Unlocking the Functional Dynamics of ERF103 in Arabidopsis thaliana: A key player in Plant Growth Regulation

Unlocking the Functional Dynamics of ERF103 in Arabidopsis thaliana: A key player in Plant Growth Regulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Unlocking the Functional Dynamics of ERF103 in Arabidopsis thaliana : A key player in Plant Growth Regulation Rahmatullah Jan, Lubna ., Saleem Asif, Zakirullah Khan, Muhammad Farooq, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7070846/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigates the role of the transcription factor AtERF013 in regulating root and shoot development, flowering time, leaf morphology, anthocyanin biosynthesis, and reproduction in Arabidopsis thaliana . AtERF013 overexpression ( 35s-AtERF013 ) enhanced vegetative growth, increasing auxin (IAA) levels in seedlings by 131%, significantly increasing root length, and accelerating lateral root development. In contrast, genome-edited ( GE-aterf013 ) lines reduced growth, with a 50% decrease in IAA levels and shorter primary and lateral roots. Overexpression also induced early flowering, accelerated stem elongation, and increased silique length by 38% and 33% compared to wild-type (Col-0) plants. In contrast, genome-edited lines delayed flowering and reduced silique length by 32% and 27%. Leaf morphology was significantly altered, with 35s-AtERF013 lines showing a 71–84% increase in leaf length and an 82–85% increase in total leaf area, while GE-etaref013 line exhibited 17–18% and 81–111% reductions in leaf length and area, respectively. AtERF013 overexpression also enhanced anthocyanin biosynthesis, increasing anthocyanin accumulation and upregulating the DFR gene seven-fold. Regarding reproductive traits, overexpression increased seed count per silique by 72–111%, while genome-edited lines showed a 50–77% decrease compared to Col-0 plants. Furthermore, GE-aterf013 lines displayed underdeveloped stigmas and a higher proportion of non-viable seeds. These findings highlight AtERF013 as a crucial regulator of plant growth, development, and metabolism, with significant implications for enhancing agronomic traits in A. thalian and other crops. auxin anthocyanin biosynthesis ERF013 Arabidopsis thaliana early flowering root and shoot growth Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Transcription factors are key regulators of gene expression, controlling key developmental processes throughout the plant life cycle, including seed germination, seedling growth, tissue morphogenesis, reproduction, and senescence. The APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily members share a common DNA-binding AP2 domain. This transcription factor family is grouped into four subfamilies based on domain differences: AP2, ERF, DEHYDRATION RESPONSE ELEMENT-BINDING ( DREB ), and RELATED TO ABSCISIC ACID-INSENSITIVE 3/Viviparous ( RAV ) [ 1 – 3 ]. Genome-wide analysis of the ERF gene family in Arabidopsis showed that the AP2/ERF superfamily comprises 147 members, accounting for 122 genes [ 2 ]. AP2/ERF transcription factors bind to specific elements such as GCC-box (GCCGCC), G-Box (CACGTG), and dehydration-responsive element/C-repeat (DRE/CRT, GCCCAC) to regulate target gene expression [ 1 , 4 ]. ERFs recognize and attach to diverse cis-elements within target gene promoters, contributing to various regulatory processes by modulating gene expression. These transcription factors regulate metabolism, growth, development, response to environmental constraints [ 5 – 7 ], flowering time [ 8 , 9 ], and seed development and yield [ 4 , 10 – 12 ] throughout the plant life cycle. They also respond to signals from auxin, cytokinins, abscisic acid, and jasmonic acid, modulating phytohormones biosynthesis and influencing agronomic traits such as plant growth, defense responses, and fruit ripening [ 13 ]. [ 14 ] compiled data highlighting the diverse roles of ERF transcription factors in plant growth, development, and stress responses, including somatic embryogenesis, root growth, shoot elongation, fruit ripening, secondary metabolism, and resilience to environmental stresses such as submergence, heavy metals, drought, high salinity, and cold. Furthermore, ERFs such as PyERF3 in Chinese pear modulate flavonoid biosynthesis by co-regulating transcription factors, forming complexes such as ERF3-MYB114-bHLH3 , which is involved in anthocyanin biosynthesis [ 15 , 16 ]. Similarly, Pp4ERF24 and PpERF96 in Red Zaosu enhance anthocyanin biosynthesis through the interaction of PpbHLH3 with PpMYB114 [ 17 ]. This study aims to investigate the ethylene-responsive transcription factor AtERF103 (AT1G77640) and its role in regulating morphological, physiological, molecular, and biochemical traits in Arabidopsis . Given the multifaceted role of ERF transcription factors in plant growth, development, and stress adaptation, we used CRISPR-Cas9-mediated genome editing and gene overexpression to investigate its regulatory role in root architecture, reproductive development, and secondary metabolite biosynthesis, focusing on anthocyanin and auxin (IAA) biosynthesis. Through transgenic Arabidopsis line analysis, we explored the molecular mechanism by which AtERF103 modulates plant growth and adaptation, providing valuable insights into its potential applications in crop improvement. Materials and Methods Generation of AtERF013 Overexpressing Arabidopsis Plants To generate Arabidopsis thaliana lines overexpressing AtERF013 , the full-length AtERF013 coding sequence (accession number PV929047) was cloned into the pCR8/GW/TOPO vector, which confers spectinomycin resistance and transformed into E. coli DH5α cells via heat shock. Positive clones were confirmed via colony PCR and sequencing. The verified entry clone was then recombined with the binary vector pEarlyGate 103 (kanamycin resistance, CaMV 35S promoter) using an LR recombination reaction, ensuring strong constative expression. All genetic constructs were validated by commercial sequencing before the Arabidopsis transformation. The final constructs, with AtERF013 under the control of the 35S promoter, were electroporated into Agrobacterium tumefaciens strain GV3101. Arabidopsis was transformed via the floral dip method [ 36 ]. AtERF103 overexpressors were selected by spraying 1-week-old seedlings with 5 µg/mL Basta. T1 plants resistant to the herbicide were identified and used for subsequent experiments. Generation of Genome-edited Arabidopsis Targeting AtERF013 To generate AtERF013 genome-edited A. thaliana lines, two single guide RNAs (sgRNAs) were designed using the CRISPR RGEN tool and integrated into the pRGEB32 vector, which carries the Cas9 gene, enabling targeted genome editing. The sgRNA sequences were cloned downstream of the U3 promoter within the pRGEB32 vector using the BsaI restriction enzyme site, as previously described [ 37 ]. Successful insertion of the sgRNA constructs was confirmed via sequencing. The verified pRGEB32 construct was then heat shock-transformed into A. tumefaciens strain EHA105 and introduced into Arabidopsis via the floral dip method. T1 plants were screened through genotypic analysis to confirm genome editing and used for subsequent experiments. The genome editing workflow is illustrated in Figure S6. Genotyping and Sequence Analysis of Edited Lines Genomic DNA was extracted from edited Arabidopsis lines using the DNeasy Plant Mini Kit (QIAGEN, Cat. 69104, Hilden, Germany), following the instruction manual. T-DNA insertion was confirmed by amplifying the hygromycin resistance gene fragment using the Hyro-F/Hygro-R primer pair. PCR amplification was performed using high-fidelity Pfu polymerase and the Genetbio PCR Master Mix kit (Yuseong-gu, Daejeon, South Korea), following the manufacturer’s instructions. To analyze genome-edited lines, the full-length CDS region of the gene was amplified and sequenced using gene-specific primers. The target sequence was aligned with the NCBI reference sequence using SnapGene software. RNA Extraction, cDNA Synthesis and qPCR Analysis Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) following the manual instructions. cDNA library was synthesized from 2 µg of total RNA using the qPCR-Bio cDNA Synthesis Kit (PCRBIOSYSTEM, Seoul, South Korea), according to the manual instructions. qPCR was performed using the cDNA template and the StepOne™ Plus RT-PCR system (Thermo Fisher Scientific, Seoul, Korea). The reaction was performed with 2x Real-Time PCR Master Mix (including SYBR Green I: BIOFACT, Daejeon, Korea). Actin was used as the reference gene, and data were analyzed via the 2 –∆∆Ct method. Indole-3-acetic acid Quantification Analysis To quantify indole-3-acetic acid (IAA), 100 mg of roots, shoots, and flowers were ground in liquid nitrogen to fine powder. IAA was extracted twice using 900 µL of extraction buffer (methanol:H₂O:acetonitrile, 90:10:1, v/v) and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Xevo TQ-S micro system (Waters, Milford, MA, USA), with a stable isotope-labeled IAA as an internal standard, as previously described [ 38 ]. Detailed analytical conditions are provided in Supplementary File 1. Extraction and Quantification of Anthocyanin Anthocyanin was extracted using an extraction buffer composed of methanol, water, formic acid, and trifluoroacetic acid in a ratio of 70:27:2:1 (v/v) as previously described [ 39 ]. Approximately 100 mg of samples collected during the flowering stage from the leaf stalk, leaf lamina, rosette junction, curved node, and normal node were ground to a fine powder in liquid nitrogen. Samples were incubated in 5 mL of extraction buffer at 37°C for 48 h. The crude extract was filtered, dried using a rotary evaporator, and reconstituted in 1 mL of HPLC-grade methanol. After dilution, 2 µL of the sample was mixed with 98 µL of 50% acetonitrile containing 0.1% formic acid and analyzed using a spectrophotometer (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland) in a 96-well plate. Absorbance was measured at A535–A650. Statistical Analyses GraphPad Prism 8 and Microsoft Office Excel (2016) were used for statistical analysis. A one-way analysis of variance was conducted to analyze the dataset, incorporating three independent biological replicates. Mean differences were assessed using Bonferroni post hoc tests, with statistical significance set at * p < 0.05, ** p < 0.01, and *** p < 0.001. Results Genetic Modulation of ERF013 Alters Root and Shoot Development in Arabidopsis To confirm the successful generation of transgenic Arabidopsis lines, plants overexpressing 35S-AtERF013 were subjected to Basta selection (10 µg/mL) (Figure S1 A). Out of 200 seeds, only 11 seedlings were resistant, confirming transformation. In contrast, genome-edited lines demonstrated higher transformation efficiency, with 27 out of 29 seeds showing successful T-DNA insertion (Figure S1 B). Phenotypic assessments revealed distinct growth patterns between transgenic lines. 