Characterization of the ddt1 Mutant in Rice and Its Impact on Plant Height Reduction and Water Use Efficiency

Rice (Oryza sativa L.), a fundamental global staple, nourishes over half of the world’s population. The identification of the ddt1 mutant in rice through EMS mutagenesis of the indica cultivar Shuhui527 revealed a dwarf phenotype, characterized by reduced plant height, smaller grain size, and decreased grain weight. Detailed phenotypic analysis and map-based cloning pinpointed the mutation to a single-base transversion in the LOC_Os03g04680 gene, encoding a cytochrome P450 enzyme, which results in a premature termination of the protein. Functional complementation tests confirmed LOC_Os03g04680 as the DDT1 gene responsible for the observed phenotype. We further demonstrated that the ddt1 mutation leads to significant alterations in gibberellic acid (GA) metabolism and signal transduction, evidenced by the differential expression of key GA-related genes such as OsGA20OX2, OsGA20OX3, and SLR1. The mutant also displayed enhanced drought tolerance, as indicated by higher survival rates, reduced water loss, and rapid stomatal closure under drought conditions. This increased drought resistance was linked to the mutant’s improved antioxidant capacity, with elevated activities of antioxidant enzymes and higher expression levels of related genes. Our findings suggest that DDT1 plays a crucial role in regulating both plant height and drought stress responses. The potential for using gene editing of DDT1 to mitigate the dwarf phenotype while retaining improved drought resistance offers promising avenues for rice improvement.


Introduction
Rice (Oryza sativa L.) stands as one of the world's most vital staple crops, sustaining over half of the global population.Plant height is a critical agronomic trait that greatly influences yield and resistance to lodging in rice.Thus, elucidating the genetic and molecular underpinnings that regulate plant height is essential for breeding high-yielding varieties.The regulation of plant height in rice results from a complex interaction between genetic factors and environmental cues.Extensive genetic mapping studies have identified numerous quantitative trait loci (QTLs) and genes associated with plant height [1][2][3][4][5][6][7][8][9].These loci frequently correspond to the genes involved in hormonal signaling, cellular elongation, and transcriptional regulation.Notably, the gibberellin (GA) pathway is a major regulator of rice plant height, with key genes such as SD1 (Semi-Dwarf 1) playing critical roles in GA biosynthesis and signaling, thus influencing internode elongation and overall stature [10][11][12].Additionally, genes in the brassinosteroid (BR) signaling pathway also significantly affect rice plant height [13][14][15][16][17][18][19][20].
Drought stress triggers a suite of molecular responses in rice, orchestrated by an intricate network involving genes, proteins, and signaling pathways.Central to the plant's response to drought is the hormone abscisic acid (ABA), which regulates stomatal closure to reduce water loss through transpiration, thereby enhancing water use efficiency under drought conditions [21][22][23].Beyond ABA signaling, rice utilizes a plethora of molecular mechanisms to withstand drought, including the synthesis and accumulation of osmoprotectants like proline, glycine betaine, and soluble sugars, which help maintain cellular osmotic balance and stabilize proteins and membranes during drought [24].Additionally, rice activates antioxidant defense systems to neutralize reactive oxygen species (ROS) and protect cells from oxidative damage induced by drought stress, with enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidases (POX) playing key roles in detoxifying ROS and maintaining cellular redox homeostasis [25].In plants, cytochrome P450 enzymes play crucial roles in diverse physiological processes, including the biosynthesis of phytohormones, secondary metabolites, and defense compounds.These enzymes are instrumental in generating a broad spectrum of specialized metabolites, such as flavonoids, alkaloids, terpenoids, and phenylpropanoids, which are essential for plant growth, development, and adaptation to environmental challenges [26][27][28][29].Notably, members of the CYP85 and CYP90 families catalyze critical steps in the biosynthesis of BR, a class of steroid hormones vital for various aspects of plant growth and development, including cell elongation, vascular differentiation, and stress responses [30,31].Cytochrome P450 enzymes also contribute to the biosynthesis of phytoalexins and defense compounds, enhancing plant defense against pathogens and environmental stresses.For example, the CYP71 and CYP81 families are involved in synthesizing various alkaloids and glucosinolates, which serve as chemical deterrents against herbivores and pathogens [32,33].Furthermore, these enzymes are integral to the production of essential plant pigments such as anthocyanins and carotenoids, which contribute to the coloration of flowers and fruits [34,35].
The integration of drought tolerance with semi-dwarfism in rice holds significant agricultural importance.Semi-dwarf varieties, exemplified by the well-known SD1 gene, have revolutionized rice cultivation by reducing lodging and increasing yield potential.However, their reduced stature can sometimes compromise their ability to withstand environmental stresses, such as drought.By combining drought tolerance traits with semidwarfism, breeders can develop varieties that not only maintain high yield potential and lodging resistance but also exhibit enhanced resilience to water scarcity.This dual trait integration addresses the evolving challenges posed by climate change and water scarcity, ensuring sustainable rice production in diverse agroecological settings.
In this study, we delineate a rice dwarf and drought tolerance 1 (ddt1) mutant and elucidate the DDT1 gene, corresponding to the previously reported P450 gene CYP96B4/SD37/ DSS1 [29,36,37].By employing genetic localization and molecular markers, we aimed to confirm the specific role of the DDT1 gene in regulating rice growth and response to drought stress.The ddt1 mutant identified in this study exemplifies this dual benefit, offering insights into enhancing both drought resilience and agronomic performance in rice breeding programs.

