways. Furthermore, anthocyanin positively influences the enzymatic activity of antioxidants and the expression of drought-responsive genes. Importantly, anthocyanin treatment promotes the accumulation of essential macronutrients, such as Na⁺, K⁺, and Ca⁺, enhancing plant resilience to drought stress. These findings collectively highlight the multifaceted role of anthocyanins in conferring drought stress tolerance in rice plants, providing valuable insights for potential applications in sustainable agricultural practices.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

SalA: Formal analysis, Software, Writing – review & editing. RJ: Methodology, Software, Validation, Writing – original draft, Writing – review & editing. SajA: Validation, Visualization, Writing – review & editing. ZK: Formal analysis, Software, Writing – review & editing. KK: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing. L: Conceptualization, Software, Validation, Writing – review & editing.