35S-AtERF013 overexpression promoted vegetative growth, whereas GE-aterf013 genome-edited plants exhibited significantly reduced growth, underscoring the role of AtERF013 in Arabidopsis development (Figure S1 C). To further investigate its function in root architecture, two genome-edited ( GE-aterf013-1 and GE-aterf013-2 ) and two overexpression ( 35S-AtERF013-1 and 35S-AtERF013-2 ) lines were analyzed alongside wild-type (Col-0) plants. After 10 days of cultivation on agar, primary and lateral root lengths were measured (Figure S2A). Quantitative analysis confirmed that ERF013 overexpression significantly enhanced root growth, while genome editing reduced primary and lateral root lengths (Figure S2B, 2C). Notably, 35S-AtERF013 overexpression substantially increased root length, whereas GE-aterf013 lines exhibited the shortest roots among all tested groups. Furthermore, morphological observations revealed distinct alterations in lateral root development, emphasizing the contrasting effects of ERF013 modulation (Figure S2D). Shoot development analysis showed that 35S-AtERF013 overexpression significantly increased hypocotyl length after 1 week of growth on simple agar media, while genome-edited plants displayed a pronounced reduction in hypocotyl length compared to wild-type plants (Fig. 1 A, B). Additionally, overexpression lines exhibited higher seed germination percentage, root fresh weight, and shoot fresh weight compared to the wild-type, whereas genome-edited lines substantially declined in these metrics (Fig. 1 C–E). These findings highlight the crucial regulatory role of ERF013 in Arabidopsis growth and development, underscoring its impact on root and shoot architecture. ERF013 Regulates Root Hair and Lateral Root Development via IAA Signaling Functional analysis of ERF013 revealed its crucial role in root hair initiation and lateral root formation. ERF013 overexpression ( 35S-atERF013 ) promoted root hair initiation within 4 days of growth, whereas the wild-type (Col-0) and genome-edited ( GE-aterf013 ) lines exhibited no root hair development (Fig. 2 A). After 1 week, root hairs were formed in Col-0 and 35S-atERF013 plants, with the latter also displaying accelerated lateral root development. In contrast, GE-aterf013 plants lacked root hairs (Fig. 2 B). Analysis of lateral root formation revealed that in Col-0 and 35S-atERF013 plants, lateral roots consistently emerged at primary root curvature points, whereas GE-aterf013 plants lacked lateral roots at any curvature (Fig. 2 C). Notably, lateral root emerged significantly faster in 35S-atERF013 than in Col-0, suggesting that ERF013 enhances IAA accumulation at root curvature sites to promote lateral root formation. Further analysis demonstrated that ERF013 selectively enhances root hair elongation at curvature points, while root hair length above these curvatures remained similar between 35S-atERF013 and Col-0 plants (Fig. 2 F–H). However, GE-aterf013 plants exhibited significantly shorter root hairs at both curvature and non-curvature compared to Col-0. At the last curvature (approximately 5 cm above the root tip), quantitative analysis revealed that 35S-atERF013 plants had significantly longer root hairs than Col-0, while GE-aterf013 plants displayed a substantial reduction (Fig. 2 F–H). These findings indicate the dual role of ERF013 in root architecture, promoting both lateral root initiation and root hair elongation. To determine whether ERF013 directly regulates IAA-dependent primary root development, we treated GE-aterf013 plants with IAA and 35S-atERF013 plants with yucasin (an IAA biosynthesis inhibitor) and compared their responses to Col-0 (Fig. 3 A). IAA treatment significantly promoted primary root elongation in GE- aterf013 plants, whereas yucasin treatment inhibited primary root growth in 35S-atERF013 plants (Fig. 3 A, B). A similar trend was observed for lateral root length (Fig. 3 C), whereas lateral root number remained unchanged (Fig. 3 D). These findings confirm ERF013 as a key regulator of IAA-mediated root development. In non-curved primary roots (5 cm above the root tip), 35S-atERF013 plants exhibited a significant increase in root hair number and length compared to Col-0, whereas GE-aterf013 plants displayed a pronounced reduction (Fig. 3 F–I). Overall, ERF013 modulates IAA-mediated root architecture by promoting lateral root emergence at curvature sites and root hair elongation. ERF013 Regulates Root Growth and Development by Modulating Auxin Biosynthesis To elucidate the role of ERF013 in root growth and development, we quantified auxin (IAA) levels in seedlings, roots, and flowers and assessed ERF013 expression in 35S-atERF013 overexpression and GE-aterf013 genome-edited lines (Fig. 4 ). IAA levels in shoots were significantly reduced by 44% and 50% in GE-aterf013-1 and GE-aterf013-2 , respectively, compared to Col-0, but significantly increased by 131% and 128% in 35S-atERF013-1 and 35S-atERF013-2 , respectively (Fig. 4 A). Peak IAA areas for all samples are shown in Figure S3. A similar trend was observed in roots, where genome-edited lines had significantly lower IAA levels, while overexpression lines showed a marked increase (Fig. 4 B). In flowers, IAA levels decreased by 80% and 65% in GE-aterf013-1 and GE-aterf013-2 , respectively, but significantly increased by 108% and 105% in 35S-atERF013-1 and 35S-atERF013-2 , respectively, compared to Col-0 (Fig. 4 C). ERF013 expression correlated with IAA biosynthesis in shoots, roots, and flowers. In GE-aterf013 plants, ERF013 expression was significantly downregulated in shoots, roots, and flowers but significantly upregulated in 35S-atERF013 plants across all three tissues compared to Col-0 (Fig. 4 D–F). These findings suggest that ERF013 promotes shoot, root, and flower development by enhancing endogenous IAA biosynthesis. ERF013 Regulates Flowering Time, Silique Morphology, and Leaf Architecture in Arabidopsis To elucidate the role of ERF013 in regulating agronomic traits in Arabidopsis , we compared genome-edited and overexpression lines. ERF013 overexpression induced early flowering, whereas genome-edited lines exhibited delayed flowering compared to Col-0 plants (Fig. 5 A). At early developmental stages, 35S-AtERF013 plants demonstrated accelerated stem elongation, while GE-aterf013 plants exhibited inhibited stem development. Additionally, inflorescence emerged earlier in 35S-AtERF013 plants but was delayed in GE-aterf013 lines compared to Col-0 (Fig. 5 D). Furthermore, ERF013 overexpression significantly increased silique length, leaf length, leaf width, and total leaf area, whereas genome-edited lines exhibited a substantial reduction in these traits (Fig. 5 ). Specifically, silique length increased by 38% and 33% in 35S-AtERF013-1 and 35S-AtERF013-2 lines, respectively, compared to Col-0, whereas GE-aterf013-1 and GE-aterf013-2 plants exhibited a 32% and 27% reduction, respectively (Fig. 5 F). Similarly, leaf length increased by 71% and 84% in 35S-AtERF013-1 and 35S-AtERF013-2 plants, but decreased by 18% and 17% in GE-aterf013-1 and GE-aterf013-2 plants, respectively, compared to Col-0 (Fig. 5 G). Total leaf area increased by 82% and 85% in 35S-AtERF013-1 and 35S-AtERF013-2 plants but decreased by 11% and 8% in GE-aterf013-1 and GE-aterf013-2 plants, respectively, compared to Col-0 plants (Fig. 5 I). Further analysis revealed that ERF013 influences silique angle. Wild-type Col-0 plants exhibited silique angles of 7–13°, genome-edited lines displayed significantly wider silique angles of 64°–77°, while overexpression lines showed silique angles between 34° and 50° (Fig. 5 E). Figure S4 demonstrates that ERF013 is involved in trichome development in Arabidopsis. ERF013 overexpression increased trichome density on leaf margins, petioles, and adaxial leaf surfaces, whereas genome-edited lines had significantly fewer trichomes than Col-0. ERF013 also modulates leaf margin morphology and internode length (Figure S5). Overexpression lines exhibited elongated internodes, whereas genome-edited lines displayed significantly shorter internodes compared to Col-0 (Figure S5B). To assess the effect of ERF013 on leaf morphology, the fifth leaf of each plant group was analyzed (Figure S5A). Col-0 plants exhibited serrated leaves, while overexpression and genome-edited plants developed lobed leaves with distinct morphological differences. GE-aterf013 plants had rounded and shallow lobes, resulting in a smooth, undulating appearance with minimal serrations. In contrast, 35S-AtERF013 plants developed deeply incised, pointed lobes, giving the leaves a more angular and pronounced appearance with serrated or toothed margins. Furthermore, their lobes were irregularly distributed, resulting in a complex and asymmetrical leaf shape. These results indicate that ERF013 plays a pivotal role in shaping Arabidopsis leaf morphology and plant architecture. ERF013 Positively Regulates Anthocyanin Biosynthesis in Arabidopsis thaliana To elucidate the role of ERF013 in anthocyanin biosynthesis, we quantified anthocyanin accumulation and analyzed key biosynthetic gene expression in Col-0, GE-aterf013 , and 35S-AtERF013 plants (Fig. 6 ). Spatiotemporal analysis of anthocyanin deposition across different tissues revealed a significant increase in 35S-AtERF013 plants and a marked reduction in GE-aterf013 plants compared to Col-0 (Fig. 6 A, B). Specifically, anthocyanin levels were highest at the curved node, followed by the normal node and leaf lamina in 35S-AtERF013 plants (Fig. 6 C–G). To further investigate the molecular basis of this phenotype, we analyzed PAL , C4H , CHS , F3H , and DFR expression, key genes involved in anthocyanin biosynthesis (Fig. 6 H–L). The transcriptional profiles aligned with anthocyanin accumulation, with significant upregulation in 35S-AtERF013 plants and downregulation in GE-aterf013 plants relative to Col-0. Among these genes, DFR exhibited the highest induction, increasing 7-fold and 6-fold in 35S-AtERF013-1 and 35S-AtERF013-2 , respectively. Similarly, F3H expression increased 6.5-fold and 6-fold, while CHS was upregulated 6.5-fold and 5.5-fold in 35S-AtERF013-1 and 35S-AtERF013-2 , respectively. These findings show that ERF013 positively regulates anthocyanin biosynthesis, likely by enhancing biosynthetic gene transcription. The substantial increase in anthocyanin accumulation in 35S-AtERF013 plants underscores the key role of ERF013 in modulating anthocyanin production in Arabidopsis. ERF013 Regulates Pollen, Seed, and Stigma Development in Arabidopsis To elucidate the role of ERF013 in seed development and fertilization, we analyzed pollen grain development, seed set, and stigma morphology in Col-0, genome-edited, and overexpressor Arabidopsis lines (Fig. 7 ). Anther visualization revealed significantly reduced pollen production in GE-aterf013 plants and higher pollen density in 35S-atERF013 plants compared to Col-0 (Fig. 7 A). These findings suggest that ERF013 is essential for normal pollen development. Analysis of seed development showed that GE-aterf013 plants had a higher proportion of infertile or underdeveloped seeds, whereas 35S-atERF013 plants showed increased seed formation compared to Col-0 (Fig. 7 B, C). Non-viable seed rates increased by 94% and 132% in GE-aterf013-1 and GE-aterf013-2 but they were significantly reduced by 71% and 61% in 35S-atERF013-1 and 35S-atERF013-2 , respectively, compared to Col-0 (Fig. 7 D). Similarly, the total seeds per silique increased by 111% and 72% in 35S-atERF013-1 and 35S-atERF013-2 and decreased by 77% and 50% in GE-aterf013-1 