Phenotypic Analysis of ddt1
The ddt1 mutant was identified through a detailed screening process following the application of EMS (ethyl methane sulfonate) mutagenesis to the indica rice cultivar Shuhui527.Relative to wild-type (WT) plants, the mutant displayed a pronounced dwarf phenotype, evident from the seedling stage through to maturity (Figure 1A,B).This reduction in height resulted from a shortened length of all internodes in the ddt1 mutant (Figure 1C,D).Additionally, the ddt1 mutant exhibited smaller grain size as well as reduced grain weight and grain weight per plant compared to WT (Figures 1E and S1).

Map-Based Cloning of DDT1
To conduct a genetic analysis of the ddt1 mutant, we crossed the mutant with the wild-type cultivar Shuhui 527.A phenotypic examination of the F1 progeny revealed characteristics similar to the wild type, indicating the recessive nature of the mutation.In the F2 population, consisting of 325 plants, 250 of them showed growth patterns similar to the wild type, while the remainder exhibited the mutant phenotype.The observed segregation ratio of 3:1 (χ²0.05= 0.24 < 3.841) supports the hypothesis that the ddt1 mutation is controlled by a single recessive allele at a nuclear locus.
To identify the gene associated with the mutant phenotype, the ddt1 mutant was crossed with the japonica rice variety Wuyunjing 7. From this cross, 210 F2 progenies manifesting the mutant phenotype were selected for further analysis.We utilized 183 simple sequence repeats (SSRs) and 41 sequence tagged sites (STSs), uniformly distributed across the 12 chromosomes of rice, for genotyping.Initial mapping efforts localized the mutant locus