and GE-aterf013-2 , respectively, compared to Col-0 (Fig. 7 E). Given the essential role of the stigma in fertilization and seed development, we examined its morphology across the three plant groups (Fig. 7 F). Notably, GE-aterf013 plants had poorly developed stigmas, whereas both Col-0 and 35S-atERF013 plants showed normal stigma formation. These findings indicate that ERF013 is required for proper stigma development in Arabidopsis. Discussion ERF013 belongs to the AP2/ERF transcription factor (TF) family, one of the largest plant-specific TF families [ 2 ]. In this study, AtERF013 was identified as a key regulator of Arabidopsis growth and development, particularly root development via IAA signaling. This study primarily focused on its role in regulating phenotypic traits in genome-edited and overexpression lines. The 35S-AtERF013 overexpression line promoted growth, whereas the GE-aterf013 genome-edited line exhibited inhibited growth compared to Col-0 plants under normal conditions. The observed increase in plant biomass resulted from enhanced root and shoot growth driven by ERF013 overexpression, whereas impaired development in genome-edited lines stemmed from the loss of ERF013 function. AAnalyses of primary roots, lateral roots, and root hairs at different time points confirmed that ERF013 plays a crucial role in promoting root development. ERF109 , a related TF, facilitates lateral root formation by linking jasmonic acid signaling with auxin biosynthesis by directly regulating ASA1 and YUC2 (key enzymes involved in auxin biosynthesis) [ 18 ]. Additionally, ERFVII TFs act as negative regulators of lateral root development, suppressing auxin-responsive genes such as LBD16 , LBD18 , and PUCHI [ 19 ]. In contrast, ERF109 and PUCHI promote lateral root development. OsERF48 regulates root growth by inducing calmodulin-like protein expression [ 20 ]. In this study, ERF013 expression significantly induced auxin biosynthesis in seedlings, primary roots, and lateral roots, while its loss in genome-edited lines led to reduced auxin biosynthesis. Moreover, ERF013 transcription was upregulated in overexpression lines and downregulated in genome-edited lines compared to control plants. Notably, exogenous auxin restored primary and lateral root development in genome-edited lines, whereas yucasin, an auxin biosynthesis inhibitor, reduced primary and lateral root length in overexpression lines. These findings suggest that ERF013 is crucial for primary and lateral root development in Arabidopsis. 35S-AtERF013 overexpression also increased root hair number and length, consistent with a recent study that reported similar results with ERF019 overexpression lines [ 18 ]. Furthermore, MPK14-mediated auxin signaling regulates lateral root development by stabilizing the ERF13 protein [ 21 ]. While this study did not directly assess the expression of auxin biosynthesis and auxin-responsive genes ( ASA1 , YUC2 , LBD , and PUCHI ), previous reports suggest that ERF013 may significantly influence root development by regulating these genes. This study provides novel insights into the regulatory role of AtERF103 in modulating flower and leaf morphology, silique size, fertility, and silique angle. AtERF103 overexpression induced early flowering, increased flower number, enhanced silique length and seed fertility, and promoted leaf elongation and expansion. In contrast, genome-edited AtERF103 lines exhibited delayed flowering, reduced flower and silique size, decreased leaf length and area, and a greater silique angle. These findings highlight the key role of AtERF103 in plant growth and reproduction. Auxin is well known for regulating plant development, particularly floral organ size, as demonstrated by the reduced flower organ size observed in ARF6/8 mutants, emphasizing its role in floral development [ 22 – 24 ]. A recent study reported that SIERF.D7 positively regulates SIARF2A/B expression, integrating auxin and ethylene signaling to modulate tomato fruit ripening [ 25 ]. Similarly, Li et al. revealed that ERF19 regulates flower number and size by activating WUS for primordia formation and SAUR33 for cell division and expansion, leading to larger floral organs in AtERF19 -overexpressing plants [ 26 ]. Conversely, the 35s::AtERF19 + SRDX mutant exhibited fewer and smaller flowers, suggesting that AtERF19 modulates floral traits through auxin-related gene regulation. Our study reveals new regulatory mechanisms governing auxin homeostasis, demonstrating that AtERF103 promotes auxin (IAA) accumulation in seedlings, roots, and flowers (Fig. 4 ). Unlike AtERF19 , which modulates floral traits via auxin-responsive genes, AtERF103 broadly regulates plant growth. Furthermore, auxin induction via AtERF103 expression is crucial for silique growth, pollen development, stigma formation, and seed development (Fig. 5 B, 6 ). These results align with previous studies demonstrating the Auxin Activation Factor enhances auxin responses, increasing silique length, flower petal expansion, seed size, and anther indehiscence [ 27 ]. The ARF6/8 double mutant further underscores the role of auxin in floral organ development, exhibiting reduced petal length and impaired stamen formation in Arabidopsis [ 22 ]. These findings highlight the intricate regulatory network governing auxin-mediated reproductive organ growth. Beyond floral and reproductive development, auxin stimulation plays a crucial role in Arabidopsis leaf morphology. This is evident in YUCCA overexpression lines, which exhibit a long, narrow, and downward-curled leaf phenotype due to elevated auxin levels, as well as in 35s-AtERF109 overexpression plants [ 18 , 28 ]. Similarly, our findings reveal that AtERF103 modulates leaf morphology, suggesting a potential link to auxin-mediated leaf development. The altered phenotypes in AtERF103 -overexpressing and genome-edited lines indicate that AtERF103 is a key regulator of auxin homeostasis, coordinating both vegetative and reproductive development in Arabidopsis. Beyond its role in auxin-mediated growth regulation, AtERF103 also influences anthocyanin biosynthesis, a crucial secondary metabolic pathway involved in plant stress responses and pigmentation. AtERF103 overexpression significantly enhanced anthocyanin accumulation in Arabidopsis leaf lamella, leaf petiole, nodes, and rosette junctions (Fig. 6 ). This observation suggests that AtERF103 functions as a positive regulator of anthocyanin biosynthesis. Conversely, genome-edited ERF103 lines exhibited reduced anthocyanin levels across these tissues, confirming its regulatory role in anthocyanin metabolism. A recent study on ERF012 revealed that ERF012 -overexpressing plants accumulated more anthocyanin, while erf012 mutants and wild-type Col-0 plants had lower anthocyanin levels, suggesting that ERF012 promotes cold adaptability [ 14 ]. Similarly, AtERF103 overexpression enhanced anthocyanin accumulation in Arabidopsis, while genome-edited lines exhibited reduced anthocyanin levels. Furthermore, our analysis of key anthocyanin biosynthetic genes ( PAL , C4H , CHS , F3H , and DFR ) revealed a strong correlation with AtERF103 expression. These genes were significantly upregulated in AtERF103 -overexpressing lines, corresponding with higher anthocyanin levels across various tissues, while their downregulation in genome-edited lines resulted in reduced anthocyanin levels. In pear fruit, ERF24 and ERF96 increased anthocyanin levels by inducing PAL , CHS , CHI , and DFR expression [ 17 ]. Recent studies suggest that AtERF19 regulates plant immunity and environmental stress responses [ 29 – 31 ]. Consistent with these findings, our study demonstrated that AtERF103 enhances anthocyanin accumulation, a known stress-mitigating compound. Although we did not directly evaluate the environmental stress response of AtERF103 overexpression lines, previous studies indicate that anthocyanin overaccumulation improves stress tolerance [ 32 , 33 ]. Anthocyanin accumulation in vacuoles protects against abiotic stress by regulating reactive oxygen species levels through enzymatic and non-enzymatic antioxidant activity [ 34 , 35 ]. Based on this, we propose that AtERF103 enhances stress resilience by regulating anthocyanin biosynthesis, highlighting the functional diversity of ERF TFs in plant adaptation. Thus, these findings provide a basis for future research on the role of AtERF103 in environmental stress resilience, its regulatory networks, and its potential application in improving crop tolerance. Conclusion This study demonstrates that the ATERF013 transcription factor is a key regulator of vegetative and reproductive development, as well as secondary metabolism, in A. thaliana . Overexpression of A tERF013 markedly enhances auxin levels, root and shoot growth, leaf size, early flowering, silique development, seed production, and anthocyanin biosynthesis, highlighting its positive role in promoting growth and yield related traits. Conversely, loss of its function impairs these processes, leading to reduced growth, delayed flowering, reduced fertility, and compromised seed viability. These findings provide valuable insights into the multifaceted role of AtERF013 in plant development and metabolism, offering a target for genetic improvement of agronomic traits in A. thaliana and potentially in crop species. Declarations Acknowledgements This work was supported by the Cooperative Research Program for Agriculture Science and Technology Development (Grant No. RS-2024-00348677), Rural Development Administration, Republic of Korea. The authors extend their appreciation to Northern Border University, Saudi Arabia, for supporting this work through project number (NBU-CRP-2025-249). Author Contributions Rahmatullah Jan, Sajjad Asaf, Lubna, and Eman R. Elsharkawy: designed the study. Rahmatullah Jan, Saleem Asif, and Zakirullah Khan: performed most of the experiments and analyzed data. Muhammad Farooq and Zakirullah Khan: performed IAA and anthocyanin analysis. Rahmatullah Jan, Sajjad Asaf, and Kyung-Min Kim: wrote the manuscript. Kyung-Min Kim: provided resources and funding acquisition. Funding This research work has not received any formal funding. Data Availability The RNA sequence has been deposited in the National Center for Biotechnology Information (NCBI) under accession number PV929047 (available at: link will be provided soon). All data generated or analyzed during this study are included in this article and its supplementary information file. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing Interest The authors declare no conflict of interest associated with this article. References Riechmann JL, Meyerowitz EM: The AP2/EREBP family of plant transcription factors . Biological chemistry 1998, 379 :633-646. Nakano T, Suzuki K, Fujimura T, Shinshi H: Genome-wide analysis of the ERF gene family in Arabidopsis and rice . Plant physiology 2006, 140 (2):411-432. 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Ni J, Bai S, Zhao Y, Qian M, Tao R, Yin L, Gao L, Teng Y: Ethylene response factors Pp4ERF24 and Pp12ERF96 regulate blue light-induced anthocyanin biosynthesis in ‘Red Zaosu’pear fruits by interacting with MYB114 . Plant Molecular Biology 2019, 99 :67-78. Cai X-T, Xu P, Zhao P-X, Liu R, Yu L-H, Xiang C-B: Arabidopsis ERF109 mediates cross-talk between jasmonic acid and auxin biosynthesis during lateral root formation . Nature Communications 2014, 5 (1):5833. Shukla V, Lombardi L, Iacopino S, Pencik A, Novak O, Perata P, Giuntoli B, Licausi F: Endogenous hypoxia in lateral root primordia controls root architecture by antagonizing auxin signaling in Arabidopsis . Molecular Plant 2019, 12 (4):538-551. Jung H, Chung PJ, Park SH, Redillas MCFR, Kim YS, Suh JW, Kim JK: Overexpression of Os ERF 48 causes regulation of Os CML 16, a calmodulin‐like protein gene that enhances root growth and drought tolerance . Plant Biotechnology Journal 2017, 15 (10):1295-1308. Lv B, Wei K, Hu K, Tian T, Zhang F, Yu Z, Zhang D, Su Y, Sang Y, Zhang X: MPK14-mediated auxin signaling controls lateral root development via ERF13-regulated very-long-chain fatty acid biosynthesis . Molecular Plant 2021, 14 (2):285-297. Nagpal P, Ellis CM, Weber H, Ploense SE, Barkawi LS, Guilfoyle TJ, Hagen G, Alonso JM, Cohen JD, Farmer EE: Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation . 2005. Reeves PH, Ellis CM, Ploense SE, Wu M-F, Yadav V, Tholl D, Chételat A, Haupt I, Kennerley BJ, Hodgens C: A regulatory network for coordinated flower maturation . PLoS genetics 2012, 8 (2):e1002506. Tabata R, Ikezaki M, Fujibe T, Aida M, Tian C-e, Ueno Y, Yamamoto KT, Machida Y, Nakamura K, Ishiguro S: Arabidopsis auxin response factor6 and 8 regulate jasmonic acid biosynthesis and floral organ development via repression of class 1 KNOX genes . Plant and Cell Physiology 2010, 51 (1):164-175. Gambhir P, Singh V, Parida A, Raghuvanshi U, Kumar R, Sharma AK: Ethylene response factor ERF. D7 activates auxin response factor 2 paralogs to regulate tomato fruit ripening . Plant Physiology 2022, 190 (4):2775-2796. Li PF, Zhan YX, Wang JC, Cheng YH, Hsu WH, Hsu HF, Chen WH, Yang CH: The AtERF19 gene regulates meristem activity and flower organ size in plants . The Plant Journal 2023, 114 (6):1338-1352. Chen WH, Hsu WH, Hsu HF, Yang CH: A tetraspanin gene regulating auxin response and affecting orchid perianth size and various plant developmental processes . Plant Direct 2019, 3 (8):e00157. Zhao Y, Christensen SK, Fankhauser C, Cashman JR, Cohen JD, Weigel D, Chory J: A role for flavin monooxygenase-like enzymes in auxin biosynthesis . Science 2001, 291 (5502):306-309. Huang P-Y, Zhang J, Jiang B, Chan C, Yu J-H, Lu Y-P, Chung K, Zimmerli L: NINJA-associated ERF19 negatively regulates Arabidopsis pattern-triggered immunity . Journal of experimental botany 2019, 70 (3):1033-1047. Lu W, Deng F, Jia J, Chen X, Li J, Wen Q, Li T, Meng Y, Shan W: The Arabidopsis thaliana gene AtERF019 negatively regulates plant resistance to Phytophthora parasitica by suppressing PAMP‐triggered immunity . Molecular Plant Pathology 2020, 21 (9):1179-1193. Scarpeci TE, Frea VS, Zanor MI, Valle EM: Overexpression of AtERF019 delays plant growth and senescence, and improves drought tolerance in Arabidopsis . Journal of experimental botany 2017, 68 (3):673-685. Naing AH, Ai TN, Lim KB, Lee IJ, Kim CK: Overexpression of Rosea1 from snapdragon enhances anthocyanin accumulation and abiotic stress tolerance in transgenic tobacco . Frontiers in Plant Science 2018, 9 :1070. Meng X, Yin B, Feng H-L, Zhang S, Liang X-Q, Meng Q-W: Overexpression of R2R3-MYB gene leads to accumulation of anthocyanin and enhanced resistance to chilling and oxidative stress . Biologia Plantarum 2014, 58 (1):121-130. Gill SS, Tuteja N: Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants . Plant physiology and biochemistry 2010, 48 (12):909-930. Hernández I, Alegre L, Van Breusegem F, Munné-Bosch S: How relevant are flavonoids as antioxidants in plants? Trends in plant science 2009, 14 (3):125-132. Clough SJ, Bent AF: Floral dip: a simplified method for Agrobacterium‐mediated transformation of Arabidopsis thaliana . The plant journal 1998, 16 (6):735-743. Park J-R, Kim E-G, Jang Y-H, Jan R, Farooq M, Ubaidillah M, Kim K-M: Applications of CRISPR/Cas9 as new strategies for short breeding to drought gene in rice . Frontiers in Plant Science 2022, 13 :850441. Liu H, Li X, Xiao J, Wang S: A convenient method for simultaneous quantification of multiple phytohormones and metabolites: application in study of rice-bacterium interaction . Plant methods 2012, 8 :1-12. Jan R, Khan MA, Asaf S, Lee I-J, Kim K-M: Modulation of sugar and nitrogen in callus induction media alter PAL pathway, SA and biomass accumulation in rice callus . Plant Cell, Tissue and Organ Culture (PCTOC) 2020, 143 :517-530. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigures.docx Supplementary File 1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7070846","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485904849,"identity":"260a6d9c-27b2-4fed-8259-1128bb99c536","order_by":0,"name":"Rahmatullah Jan","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Rahmatullah","middleName":"","lastName":"Jan","suffix":""},{"id":485904850,"identity":"acf140dc-d85c-4134-9f2e-c150190e8c52","order_by":1,"name":"Lubna .","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Lubna","middleName":"","lastName":".","suffix":""},{"id":485904851,"identity":"f08e8076-106f-429c-b524-8142ccd8444c","order_by":2,"name":"Saleem Asif","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Saleem","middleName":"","lastName":"Asif","suffix":""},{"id":485904852,"identity":"0a377170-4a6f-4571-8139-c88351fafb91","order_by":3,"name":"Zakirullah Khan","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Zakirullah","middleName":"","lastName":"Khan","suffix":""},{"id":485904853,"identity":"0507e8e7-6c76-41e1-8aca-c2c72734de6f","order_by":4,"name":"Muhammad Farooq","email":"","orcid":"","institution":"Jeon-buk National University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Farooq","suffix":""},{"id":485904854,"identity":"af7039fd-e4e3-4ca9-94a1-5828c6886a82","order_by":5,"name":"Eman R. Elsharkawy","email":"","orcid":"","institution":"Northern Border University-ARAR","correspondingAuthor":false,"prefix":"","firstName":"Eman","middleName":"R.","lastName":"Elsharkawy","suffix":""},{"id":485904855,"identity":"799422d7-3f82-41c4-9053-bf45c861a800","order_by":6,"name":"Sajjad Asaf","email":"","orcid":"","institution":"University of Nizwa","correspondingAuthor":false,"prefix":"","firstName":"Sajjad","middleName":"","lastName":"Asaf","suffix":""},{"id":485904856,"identity":"5e9e0a08-9973-495f-a308-d0c24b21575c","order_by":7,"name":"Kyung-Min Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoUlEQVRIiWNgGAWjYBACCR4GxocNEDYz0VqYDUnWwiZJmhbJntNplTN3HGbgbz/AbFxBjBZp3t5tNzeeOcwgcSaBOfEMMVrk+Hm33XzYdpiB4QYD88EGYrUUgrTIE60F5DDGjUAtBkAtiURpkew5u1lyZls6j+GZxGZYaOMHEmdyN37sbbOWkzt++DAstIkDPAwMjCRpGAWjYBSMglGADwAAlzgwAwJet3sAAAAASUVORK5CYII=","orcid":"","institution":"Kyungpook National University","correspondingAuthor":true,"prefix":"","firstName":"Kyung-Min","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-07-08 05:38:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7070846/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7070846/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86938517,"identity":"8041f929-8b14-48d2-bdb0-d3c35378dd44","added_by":"auto","created_at":"2025-07-17 11:23:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":279349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtERF013\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e regulates plant growth and development.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of Arabidopsis thaliana seedlings illustrating root and shoot length evaluation. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of hypocotyl length. \u003cstrong\u003e(C–E)\u003c/strong\u003e Graphs depicting germination percentage \u003cstrong\u003e(C)\u003c/strong\u003e, root fresh weight \u003cstrong\u003e(D)\u003c/strong\u003e, and shoot fresh weight \u003cstrong\u003e(E)\u003c/strong\u003e. Seedlings were grown on agar under normal conditions. Overall, 50 seedlings were analyzed per parameter, with triplicate measurements. Data are presented as mean ± standard deviation (SD). Statistical significance was determined using multiple post hoc tests, with significance levels denoted as \u003cem\u003e*P \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, and \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7070846/v1/52815bb5a7212f3f4d20f1ec.png"},{"id":86938519,"identity":"216f9844-541c-4dd7-afce-f2ab2cced25b","added_by":"auto","created_at":"2025-07-17 11:23:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":594955,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtERF013\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e modulates root and root hair development in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Representative images showing root and root hair development in Col-0, \u003cem\u003eGE-aterf103\u003c/em\u003e, and \u003cem\u003e35S-atERF103 \u003c/em\u003eseedlings after 4 days of growth on agar under standard conditions. \u003cstrong\u003e(B) \u003c/strong\u003eRoot hair development in Col-0, \u003cem\u003eGE-aterf103\u003c/em\u003e, and \u003cem\u003e35S atERF103 \u003c/em\u003eseedlings after 1 week of growth under standard conditions. Red lines indicate root hairs. \u003cstrong\u003e(C)\u003c/strong\u003e Lateral root initiation at the primary root curvature in Col-0, \u003cem\u003eGE-aterf103\u003c/em\u003e, and \u003cem\u003e35S-atERF103\u003c/em\u003e seedlings after 1 week of growth. Red arrows denote lateral root initiation. \u003cstrong\u003e(D)\u003c/strong\u003e Cross-section of a \u003cem\u003e35S-atERF103\u003c/em\u003e root illustrating lateral root initiation. \u003cem\u003e(E)\u003c/em\u003eMicroscopic image of root hair emergence from a single epidermal cell in \u003cem\u003e35S-atERF103 \u003c/em\u003eseedlings after 1 week. Red arrows indicate lateral root formation sites \u003cstrong\u003e(D) \u003c/strong\u003eand root hair development \u003cstrong\u003e(E)\u003c/strong\u003e. \u003cstrong\u003e(F, G)\u003c/strong\u003e Root hair length at the final root curvature and above the last curvature in Col-0, \u003cem\u003eGE-aterf103\u003c/em\u003e, and \u003cem\u003e35S-atERF103\u003c/em\u003e seedlings. \u003cstrong\u003e(F)\u003c/strong\u003e Original root hair images and \u003cstrong\u003e(G) \u003c/strong\u003eImageJ-processed images, with yellow lines indicating measured root hairs. Measurements were taken after 1 week of growth under standard conditions. \u003cstrong\u003e(H, I)\u003c/strong\u003eQuantification of root hair length above the curvature \u003cstrong\u003e(H)\u003c/strong\u003e and at the final root curvature \u003cstrong\u003e(I)\u003c/strong\u003e. \u003cstrong\u003e(J)\u003c/strong\u003e Schematic of seedling regions used for root hair length measurements. \u003cstrong\u003e(K)\u003c/strong\u003e Cross-sections of roots showing lateral root development after 1 week of growth. \u003cem\u003e35S-atERF103\u003c/em\u003e seedlings exhibited well-developed lateral roots, while Col-0 plants showed lateral root initiation. In contrast, \u003cem\u003eGE-aterf103\u003c/em\u003e plants failed to develop lateral roots after 1 week. Data in \u003cem\u003e(H)\u003c/em\u003e and \u003cem\u003e(I)\u003c/em\u003e are presented as mean ± standard deviation (SD). Statistical