Map-Based Cloning of DDT1
To conduct a genetic analysis of the ddt1 mutant, we crossed the mutant with the wild-type cultivar Shuhui 527.A phenotypic examination of the F 1 progeny revealed characteristics similar to the wild type, indicating the recessive nature of the mutation.In the F 2 population, consisting of 325 plants, 250 of them showed growth patterns similar to the wild type, while the remainder exhibited the mutant phenotype.The observed segregation ratio of 3:1 (χ² 0.05 = 0.24 < 3.841) supports the hypothesis that the ddt1 mutation is controlled by a single recessive allele at a nuclear locus.
To identify the gene associated with the mutant phenotype, the ddt1 mutant was crossed with the japonica rice variety Wuyunjing 7. From this cross, 210 F 2 progenies mani-festing the mutant phenotype were selected for further analysis.We utilized 183 simple sequence repeats (SSRs) and 41 sequence tagged sites (STSs), uniformly distributed across the 12 chromosomes of rice, for genotyping.Initial mapping efforts localized the mutant locus between markers B3-1 and B3-3 on chromosome 3, as determined by analyzing 22 F 2 plants (Figure 2A).Further fine mapping with a larger set of 210 F 2 mutants refined the locus to a specific interval flanked by markers P3 and P4 (Figure 2A).According to data from the Rice Genome Annotation Project (RGAP), this interval contains six predicted open reading frames (ORFs).Sequencing of this region in the ddt1 mutant revealed a transversion mutation from A to T within an exon of LOC_Os03g04680, encoding a cytochrome P450 enzyme, which resulted in a premature termination of the protein (Figure 2A).Therefore, LOC_Os03g04680 was identified as the candidate gene responsible for the observed mutant phenotype.
(ORFs).Sequencing of this region in the ddt1 mutant revealed a transversion mutation fr A to T within an exon of LOC_Os03g04680, encoding a cytochrome P450 enzyme, which sulted in a premature termination of the protein (Figure 2A).Therefore, LOC_Os03g04 was identified as the candidate gene responsible for the observed mutant phenotype.
To definitively confirm that DDT1 corresponds to LOC_Os03g04680, we carefu inserted a genomic fragment of LOC_Os03g04680 into the binary vector pCAMBIA1 and introduced it into the ddt1 mutant through a transformation.This yielded 12 in pendent T0 transformants.Detailed phenotypic analysis (COM-1, COM-2) demonstra that each transformant effectively mitigated the ddt1 mutant phenotype, which was denced by enhanced plant height (Figure 2B,C).These findings provide compelling dence supporting the hypothesis that the dwarf phenotype observed in the ddt1 mutan due to a single-base substitution mutation within the LOC_Os03g04680 gene.
Through qRT-PCR analysis, our study revealed a broad expression spectrum of DDT1 gene across various plant tissues, including the roots, culms, leaves, leaf shea and panicles.Notably, DDT1 showed its highest expression level in leaf tissue, while lowest expression was observed in the panicle (Figure 2C).To definitively confirm that DDT1 corresponds to LOC_Os03g04680, we carefully inserted a genomic fragment of LOC_Os03g04680 into the binary vector pCAMBIA1300 and introduced it into the ddt1 mutant through a transformation.This yielded 12 independent T 0 transformants.Detailed phenotypic analysis (COM-1, COM-2) demonstrated that each transformant effectively mitigated the ddt1 mutant phenotype, which was evidenced by enhanced plant height (Figure 2B,C).These findings provide compelling evidence supporting the hypothesis that the dwarf phenotype observed in the ddt1 mutant is due to a single-base substitution mutation within the LOC_Os03g04680 gene.
Through qRT-PCR analysis, our study revealed a broad expression spectrum of the DDT1 gene across various plant tissues, including the roots, culms, leaves, leaf sheaths, and panicles.Notably, DDT1 showed its highest expression level in leaf tissue, while its lowest expression was observed in the panicle (Figure 2C).

Subcellular Localization of DDT1
To elucidate the subcellular localization of DDT1, our study utilized a strategic approach by creating a recombinant expression vector (p35S::DDT1-GFP).This vector was specifically designed to fuse the DDT1 coding sequence with the green fluorescent protein (GFP) marker, enabling visual tracking of the protein within cells.The construct was then introduced into rice protoplasts, which served as an effective model system for plant cell biology studies.Employing advanced confocal microscopy techniques, we carefully observed the distribution of the DDT1-GFP fusion protein within these cells.Our findings clearly demonstrate that DDT1 predominantly localizes to the endoplasmic reticulum (Figure 3), providing significant insights into its functional role in cellular processes.

Subcellular Localization of DDT1
To elucidate the subcellular localization of DDT1, our study utilized a strategic approach by creating a recombinant expression vector (p35S::DDT1-GFP).This vector was specifically designed to fuse the DDT1 coding sequence with the green fluorescent protein (GFP) marker, enabling visual tracking of the protein within cells.The construct was then introduced into rice protoplasts, which served as an effective model system for plant cell biology studies.Employing advanced confocal microscopy techniques, we carefully observed the distribution of the DDT1-GFP fusion protein within these cells.Our findings clearly demonstrate that DDT1 predominantly localizes to the endoplasmic reticulum (Figure 3), providing significant insights into its functional role in cellular processes.

Unveiling the Role of DDT1 in Plant Height Regulation
We investigated the expression patterns of genes involved in gibberellic acid (GA) synthesis and degradation in the wild type (WT), ddt1 mutant, and complementary lines.Our findings demonstrated significant alterations in the expression levels of key genes: OsGA20OX3 was notably increased, while OsGA20OX2 was significantly decreased in the ddt1 mutant compared to WT and complementary lines (Figure 4A,B).Furthermore, SLR1, a critical gene in GA signal transduction, was significantly upregulated (Figure 4C), indicating that the reduced plant height observed in ddt1 mutants might be due to decreased GA content.To support these findings, we evaluated the protein levels of SLR1, which reflected the changes observed at the mRNA level (Figure 4D).Then, we cloned DDT1 into a pGBKT7 expression vector and SSLR1 into a pGADT7 expression vector, followed by co-transformation into yeast cells.However, yeast two-hybrid assays revealed no direct interaction between SLR1 and DDT1 (Figure 4E), suggesting that DDT1 might indirectly influence SLR1 expression.