significance was determined using multiple post hoc tests, with significance levels denoted as follows: \u003cem\u003e*P \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, and \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7070846/v1/8d03031ef425a1102b86fcde.png"},{"id":86937299,"identity":"cc2c6a45-bdca-43bb-abbb-65032295f4d9","added_by":"auto","created_at":"2025-07-17 11:07:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":458413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtERF013\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Modulates Root Development via IAA-Mediated Signaling\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003eA. thaliana\u003c/em\u003e root architecture in response to exogenous IAA and yucasin treatments. \u003cem\u003e35s-atERF103\u003c/em\u003e seedlings were cultivated on agar supplemented with 30 µM yucasin, \u003cem\u003eGE-aterf103 \u003c/em\u003eseedlings on 10 mM IAA-supplemented agar medium, and Col-0 seedlings (wild-type control) on standard agar. All seedlings were maintained under identical growth conditions for 1 week. \u003cstrong\u003e(B–D)\u003c/strong\u003e Morphometric analysis of primary root length \u003cstrong\u003e(B)\u003c/strong\u003e, lateral root length \u003cstrong\u003e(C)\u003c/strong\u003e, and lateral root number \u003cstrong\u003e(D)\u003c/strong\u003ein seedlings subjected to the indicated treatments. \u003cstrong\u003e(E)\u003c/strong\u003e Representative root images depicting root hair formation in seedlings grown under standard conditions for 1 week. The region analyzed is 5 cm proximal to the root tip. \u003cstrong\u003e(F)\u003c/strong\u003eRoot hair length visualized using yellow line demarcations. \u003cstrong\u003e(G)\u003c/strong\u003e Root hair density annotated with yellow dots. \u003cstrong\u003e(H, I)\u003c/strong\u003e Quantification of root hair frequency \u003cstrong\u003e(H)\u003c/strong\u003e and root hair length \u003cstrong\u003e(I)\u003c/strong\u003e. Data are represented as mean ± standard deviation (SD). Statistical significance was evaluated using multiple post hoc tests, with significance thresholds denoted as \u003cem\u003e*P \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, and \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7070846/v1/e61c58267e271f6311ed3f23.png"},{"id":86938037,"identity":"b2eef2c5-2c83-4de7-8d90-5d1b60bd2900","added_by":"auto","created_at":"2025-07-17 11:15:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":251065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndogenous levels of IAA and expression profile of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtERF013 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene in various tissues of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A–C)\u003c/strong\u003e IAA concentrations in shoots \u003cstrong\u003e(A)\u003c/strong\u003e, roots \u003cstrong\u003e(B)\u003c/strong\u003e, and flowers \u003cstrong\u003e(C)\u003c/strong\u003e. \u003cstrong\u003e(D–F)\u003c/strong\u003e \u003cem\u003eAtERF013 \u003c/em\u003eexpression levels in shoots \u003cstrong\u003e(D)\u003c/strong\u003e, roots \u003cstrong\u003e(E)\u003c/strong\u003e, and flowers \u003cstrong\u003e(F)\u003c/strong\u003e. Data are presented as mean ± standard deviation (SD). Statistical significance was determined through multiple post hoc comparisons, with significance levels set at \u003cem\u003e*P \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, and \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7070846/v1/434526b44f71392108810890.png"},{"id":86938035,"identity":"afd46454-d9d4-4d79-a9f7-4103012511a0","added_by":"auto","created_at":"2025-07-17 11:15:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":642653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtERF013\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-mediated phenotypic variations in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Representative images of floral development across different genotypes. Red arrows indicate early flowering in \u003cem\u003e35s-atERF013 \u003c/em\u003eplants, followed by Col-0, whereas \u003cem\u003eGE-aterf013\u003c/em\u003e plants exhibited delayed flowering. \u003cstrong\u003e(B–D)\u003c/strong\u003e Morphological comparison of seeds \u003cstrong\u003e(B)\u003c/strong\u003e, leaves \u003cstrong\u003e(C)\u003c/strong\u003e, and flowers \u003cstrong\u003e(D)\u003c/strong\u003e across different genotypes. \u003cstrong\u003e(E)\u003c/strong\u003e \u003cem\u003eAtERF013\u003c/em\u003e-induced silique angle variations measured using a standard protractor. \u003cstrong\u003e(F–I)\u003c/strong\u003eQuantification of silique length \u003cstrong\u003e(F)\u003c/strong\u003e, leaf length \u003cstrong\u003e(G)\u003c/strong\u003e, leaf width \u003cstrong\u003e(H)\u003c/strong\u003e, and leaf area (I). The fifth leaf was selected from each genotype grown under identical soil conditions and analyzed. Data represent means from 10 biological replicates and are presented as mean ± standard deviation (SD). Statistical significance was determined using multiple post hoc tests, with significance levels denoted as \u003cem\u003e*P \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, and \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7070846/v1/49d310afa9be2021f53c79cd.png"},{"id":86937304,"identity":"a03e7d1a-266e-4afd-a03f-960b2ca2283c","added_by":"auto","created_at":"2025-07-17 11:07:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":610186,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtERF103\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promotes anthocyanin biosynthesis in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Anthocyanin accumulation in various plant tissues. The upper, middle, and lower panels show accumulation at the rosette junction, leaf lamina, and petiole, respectively. \u003cstrong\u003e(B)\u003c/strong\u003e Anthocyanin distribution in \u003cem\u003eA. thaliana\u003c/em\u003e nodes, with the upper and lower panels displaying normal and curved nodes, respectively. \u003cstrong\u003e(C–G)\u003c/strong\u003e Quantification of anthocyanin levels in the leaf lamina, petiole, normal nodes, curved nodes, and rosette junction. \u003cstrong\u003e(H–L)\u003c/strong\u003eExpression of key anthocyanin biosynthetic genes (\u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003eC4H\u003c/em\u003e, \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eF3H\u003c/em\u003e, and \u003cem\u003eDFR\u003c/em\u003e). Curly braces and circles indicate the regions of interest for anthocyanin accumulation. Data are presented as mean ± standard deviation (SD) from multiple biological replicates. Statistical significance was determined using multiple post hoc comparisons, with significance levels indicated as \u003cem\u003e*P \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, and \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7070846/v1/e7fa57ca88851b8d349bd23b.png"},{"id":86937315,"identity":"349e3821-8bdc-4920-bed8-ff8b8fc44d76","added_by":"auto","created_at":"2025-07-17 11:07:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":577869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtERF103 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePromotes Pollen, Seed, and Stigma Development in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. Thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Pollen grain development within the anther collected 1 day before anthesis. \u003cstrong\u003e(B)\u003c/strong\u003e Mature seed development across genotypes. \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of total seed number per silique for each genotype. \u003cstrong\u003e(D)\u003c/strong\u003e The proportion of non-viable (non-fertile) seeds per silique in each genotype. \u003cstrong\u003e(E)\u003c/strong\u003e Comparative analysis of total seed production per silique. \u003cstrong\u003e(F)\u003c/strong\u003e Stigma morphology and development a across genotypes, evaluated on the day of anthesis. Anther and stigma structures were imaged using an inverted microscope. Data are presented as mean ± standard deviation (SD) from multiple biological replicates. Statistical significance was determined using post hoc multiple comparisons, with significance levels denoted as \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e, \u003cem\u003e***P \u0026lt; 0.001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7070846/v1/e05edac4566c9ac9f59e684d.png"},{"id":92565273,"identity":"24187edf-0181-4528-94fb-0d59705e02db","added_by":"auto","created_at":"2025-10-01 06:10:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6309223,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7070846/v1/39b42c6e-3016-4b51-9c00-4f674e95d6a7.pdf"},{"id":86938034,"identity":"cf807216-ceea-4532-b6d4-887d11ed9bf2","added_by":"auto","created_at":"2025-07-17 11:15:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2123319,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary File 1\u003c/p\u003e","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7070846/v1/a309d8088c906c26bcf46919.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eUnlocking the Functional Dynamics of \u003cem\u003eERF103\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e: A key player in Plant Growth Regulation\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTranscription factors are key regulators of gene expression, controlling key developmental processes throughout the plant life cycle, including seed germination, seedling growth, tissue morphogenesis, reproduction, and senescence. The APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily members share a common DNA-binding AP2 domain. This transcription factor family is grouped into four subfamilies based on domain differences: AP2, ERF, DEHYDRATION RESPONSE ELEMENT-BINDING (\u003cem\u003eDREB\u003c/em\u003e), and RELATED TO ABSCISIC ACID-INSENSITIVE 3/Viviparous (\u003cem\u003eRAV\u003c/em\u003e) [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Genome-wide analysis of the ERF gene family in \u003cem\u003eArabidopsis\u003c/em\u003e showed that the AP2/ERF superfamily comprises 147 members, accounting for 122 genes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. AP2/ERF transcription factors bind to specific elements such as GCC-box (GCCGCC), G-Box (CACGTG), and dehydration-responsive element/C-repeat (DRE/CRT, GCCCAC) to regulate target gene expression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. ERFs recognize and attach to diverse cis-elements within target gene promoters, contributing to various regulatory processes by modulating gene expression.\u003c/p\u003e\u003cp\u003eThese transcription factors regulate metabolism, growth, development, response to environmental constraints [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], flowering time [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and seed development and yield [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] throughout the plant life cycle. They also respond to signals from auxin, cytokinins, abscisic acid, and jasmonic acid, modulating phytohormones biosynthesis and influencing agronomic traits such as plant growth, defense responses, and fruit ripening [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] compiled data highlighting the diverse roles of ERF transcription factors in plant growth, development, and stress responses, including somatic embryogenesis, root growth, shoot elongation, fruit ripening, secondary metabolism, and resilience to environmental stresses such as submergence, heavy metals, drought, high salinity, and cold. Furthermore, ERFs such as \u003cem\u003ePyERF3\u003c/em\u003e in Chinese pear modulate flavonoid biosynthesis by co-regulating transcription factors, forming complexes such as \u003cem\u003eERF3-MYB114-bHLH3\u003c/em\u003e, which is involved in anthocyanin biosynthesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similarly, \u003cem\u003ePp4ERF24\u003c/em\u003e and \u003cem\u003ePpERF96\u003c/em\u003e in Red Zaosu enhance anthocyanin biosynthesis through the interaction of \u003cem\u003ePpbHLH3\u003c/em\u003e with \u003cem\u003ePpMYB114\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study aims to investigate the ethylene-responsive transcription factor \u003cem\u003eAtERF103\u003c/em\u003e (AT1G77640) and its role in regulating morphological, physiological, molecular, and biochemical traits in \u003cem\u003eArabidopsis\u003c/em\u003e. Given the multifaceted role of ERF transcription factors in plant growth, development, and stress adaptation, we used CRISPR-Cas9-mediated genome editing and gene overexpression to investigate its regulatory role in root architecture, reproductive development, and secondary metabolite biosynthesis, focusing on anthocyanin and auxin (IAA) biosynthesis. Through transgenic Arabidopsis line analysis, we explored the molecular mechanism by which \u003cem\u003eAtERF103\u003c/em\u003e modulates plant growth and adaptation, providing valuable insights into its potential applications in crop improvement.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eGeneration of\u003c/b\u003e \u003cb\u003eAtERF013\u003c/b\u003e \u003cb\u003eOverexpressing Arabidopsis Plants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo generate \u003cem\u003eArabidopsis thaliana\u003c/em\u003e lines overexpressing \u003cem\u003eAtERF013\u003c/em\u003e, the full-length \u003cem\u003eAtERF013\u003c/em\u003e coding sequence (accession number PV929047) was cloned into the pCR8/GW/TOPO vector, which confers spectinomycin resistance and transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α cells via heat shock. Positive clones were confirmed via colony PCR and sequencing. The verified entry clone was then recombined with the binary vector pEarlyGate 103 (kanamycin resistance, CaMV 35S promoter) using an LR recombination reaction, ensuring strong constative expression. All genetic constructs were validated by commercial sequencing before the Arabidopsis transformation. The final constructs, with \u003cem\u003eAtERF013\u003c/em\u003e under the control of the 35S promoter, were electroporated into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101. Arabidopsis was transformed via the floral dip method [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. \u003cem\u003eAtERF103\u003c/em\u003e overexpressors were selected by spraying 1-week-old seedlings with 5 \u0026micro;g/mL Basta. T1 plants resistant to the herbicide were identified and used for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGeneration of Genome-edited Arabidopsis Targeting\u003c/b\u003e \u003cb\u003eAtERF013\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo generate \u003cem\u003eAtERF013\u003c/em\u003e genome-edited \u003cem\u003eA. thaliana\u003c/em\u003e lines, two single guide RNAs (sgRNAs) were designed using the CRISPR RGEN tool and integrated into the pRGEB32 vector, which carries the Cas9 gene, enabling targeted genome editing. The sgRNA sequences were cloned downstream of the U3 promoter within the pRGEB32 vector using the BsaI restriction enzyme site, as previously described [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Successful insertion of the sgRNA constructs was confirmed via sequencing. The verified pRGEB32 construct was then heat shock-transformed into \u003cem\u003eA. tumefaciens\u003c/em\u003e strain EHA105 and introduced into \u003cem\u003eArabidopsis\u003c/em\u003e via the floral dip method. T1 plants were screened through genotypic analysis to confirm genome editing and used for subsequent experiments. The genome editing workflow is illustrated in Figure S6.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGenotyping and Sequence Analysis of Edited Lines\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from edited \u003cem\u003eArabidopsis\u003c/em\u003e lines using the DNeasy Plant Mini Kit (QIAGEN, Cat. 69104, Hilden, Germany), following the instruction manual. T-DNA insertion was confirmed by amplifying the hygromycin resistance gene fragment using the Hyro-F/Hygro-R primer pair. PCR amplification was performed using high-fidelity Pfu polymerase and the Genetbio PCR Master Mix kit (Yuseong-gu, Daejeon, South Korea), following the manufacturer\u0026rsquo;s instructions. To analyze genome-edited lines, the full-length CDS region of the gene was amplified and sequenced using gene-specific primers. The target sequence was aligned with the NCBI reference sequence using SnapGene software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA Extraction, cDNA Synthesis and qPCR Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) following the manual instructions. cDNA library was synthesized from 2 \u0026micro;g of total RNA using the qPCR-Bio cDNA Synthesis Kit (PCRBIOSYSTEM, Seoul, South Korea), according to the manual instructions. qPCR was performed using the cDNA template and the StepOne\u0026trade; Plus RT-PCR system (Thermo Fisher Scientific, Seoul, Korea). The reaction was performed with 2x Real-Time PCR Master Mix (including SYBR Green I: BIOFACT, Daejeon, Korea). \u003cem\u003eActin\u003c/em\u003e was used as the reference gene, and data were analyzed via the 2\u003csup\u003e\u0026ndash;∆∆Ct\u003c/sup\u003e method.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIndole-3-acetic acid Quantification Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo quantify indole-3-acetic acid (IAA), 100 mg of roots, shoots, and flowers were ground in liquid nitrogen to fine powder. IAA was extracted twice using 900 \u0026micro;L of extraction buffer (methanol:H₂O:acetonitrile, 90:10:1, v/v) and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Xevo TQ-S micro system (Waters, Milford, MA, USA), with a stable isotope-labeled IAA as an internal standard, as previously described [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Detailed analytical conditions are provided in Supplementary File 1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExtraction and Quantification of Anthocyanin\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnthocyanin was extracted using an extraction buffer composed of methanol, water, formic acid, and trifluoroacetic acid in a ratio of 70:27:2:1 (v/v) as previously described [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Approximately 100 mg of samples collected during the flowering stage from the leaf stalk, leaf lamina, rosette junction, curved node, and normal node were ground to a fine powder in liquid nitrogen. Samples were incubated in 5 mL of extraction buffer at 37\u0026deg;C for 48 h. The crude extract was filtered, dried using a rotary evaporator, and reconstituted in 1 mL of HPLC-grade methanol. After dilution, 2 \u0026micro;L of the sample was mixed with 98 \u0026micro;L of 50% acetonitrile containing 0.1% formic acid and analyzed using a spectrophotometer (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland) in a 96-well plate. Absorbance was measured at A535\u0026ndash;A650.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGraphPad Prism 8 and Microsoft Office Excel (2016) were used for statistical analysis. A one-way analysis of variance was conducted to analyze the dataset, incorporating three independent biological replicates. Mean differences were assessed using Bonferroni post hoc tests, with statistical significance set at * \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05, ** \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01, and *** \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eGenetic Modulation of\u003c/b\u003e \u003cb\u003eERF013\u003c/b\u003e \u003cb\u003eAlters Root and Shoot Development in Arabidopsis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo confirm the successful generation of transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines, plants overexpressing \u003cem\u003e35S-AtERF013\u003c/em\u003e were subjected to Basta selection (10 \u0026micro;g/mL) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Out of 200 seeds, only 11 seedlings were resistant, confirming transformation. In contrast, genome-edited lines demonstrated higher transformation efficiency, with 27 out of 29 seeds showing successful T-DNA insertion (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Phenotypic assessments revealed distinct growth patterns between transgenic lines. \u003cem\u003e35S-AtERF013\u003c/em\u003e overexpression promoted vegetative growth, whereas \u003cem\u003eGE-aterf013\u003c/em\u003e genome-edited plants exhibited significantly reduced growth, underscoring the role of \u003cem\u003eAtERF013\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e development (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). To further investigate its function in root architecture, two genome-edited (\u003cem\u003eGE-aterf013-1\u003c/em\u003e and \u003cem\u003eGE-aterf013-2\u003c/em\u003e) and two overexpression (\u003cem\u003e35S-AtERF013-1\u003c/em\u003e and \u003cem\u003e35S-AtERF013-2\u003c/em\u003e) lines were analyzed alongside wild-type (Col-0) plants. After 10 days of cultivation on agar, primary and lateral root lengths were measured (Figure S2A).\u003c/p\u003e\u003cp\u003eQuantitative analysis confirmed that \u003cem\u003eERF013\u003c/em\u003e overexpression significantly enhanced root growth, while genome editing reduced primary and lateral root lengths (Figure S2B, 2C). Notably, \u003cem\u003e35S-AtERF013\u003c/em\u003e overexpression substantially increased root length, whereas \u003cem\u003eGE-aterf013\u003c/em\u003e lines exhibited the shortest roots among all tested groups. Furthermore, morphological observations revealed distinct alterations in lateral root development, emphasizing the contrasting effects of \u003cem\u003eERF013\u003c/em\u003e modulation (Figure S2D). Shoot development analysis showed that \u003cem\u003e35S-AtERF013\u003c/em\u003e overexpression significantly increased hypocotyl length after 1 week of growth on simple agar media, while genome-edited plants displayed a pronounced reduction in hypocotyl length compared to wild-type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Additionally, overexpression lines exhibited higher seed germination percentage, root fresh weight, and shoot fresh weight compared to the wild-type, whereas genome-edited lines substantially declined in these metrics (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;E). These findings highlight the crucial regulatory role of \u003cem\u003eERF013\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e growth and development, underscoring its impact on root and shoot architecture.