Unveiling the Role of DDT1 in Plant Height Regulation
We investigated the expression patterns of genes involved in gibberellic acid (GA) synthesis and degradation in the wild type (WT), ddt1 mutant, and complementary lines.Our findings demonstrated significant alterations in the expression levels of key genes: OsGA20OX3 was notably increased, while OsGA20OX2 was significantly decreased in the ddt1 mutant compared to WT and complementary lines (Figure 4A,B).Furthermore, SLR1, a critical gene in GA signal transduction, was significantly upregulated (Figure 4C), indicating that the reduced plant height observed in ddt1 mutants might be due to decreased GA content.To support these findings, we evaluated the protein levels of SLR1, which reflected the changes observed at the mRNA level (Figure 4D).Then, we cloned DDT1 into a pGBKT7 expression vector and SSLR1 into a pGADT7 expression vector, followed by co-transformation into yeast cells.However, yeast two-hybrid assays revealed no direct interaction between SLR1 and DDT1 (Figure 4E), suggesting that DDT1 might indirectly influence SLR1 expression.

Role of the DDT1 in Enhancing Drought Stress Tolerance in Rice
In plants, cytochrome P450 enzymes play crucial roles in stress responses [38].The DDT1 gene is a member of the cytochrome P450 family.Hence, we examined the expression levels of DDT1 after subjecting wild-type plants to cold, heat, salt, and drought treatments.The results indicate that salt and drought stress significantly induce the expression of the DDT1 gene, reaching a peak at 5 h under salt stress and 4 h under drought stress (Figure 5C,D).In contrast, there were no significant changes under cold and heat stress (Figure 5A,B).These experimental findings suggest that the DDT1 gene may play a role in regulating the response to drought and salt stress in rice.

Role of the DDT1 in Enhancing Drought Stress Tolerance in Rice
In plants, cytochrome P450 enzymes play crucial roles in stress responses [38].The DDT1 gene is a member of the cytochrome P450 family.Hence, we examined the expression levels of DDT1 after subjecting wild-type plants to cold, heat, salt, and drought treatments.The results indicate that salt and drought stress significantly induce the expression of the DDT1 gene, reaching a peak at 5 h under salt stress and 4 h under drought stress (Figure 5C,D).In contrast, there were no significant changes under cold and heat stress (Figure 5A,B).These experimental findings suggest that the DDT1 gene may play a role in regulating the response to drought and salt stress in rice.To further investigate the impact of the DDT1 gene on drought resistance, we the wild-type and ddt1 mutant rice plants with 20% PEG.The results showed t leaves of the wild-type plants exhibited severe curling, whereas the mutant leav played no significant changes.After a 7-day recovery period, the survival rate of t tants was found to be over 60%, compared to less than 20% for the wild type (Figu This suggests that the mutation in the DDT1 gene enhances drought tolerance in r further confirm the drought resistance of the mutants, both wild-type and ddt1 plan subjected to mannitol treatment in tissue culture, which demonstrated more pron growth inhibition in the wild type (Figure 6B).Additionally, we measured the wa rate of both genotypes, finding that the wild type exhibited a significantly higher water loss compared to the mutant (Figure 6C).We also examined and quantified havior of stomata in leaves of both genotypes before and after drought treatment.scopic observations indicated that post-drought, the number of fully open and p open stomata was significantly lower in the mutant, while the number of fully clos mata was significantly higher compared to the wild type (Figure 6D,E).Furthermo analyzed leaf length, leaf width, and stomatal density in both the WT and ddt1 plan findings indicate that leaf length and width were significantly reduced in ddt1 comp the wild type, while stomatal density showed no significant changes (Figure 6F-H To further investigate the impact of the DDT1 gene on drought resistance, we treated the wild-type and ddt1 mutant rice plants with 20% PEG.The results showed that the leaves of the wild-type plants exhibited severe curling, whereas the mutant leaves displayed no significant changes.After a 7-day recovery period, the survival rate of the mutants was found to be over 60%, compared to less than 20% for the wild type (Figure 6A).This suggests that the mutation in the DDT1 gene enhances drought tolerance in rice.To further confirm the drought resistance of the mutants, both wild-type and ddt1 plants were subjected to mannitol treatment in tissue culture, which demonstrated more pronounced growth inhibition in the wild type (Figure 6B).Additionally, we measured the water loss rate of both genotypes, finding that the wild type exhibited a significantly higher rate of water loss compared to the mutant (Figure 6C).We also examined and quantified the behavior of stomata in leaves of both genotypes before and after drought treatment.Microscopic observations indicated that post-drought, the number of fully open and partially open stomata was significantly lower in the mutant, while the number of fully closed stomata was significantly higher compared to the wild type (Figure 6D,E).Furthermore, we analyzed leaf length, leaf width, and stomatal density in both the WT and ddt1 plants.Our findings indicate that leaf length and width were significantly reduced in ddt1 compared to the wild type, while stomatal density showed no significant changes (Figure 6F-H).This suggests that the reduced water loss in the mutant under drought conditions may be associated with rapid stomatal closure induced by the drought.
suggests that the reduced water loss in the mutant under drought conditions may b ciated with rapid stomatal closure induced by the drought.