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eERF013\u003c/b\u003e \u003cb\u003eRegulates Root Hair and Lateral Root Development via IAA Signaling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFunctional analysis of \u003cem\u003eERF013\u003c/em\u003e revealed its crucial role in root hair initiation and lateral root formation. \u003cem\u003eERF013\u003c/em\u003e overexpression (\u003cem\u003e35S-atERF013\u003c/em\u003e) promoted root hair initiation within 4 days of growth, whereas the wild-type (Col-0) and genome-edited (\u003cem\u003eGE-aterf013\u003c/em\u003e) lines exhibited no root hair development (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). After 1 week, root hairs were formed in Col-0 and \u003cem\u003e35S-atERF013\u003c/em\u003e plants, with the latter also displaying accelerated lateral root development. In contrast, \u003cem\u003eGE-aterf013\u003c/em\u003e plants lacked root hairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Analysis of lateral root formation revealed that in Col-0 and \u003cem\u003e35S-atERF013\u003c/em\u003e plants, lateral roots consistently emerged at primary root curvature points, whereas \u003cem\u003eGE-aterf013\u003c/em\u003e plants lacked lateral roots at any curvature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Notably, lateral root emerged significantly faster in \u003cem\u003e35S-atERF013\u003c/em\u003e than in Col-0, suggesting that \u003cem\u003eERF013\u003c/em\u003e enhances IAA accumulation at root curvature sites to promote lateral root formation. Further analysis demonstrated that \u003cem\u003eERF013\u003c/em\u003e selectively enhances root hair elongation at curvature points, while root hair length above these curvatures remained similar between \u003cem\u003e35S-atERF013\u003c/em\u003e and Col-0 plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u0026ndash;H). However, \u003cem\u003eGE-aterf013\u003c/em\u003e plants exhibited significantly shorter root hairs at both curvature and non-curvature compared to Col-0. At the last curvature (approximately 5 cm above the root tip), quantitative analysis revealed that \u003cem\u003e35S-atERF013\u003c/em\u003e plants had significantly longer root hairs than Col-0, while \u003cem\u003eGE-aterf013\u003c/em\u003e plants displayed a substantial reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u0026ndash;H). These findings indicate the dual role of \u003cem\u003eERF013\u003c/em\u003e in root architecture, promoting both lateral root initiation and root hair elongation.\u003c/p\u003e\u003cp\u003eTo determine whether \u003cem\u003eERF013\u003c/em\u003e directly regulates IAA-dependent primary root development, we treated \u003cem\u003eGE-aterf013\u003c/em\u003e plants with IAA and \u003cem\u003e35S-atERF013\u003c/em\u003e plants with yucasin (an IAA biosynthesis inhibitor) and compared their responses to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). IAA treatment significantly promoted primary root elongation in GE-\u003cem\u003eaterf013\u003c/em\u003e plants, whereas yucasin treatment inhibited primary root growth in \u003cem\u003e35S-atERF013\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). A similar trend was observed for lateral root length (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), whereas lateral root number remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These findings confirm \u003cem\u003eERF013\u003c/em\u003e as a key regulator of IAA-mediated root development. In non-curved primary roots (5 cm above the root tip), \u003cem\u003e35S-atERF013\u003c/em\u003e plants exhibited a significant increase in root hair number and length compared to Col-0, whereas \u003cem\u003eGE-aterf013\u003c/em\u003e plants displayed a pronounced reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u0026ndash;I). Overall, \u003cem\u003eERF013\u003c/em\u003e modulates IAA-mediated root architecture by promoting lateral root emergence at curvature sites and root hair elongation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eERF013\u003c/b\u003e \u003cb\u003eRegulates Root Growth and Development by Modulating Auxin Biosynthesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the role of \u003cem\u003eERF013\u003c/em\u003e in root growth and development, we quantified auxin (IAA) levels in seedlings, roots, and flowers and assessed \u003cem\u003eERF013\u003c/em\u003e expression in \u003cem\u003e35S-atERF013\u003c/em\u003e overexpression and \u003cem\u003eGE-aterf013\u003c/em\u003e genome-edited lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). IAA levels in shoots were significantly reduced by 44% and 50% in \u003cem\u003eGE-aterf013-1\u003c/em\u003e and \u003cem\u003eGE-aterf013-2\u003c/em\u003e, respectively, compared to Col-0, but significantly increased by 131% and 128% in \u003cem\u003e35S-atERF013-1\u003c/em\u003e and \u003cem\u003e35S-atERF013-2\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Peak IAA areas for all samples are shown in Figure S3. A similar trend was observed in roots, where genome-edited lines had significantly lower IAA levels, while overexpression lines showed a marked increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In flowers, IAA levels decreased by 80% and 65% in \u003cem\u003eGE-aterf013-1\u003c/em\u003e and \u003cem\u003eGE-aterf013-2\u003c/em\u003e, respectively, but significantly increased by 108% and 105% in \u003cem\u003e35S-atERF013-1\u003c/em\u003e and \u003cem\u003e35S-atERF013-2\u003c/em\u003e, respectively, compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). \u003cem\u003eERF013\u003c/em\u003e expression correlated with IAA biosynthesis in shoots, roots, and flowers. In \u003cem\u003eGE-aterf013\u003c/em\u003e plants, \u003cem\u003eERF013\u003c/em\u003e expression was significantly downregulated in shoots, roots, and flowers but significantly upregulated in \u003cem\u003e35S-atERF013\u003c/em\u003e plants across all three tissues compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;F). These findings suggest that \u003cem\u003eERF013\u003c/em\u003e promotes shoot, root, and flower development by enhancing endogenous IAA biosynthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eERF013\u003c/b\u003e \u003cb\u003eRegulates Flowering Time, Silique Morphology, and Leaf Architecture in Arabidopsis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the role of \u003cem\u003eERF013\u003c/em\u003e in regulating agronomic traits in \u003cem\u003eArabidopsis\u003c/em\u003e, we compared genome-edited and overexpression lines. \u003cem\u003eERF013\u003c/em\u003e overexpression induced early flowering, whereas genome-edited lines exhibited delayed flowering compared to Col-0 plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At early developmental stages, \u003cem\u003e35S-AtERF013\u003c/em\u003e plants demonstrated accelerated stem elongation, while GE-aterf013 plants exhibited inhibited stem development. Additionally, inflorescence emerged earlier in \u003cem\u003e35S-AtERF013\u003c/em\u003e plants but was delayed in GE-aterf013 lines compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, \u003cem\u003eERF013\u003c/em\u003e overexpression significantly increased silique length, leaf length, leaf width, and total leaf area, whereas genome-edited lines exhibited a substantial reduction in these traits (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Specifically, silique length increased by 38% and 33% in \u003cem\u003e35S-AtERF013-1\u003c/em\u003e and \u003cem\u003e35S-AtERF013-2\u003c/em\u003e lines, respectively, compared to Col-0, whereas \u003cem\u003eGE-aterf013-1\u003c/em\u003e and \u003cem\u003eGE-aterf013-2\u003c/em\u003e plants exhibited a 32% and 27% reduction, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Similarly, leaf length increased by 71% and 84% in \u003cem\u003e35S-AtERF013-1\u003c/em\u003e and \u003cem\u003e35S-AtERF013-2\u003c/em\u003e plants, but decreased by 18% and 17% in \u003cem\u003eGE-aterf013-1\u003c/em\u003e and \u003cem\u003eGE-aterf013-2\u003c/em\u003e plants, respectively, compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Total leaf area increased by 82% and 85% in \u003cem\u003e35S-AtERF013-1\u003c/em\u003e and \u003cem\u003e35S-AtERF013-2\u003c/em\u003e plants but decreased by 11% and 8% in \u003cem\u003eGE-aterf013-1\u003c/em\u003e and \u003cem\u003eGE-aterf013-2\u003c/em\u003e plants, respectively, compared to Col-0 plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eFurther analysis revealed that \u003cem\u003eERF013\u003c/em\u003e influences silique angle. Wild-type Col-0 plants exhibited silique angles of 7\u0026ndash;13\u0026deg;, genome-edited lines displayed significantly wider silique angles of 64\u0026deg;\u0026ndash;77\u0026deg;, while overexpression lines showed silique angles between 34\u0026deg; and 50\u0026deg; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Figure S4 demonstrates that \u003cem\u003eERF013\u003c/em\u003e is involved in trichome development in Arabidopsis. \u003cem\u003eERF013\u003c/em\u003e overexpression increased trichome density on leaf margins, petioles, and adaxial leaf surfaces, whereas genome-edited lines had significantly fewer trichomes than Col-0. \u003cem\u003eERF013\u003c/em\u003e also modulates leaf margin morphology and internode length (Figure S5). Overexpression lines exhibited elongated internodes, whereas genome-edited lines displayed significantly shorter internodes compared to Col-0 (Figure S5B).\u003c/p\u003e\u003cp\u003eTo assess the effect of \u003cem\u003eERF013\u003c/em\u003e on leaf morphology, the fifth leaf of each plant group was analyzed (Figure S5A). Col-0 plants exhibited serrated leaves, while overexpression and genome-edited plants developed lobed leaves with distinct morphological differences. \u003cem\u003eGE-aterf013\u003c/em\u003e plants had rounded and shallow lobes, resulting in a smooth, undulating appearance with minimal serrations. In contrast, \u003cem\u003e35S-AtERF013\u003c/em\u003e plants developed deeply incised, pointed lobes, giving the leaves a more angular and pronounced appearance with serrated or toothed margins. Furthermore, their lobes were irregularly distributed, resulting in a complex and asymmetrical leaf shape. These results indicate that \u003cem\u003eERF013\u003c/em\u003e plays a pivotal role in shaping Arabidopsis leaf morphology and plant architecture.