Enhancing Drought Tolerance through Antioxidant Capacity Modulation in ddt1 Mu
To investigate whether mutations in the DDT1 gene enhance drought tolera affecting the plant's antioxidant capacity, this study measured the levels of supe anions, hydrogen peroxide, and malondialdehyde (MDA) in wild-type and ddt1 m plants before and after drought treatment.The results showed that after drought the levels of superoxide anions, hydrogen peroxide, and MDA were significantly in the wild type than in the mutant (Figure 7A-C).Additionally, this study exa changes in proline content in both genotypes before and after drought treatme vealing a significant increase in proline levels in the mutant compared to the wil (Figure 7D).Furthermore, the activities of antioxidant enzymes such as ascorb roxidase (APX), peroxidase (POD), superoxide dismutase (SOD), and catalase were assessed, with results indicating significantly higher activities in the ddt1 m after drought stress (Figure 7E-H).To further validate whether the enhanced d

Enhancing Drought Tolerance through Antioxidant Capacity Modulation in ddt1 Mutant
To investigate whether mutations in the DDT1 gene enhance drought tolerance by affecting the plant's antioxidant capacity, this study measured the levels of superoxide anions, hydrogen peroxide, and malondialdehyde (MDA) in wild-type and ddt1 mutant plants before and after drought treatment.The results showed that after drought stress, the levels of superoxide anions, hydrogen peroxide, and MDA were significantly higher in the wild type than in the mutant (Figure 7A-C).Additionally, this study examined changes in proline content in both genotypes before and after drought treatment, revealing a significant increase in proline levels in the mutant compared to the wild type (Figure 7D).Furthermore, the activities of antioxidant enzymes such as ascorbate peroxidase (APX), peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) were assessed, with results indicating significantly higher activities in the ddt1 mutant after drought stress (Figure 7E-H).To further validate whether the enhanced drought tolerance of the ddt1 mutant is mediated through its antioxidant capacity, the expression levels of antioxidantrelated genes, including APX1, POD1, FeSOD, and CATB, were measured before and after drought treatment.The expression levels of these genes were significantly higher in the mutant compared to the wild type after drought treatment (Figure 7I-L).Collectively, these findings suggest that the mutation in the DDT1 gene enhances drought tolerance by impacting the plant's antioxidant capacity.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 9 of 14 tolerance of the ddt1 mutant is mediated through its antioxidant capacity, the expression levels of antioxidant-related genes, including APX1, POD1, FeSOD, and CATB, were measured before and after drought treatment.The expression levels of these genes were significantly higher in the mutant compared to the wild type after drought treatment (Figure 7I-L).Collectively, these findings suggest that the mutation in the DDT1 gene enhances drought tolerance by impacting the plant's antioxidant capacity.

Discussion
The significance of rice as a staple food for over half of the global population underscores the critical need to understand and enhance its agronomic traits, such as plant height, which significantly influences yield and lodging resistance.Recent research has illuminated the complex genetic and molecular networks that regulate plant height, focusing on identifying QTLs and genes responsible for this trait.Among these, GA and BR signaling pathways have emerged as key regulatory mechanisms [39].Moreover, rice's response to abiotic stresses, such as drought, involves a sophisticated network of molecular mechanisms, where the hormone ABA plays a central role in regulating plants' water use efficiency and osmotic balance.The synthesis and accumulation of osmoprotectants and the activation of antioxidant defense systems further help the plant mitigate the impacts of stress [24,40].In this context, the characterization of the rice dwarf and drought tolerance 1 (ddt1) mutant, which corresponds to the previously reported P450 gene CYP96B4/SD37/DSS1 [29,36,37], provides in-

Discussion
The significance of rice as a staple food for over half of the global population underscores the critical need to understand and enhance its agronomic traits, such as plant height, which significantly influences yield and lodging resistance.Recent research has illuminated the complex genetic and molecular networks that regulate plant height, focusing on identifying QTLs and genes responsible for this trait.Among these, GA and BR signaling pathways have emerged as key regulatory mechanisms [39].Moreover, rice's response to abiotic stresses, such as drought, involves a sophisticated network of molecular mechanisms, where the hormone ABA plays a central role in regulating plants' water use efficiency and osmotic balance.The synthesis and accumulation of osmoprotectants and the activation of antioxidant defense systems further help the plant mitigate the impacts of stress [24,40].In this context, the characterization of the rice dwarf and drought tolerance 1 (ddt1) mutant, which corresponds to the previously reported P450 gene CYP96B4/SD37/DSS1 [29,36,37], provides insights into both the regulation of plant height and stress responses.Previous studies have shown that cytochrome P450 enzymes play critical roles in plant growth and stress responses.For instance, cytochrome P450 enzymes are involved in the biosynthesis of various phytohormones, including gibberellins, which are essential for regulating plant height [41,42].Mutations in cytochrome P450 genes have been linked to altered plant architecture and improved stress tolerance in several plant species.For example, the cytochrome P450 gene CYP78A5 has been implicated in controlling organ size and stress responses in Arabidopsis [43].Similarly, the CYP714B1 and CYP714B2 genes in rice are involved in gibberellin deactivation, affecting plant height and stress tolerance [44].The ddt1 mutant exhibits a distinct dwarf phenotype along with enhanced drought tolerance, which are attributed to changes in GA metabolism and stress response pathways.
The identification of the DDT1 gene within a critical region influencing plant height and stress responses illustrates the multifunctional nature of some genetic elements in rice.This gene, through its involvement in the biosynthesis and signaling pathways of phytohormones, particularly GA and ABA, illustrates a mechanistic overlap between growth regulation and stress response [45].The response of ddt1 mutants to drought stress, particularly through the modulation of antioxidant mechanisms, highlights an adaptive strategy employed by rice.The increased expression of antioxidant enzymes and genes in the mutant suggests an enhanced capacity to mitigate oxidative stress, which is commonly exacerbated under drought conditions [46].Cytochrome P450 enzymes have been shown to participate in the detoxification of reactive oxygen species (ROS) and the synthesis of secondary metabolites involved in stress defense [47].The findings that mutants maintain lower levels of reactive oxygen species (ROS) and higher levels of protective osmoprotectants such as proline during stress conditions reflect an intrinsic modification of the cellular defense mechanisms, likely conferred by the mutation in the DDT1 gene.
Understanding the genetic basis of traits like plant height and drought tolerance provides critical insights for rice breeding programs.The reduced plant height and altered water use efficiency (WUE) are primary factors contributing to the improved drought tolerance in the ddt1 mutant.This validation supports the potential for using targeted gene editing technologies, such as CRISPR/Cas9, to manipulate DDT1 function.CRISPR/Cas9 has been successfully employed to edit genes involved in abiotic stress tolerance and growth regulation in various crops [48].By precisely editing the DDT1 gene, it may be possible to mitigate the dwarf phenotype while retaining or even enhancing drought tolerance, which could be instrumental in breeding programs aimed at developing high-yielding, stress-resilient rice varieties.The ddt1 mutant not only offers a potential candidate gene for manipulating plant height but also suggests a link between dwarfism and stress tolerance that could be exploited to develop rice varieties suited for environments prone to drought stress.This dual benefit could significantly impact rice productivity and sustainability, particularly in regions facing increasing climatic variability.
Further research should aim to elucidate the detailed molecular mechanisms by which the DDT1 gene influences both growth and stress response pathways.Such studies could involve comprehensive analyses of downstream targets of DDT1, interactions with other hormonal pathways, and its role in cellular signaling networks.Additionally, field trials and phenotypic analyses under varied environmental conditions would be essential to confirm the laboratory findings and assess their practical implications for crop improvement [49].

Plant Material and Growth Conditions
This study utilized the indica rice cultivar Shuhui527 as the wild type (WT) and its corresponding mutant line, ddt1, which was generated through ethyl methane sulfonate (EMS) mutagenesis.Both the WT and mutant seeds were germinated and grown under natural field conditions in Fuyang (Zhejiang province, coordinates: 119 • 95 ′ E, 30 • 05 ′ N) and Lingshui (Hainan province, coordinates: 110 • 02 ′ E, 18 • 48 ′ N), exploiting the optimal growth periods across two distinct climatic zones during the rice growing season.

Genetic Mapping and Mutant Analysis
To identify the genetic basis of the ddt1 phenotype, we first crossed ddt1 mutants with the wild type, Shuhui527.F 1 progenies were backcrossed to the parent ddt1 to generate an F 2 population, which was then used for segregation analysis and mapping.A total of 210 F 2 plants were phenotypically screened, and genotyping was performed using simple sequence repeats (SSRs) and sequence tagged site (STS) markers covering all 12 rice chromosomes.The initial mapping localized the mutation to a region on chromosome 3, which was further refined through additional crosses and finer mapping strategies.The specific primer sequences employed in this study are detailed in Supplementary Table S1.

Characterization of DDT1
The candidate region identified was sequenced to pinpoint mutations affecting gene function.LOC_Os03g04680 was identified as the gene disrupted in the ddt1 mutant.This gene encodes a cytochrome P450 enzyme.To confirm the function of LOC_Os03g04680, we cloned a wild-type copy of the gene into the binary vector pCAMBIA1300 [50] and transformed it into ddt1 mutants using Agrobacterium-mediated transformation.Transgenic plants were assessed for phenotypic restoration to wild type.

Phenotypic Assessments
Detailed phenotypic analyses were conducted on WT, ddt1 mutant, and transgenic lines.Measurements included plant height, internode length, grain size, and grain weight.Seeds were soaked at room temperature for 48 h until germination, followed by sowing them in a 96-well plate without bottoms.Seedlings were cultured under a 13 h light/11 h dark photoperiod.For PEG treatment, 14-day-old seedlings were transferred into a culture solution containing 18% (w/v) PEG6000.After 7-14 days of treatment, recovery was initiated, and physiological responses such as survival rate, water loss rate, and stomatal conductance were recorded.

qRT-PCR Analyses
Quantitative real-time PCR (qRT-PCR) was used to assess the expression levels of DDT1 and other stress-related genes in various tissues under normal and stress conditions.Total RNA was isolated from various organs using an RNA extraction kit (Cowin Biotech, Taizhou, China).gDNase-treated RNA was reverse transcribed to generate first-strand cDNA using the one step RT Kit (Cowin Biotech).qRT-PCR was performed using SYBR Premix Ex Taq II (TaKaRa Bio, Shiga, Japan) with the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).The qPCR conditions were as follows: 95 • C for 3 min and 40 cycles of 94 • C for 30 s, 55 • C for 30 s, and 72 • C for 40 s.The rice Actin gene (LOC_Os03g50885) was used as an internal control.

Subcellular Localization of DDT1
For subcellular localization studies, the DDT1 coding sequence was fused to the green fluorescent protein (GFP) under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S) in the vector p35S::DDT1-GFP.The construct was transiently expressed in rice protoplasts isolated from young leaves of wild-type plants.Protoplasts were prepared by enzymatic digestion of leaf tissue followed by purification through a sucrose gradient.Transfection of the protoplasts was performed using polyethylene glycol (PEG)-mediated transformation.Twenty hours post-transfection, the protoplasts were observed under a confocal laser scanning microscope to examine the fluorescence emitted by the GFP fusion protein.The specific subcellular localization of the DDT1-GFP fusion protein was determined based on co-localization with organelle-specific markers for the endoplasmic reticulum.

Yeast Two-Hybrid Assay
Clone the full-length DDT1 into the pGBKDT7 vector.Clone the full-length SLR1 into the pGADT7 vector.Co-transform the yeast strain AH109 with DDT1-pGBKT7 and SLR1-pGADT7.Grow yeast cells on -Leu/-Trp or -Leu/-Trp/-His/-Ade media for 4 days.

Conclusions
The intricate relationship between plant height, yield, and stress tolerance in rice (as mediated by the DDT1 gene) offers promising avenues for enhancing crop resilience through genetic interventions.By leveraging advanced genetic and molecular tools, researchers can potentially unlock new dimensions of crop improvement that align with the global food security goals.

Figure 1 .
Figure 1.Phenotypic analysis of ddt1.(A) Plant phenotype of WT and ddt1 mutant during the seedling stage.Scale bar = 5 cm.(B) Whole plant phenotype of WT and ddt1 mutant during the heading stage.Scale bar = 10 cm.(C) Phenotypic comparison of panicles and internodes in WT and ddt1.P, I, II, III, IV, and V indicate the panicle, the first, the second, the third, the fourth, and the fifth internode, respectively.Scale bar = 5 cm.(D) Statistical analysis of the lengths of panicles and internodes in WT and ddt1.(E) Grain phenotype, scale bar = 1 cm.** Indicates statistical significance determined by student t-test.

Figure 1 .
Figure 1.Phenotypic analysis of ddt1.(A) Plant phenotype of WT and ddt1 mutant during the seedling stage.Scale bar = 5 cm.(B) Whole plant phenotype of WT and ddt1 mutant during the heading stage.Scale bar = 10 cm.(C) Phenotypic comparison of panicles and internodes in WT and ddt1.P, I, II, III, IV, and V indicate the panicle, the first, the second, the third, the fourth, and the fifth internode, respectively.Scale bar = 5 cm.(D) Statistical analysis of the lengths of panicles and internodes in WT and ddt1.(E) Grain phenotype, scale bar = 1 cm.** Indicates statistical significance determined by student t-test.

Figure 2 .
Figure 2. Map-based cloning of OsDDT1.(A) Map-based cloning of OsDDT1.(B) Plant phenot of WT, ddt1, and complementary plants during the heading stage.Scale bar = 10 cm.(C) P height of WT, ddt1, and complementary plants during the heading stage.Different letters indi significant differences.(D) Expression patterns of DDT1.Data are presented as mean ± SD (n Different letters denote significant differences at p < 0.05 (one-way ANOVA, Duncan's mult range test).All experiments were repeated at least three times with similar results.

Figure 2 .
Figure 2. Map-based cloning of OsDDT1.(A) Map-based cloning of OsDDT1.(B) Plant phenotype of WT, ddt1, and complementary plants during the heading stage.Scale bar = 10 cm.(C) Plant height of WT, ddt1, and complementary plants during the heading stage.Different letters indicate significant differences.(D) Expression patterns of DDT1.Data are presented as mean ± SD (n = 3).Different letters denote significant differences at p < 0.05 (one-way ANOVA, Duncan's multiple range test).All experiments were repeated at least three times with similar results.

Figure 5 .
Figure 5. Relative expression of DDT1 in stress responses.(A-D) RT-qPCR analysis of OsD rice seedlings treated with 4 °C, 45 °C, 180 mM NaCl and 20％PEG.Data are presented as SD (n = 3).Different letters denote significant differences at p < 0.05 (one-way ANOVA, D multiple range test).N. S. indicates no significant difference.

Figure 5 .
Figure 5. Relative expression of DDT1 in stress responses.(A-D) RT-qPCR analysis of OsDTT1 in rice seedlings treated with 4 • C, 45 • C, 180 mM NaCl and 20%PEG.Data are presented as mean ± SD (n = 3).Different letters denote significant differences at p < 0.05 (one-way ANOVA, Duncan's multiple range test).N. S. indicates no significant difference.

Figure 6 .
Figure 6.Enhancing drought stress tolerance in ddt1 mutant.(A) Phenotype and survival WT and ddt1 under drought treatment.(B) Phenotype and statistical analysis of plant he tween WT and ddt1 under mannitol treatment.(C) Water loss rate of WT and ddt1.(D) S observation of WT and ddt1.(E) Statistical of stomatal opening in WT and ddt1.CO, com open; PO, partially open; CC, completely closed.Leaf length (F), leaf width (G), and stoma sity (H) in WT and ddt1 plants.Data are presented as means ± SD (n = 6), with statistical cance evaluated via Student's t-test (* p < 0.05, ** p < 0.01).

Figure 6 .
Figure 6.Enhancing drought stress tolerance in ddt1 mutant.(A) Phenotype and survival rate of WT and ddt1 under drought treatment.(B) Phenotype and statistical analysis of plant height between WT and ddt1 under mannitol treatment.(C) Water loss rate of WT and ddt1.(D) Stomatal observation of WT and ddt1.(E) Statistical of stomatal opening in WT and ddt1.CO, completely open; PO, partially open; CC, completely closed.Leaf length (F), leaf width (G), and stomatal density (H) in WT and ddt1 plants.Data are presented as means ± SD (n = 6), with statistical significance evaluated via Student's t-test (* p < 0.05, ** p < 0.01).

Figure 7 .
Figure 7. Analysis of antioxidant capacity of ddt1.(A-D) ROS and proline content.(E-H) Antioxidant enzyme activity.(I-L) The expression of antioxidase-related genes.Data are presented as mean ± SD (n = 3).Different letters denote significant differences at p < 0.05 (one-way ANOVA, Duncan's multiple range test).

Figure 7 .
Figure 7. Analysis of antioxidant capacity of ddt1.(A-D) ROS and proline content.(E-H) Antioxidant enzyme activity.(I-L) The expression of antioxidase-related genes.Data are presented as mean ± SD (n = 3).Different letters denote significant differences at p < 0.05 (one-way ANOVA, Duncan's multiple range test).