\u003c/p\u003e\u003cp\u003e\u003cb\u003eERF013\u003c/b\u003e \u003cb\u003ePositively Regulates Anthocyanin Biosynthesis in\u003c/b\u003e \u003cb\u003eArabidopsis thaliana\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the role of \u003cem\u003eERF013\u003c/em\u003e in anthocyanin biosynthesis, we quantified anthocyanin accumulation and analyzed key biosynthetic gene expression in Col-0, \u003cem\u003eGE-aterf013\u003c/em\u003e, and \u003cem\u003e35S-AtERF013\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Spatiotemporal analysis of anthocyanin deposition across different tissues revealed a significant increase in \u003cem\u003e35S-AtERF013\u003c/em\u003e plants and a marked reduction in \u003cem\u003eGE-aterf013\u003c/em\u003e plants compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Specifically, anthocyanin levels were highest at the curved node, followed by the normal node and leaf lamina in \u003cem\u003e35S-AtERF013\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026ndash;G). To further investigate the molecular basis of this phenotype, we analyzed \u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003eC4H\u003c/em\u003e, \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eF3H\u003c/em\u003e, and \u003cem\u003eDFR\u003c/em\u003e expression, key genes involved in anthocyanin biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH\u0026ndash;L). The transcriptional profiles aligned with anthocyanin accumulation, with significant upregulation in \u003cem\u003e35S-AtERF013\u003c/em\u003e plants and downregulation in \u003cem\u003eGE-aterf013\u003c/em\u003e plants relative to Col-0. Among these genes, \u003cem\u003eDFR\u003c/em\u003e exhibited the highest induction, increasing 7-fold and 6-fold in \u003cem\u003e35S-AtERF013-1\u003c/em\u003e and \u003cem\u003e35S-AtERF013-2\u003c/em\u003e, respectively. Similarly, \u003cem\u003eF3H\u003c/em\u003e expression increased 6.5-fold and 6-fold, while \u003cem\u003eCHS\u003c/em\u003e was upregulated 6.5-fold and 5.5-fold in \u003cem\u003e35S-AtERF013-1\u003c/em\u003e and \u003cem\u003e35S-AtERF013-2\u003c/em\u003e, respectively. These findings show that \u003cem\u003eERF013\u003c/em\u003e positively regulates anthocyanin biosynthesis, likely by enhancing biosynthetic gene transcription. The substantial increase in anthocyanin accumulation in \u003cem\u003e35S-AtERF013\u003c/em\u003e plants underscores the key role of \u003cem\u003eERF013\u003c/em\u003e in modulating anthocyanin production in Arabidopsis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eERF013\u003c/b\u003e \u003cb\u003eRegulates Pollen, Seed, and Stigma Development in Arabidopsis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the role of \u003cem\u003eERF013\u003c/em\u003e in seed development and fertilization, we analyzed pollen grain development, seed set, and stigma morphology in Col-0, genome-edited, and overexpressor Arabidopsis lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Anther visualization revealed significantly reduced pollen production in \u003cem\u003eGE-aterf013\u003c/em\u003e plants and higher pollen density in \u003cem\u003e35S-atERF013\u003c/em\u003e plants compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). These findings suggest that \u003cem\u003eERF013\u003c/em\u003e is essential for normal pollen development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of seed development showed that \u003cem\u003eGE-aterf013\u003c/em\u003e plants had a higher proportion of infertile or underdeveloped seeds, whereas \u003cem\u003e35S-atERF013\u003c/em\u003e plants showed increased seed formation compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). Non-viable seed rates increased by 94% and 132% in \u003cem\u003eGE-aterf013-1\u003c/em\u003e and \u003cem\u003eGE-aterf013-2\u003c/em\u003e but they were significantly reduced by 71% and 61% in \u003cem\u003e35S-atERF013-1\u003c/em\u003e and \u003cem\u003e35S-atERF013-2\u003c/em\u003e, respectively, compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Similarly, the total seeds per silique increased by 111% and 72% in \u003cem\u003e35S-atERF013-1\u003c/em\u003e and \u003cem\u003e35S-atERF013-2\u003c/em\u003e and decreased by 77% and 50% in \u003cem\u003eGE-aterf013-1\u003c/em\u003e and \u003cem\u003eGE-aterf013-2\u003c/em\u003e, respectively, compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Given the essential role of the stigma in fertilization and seed development, we examined its morphology across the three plant groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Notably, \u003cem\u003eGE-aterf013\u003c/em\u003e plants had poorly developed stigmas, whereas both Col-0 and \u003cem\u003e35S-atERF013\u003c/em\u003e plants showed normal stigma formation. These findings indicate that \u003cem\u003eERF013\u003c/em\u003e is required for proper stigma development in Arabidopsis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eERF013\u003c/em\u003e belongs to the AP2/ERF transcription factor (TF) family, one of the largest plant-specific TF families [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In this study, \u003cem\u003eAtERF013\u003c/em\u003e was identified as a key regulator of Arabidopsis growth and development, particularly root development via IAA signaling. This study primarily focused on its role in regulating phenotypic traits in genome-edited and overexpression lines.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003e35S-AtERF013\u003c/em\u003e overexpression line promoted growth, whereas the \u003cem\u003eGE-aterf013\u003c/em\u003e genome-edited line exhibited inhibited growth compared to Col-0 plants under normal conditions. The observed increase in plant biomass resulted from enhanced root and shoot growth driven by \u003cem\u003eERF013\u003c/em\u003e overexpression, whereas impaired development in genome-edited lines stemmed from the loss of \u003cem\u003eERF013\u003c/em\u003e function. AAnalyses of primary roots, lateral roots, and root hairs at different time points confirmed that \u003cem\u003eERF013\u003c/em\u003e plays a crucial role in promoting root development. \u003cem\u003eERF109\u003c/em\u003e, a related TF, facilitates lateral root formation by linking jasmonic acid signaling with auxin biosynthesis by directly regulating \u003cem\u003eASA1\u003c/em\u003e and \u003cem\u003eYUC2\u003c/em\u003e (key enzymes involved in auxin biosynthesis) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, ERFVII TFs act as negative regulators of lateral root development, suppressing auxin-responsive genes such as \u003cem\u003eLBD16\u003c/em\u003e, \u003cem\u003eLBD18\u003c/em\u003e, and \u003cem\u003ePUCHI\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In contrast, \u003cem\u003eERF109\u003c/em\u003e and \u003cem\u003ePUCHI\u003c/em\u003e promote lateral root development. OsERF48 regulates root growth by inducing calmodulin-like protein expression [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, \u003cem\u003eERF013\u003c/em\u003e expression significantly induced auxin biosynthesis in seedlings, primary roots, and lateral roots, while its loss in genome-edited lines led to reduced auxin biosynthesis. Moreover, \u003cem\u003eERF013\u003c/em\u003e transcription was upregulated in overexpression lines and downregulated in genome-edited lines compared to control plants. Notably, exogenous auxin restored primary and lateral root development in genome-edited lines, whereas yucasin, an auxin biosynthesis inhibitor, reduced primary and lateral root length in overexpression lines. These findings suggest that \u003cem\u003eERF013\u003c/em\u003e is crucial for primary and lateral root development in Arabidopsis. \u003cem\u003e35S-AtERF013\u003c/em\u003e overexpression also increased root hair number and length, consistent with a recent study that reported similar results with \u003cem\u003eERF019\u003c/em\u003e overexpression lines [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, MPK14-mediated auxin signaling regulates lateral root development by stabilizing the \u003cem\u003eERF13\u003c/em\u003e protein [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. While this study did not directly assess the expression of auxin biosynthesis and auxin-responsive genes (\u003cem\u003eASA1\u003c/em\u003e, \u003cem\u003eYUC2\u003c/em\u003e, \u003cem\u003eLBD\u003c/em\u003e, and \u003cem\u003ePUCHI\u003c/em\u003e), previous reports suggest that \u003cem\u003eERF013\u003c/em\u003e may significantly influence root development by regulating these genes.\u003c/p\u003e\u003cp\u003eThis study provides novel insights into the regulatory role of \u003cem\u003eAtERF103\u003c/em\u003e in modulating flower and leaf morphology, silique size, fertility, and silique angle. \u003cem\u003eAtERF103\u003c/em\u003e overexpression induced early flowering, increased flower number, enhanced silique length and seed fertility, and promoted leaf elongation and expansion. In contrast, genome-edited \u003cem\u003eAtERF103\u003c/em\u003e lines exhibited delayed flowering, reduced flower and silique size, decreased leaf length and area, and a greater silique angle. These findings highlight the key role of \u003cem\u003eAtERF103\u003c/em\u003e in plant growth and reproduction. Auxin is well known for regulating plant development, particularly floral organ size, as demonstrated by the reduced flower organ size observed in \u003cem\u003eARF6/8\u003c/em\u003e mutants, emphasizing its role in floral development [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A recent study reported that \u003cem\u003eSIERF.D7\u003c/em\u003e positively regulates \u003cem\u003eSIARF2A/B\u003c/em\u003e expression, integrating auxin and ethylene signaling to modulate tomato fruit ripening [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Similarly, Li et al. revealed that \u003cem\u003eERF19\u003c/em\u003e regulates flower number and size by activating \u003cem\u003eWUS\u003c/em\u003e for primordia formation and \u003cem\u003eSAUR33\u003c/em\u003e for cell division and expansion, leading to larger floral organs in \u003cem\u003eAtERF19\u003c/em\u003e-overexpressing plants [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Conversely, the \u003cem\u003e35s::AtERF19\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eSRDX\u003c/em\u003e mutant exhibited fewer and smaller flowers, suggesting that \u003cem\u003eAtERF19\u003c/em\u003e modulates floral traits through auxin-related gene regulation.\u003c/p\u003e\u003cp\u003eOur study reveals new regulatory mechanisms governing auxin homeostasis, demonstrating that \u003cem\u003eAtERF103\u003c/em\u003e promotes auxin (IAA) accumulation in seedlings, roots, and flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Unlike \u003cem\u003eAtERF19\u003c/em\u003e, which modulates floral traits via auxin-responsive genes, \u003cem\u003eAtERF103\u003c/em\u003e broadly regulates plant growth. Furthermore, auxin induction via \u003cem\u003eAtERF103\u003c/em\u003e expression is crucial for silique growth, pollen development, stigma formation, and seed development (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results align with previous studies demonstrating the Auxin Activation Factor enhances auxin responses, increasing silique length, flower petal expansion, seed size, and anther indehiscence [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The \u003cem\u003eARF6/8\u003c/em\u003e double mutant further underscores the role of auxin in floral organ development, exhibiting reduced petal length and impaired stamen formation in Arabidopsis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These findings highlight the intricate regulatory network governing auxin-mediated reproductive organ growth. Beyond floral and reproductive development, auxin stimulation plays a crucial role in Arabidopsis leaf morphology. This is evident in \u003cem\u003eYUCCA\u003c/em\u003e overexpression lines, which exhibit a long, narrow, and downward-curled leaf phenotype due to elevated auxin levels, as well as in \u003cem\u003e35s-AtERF109\u003c/em\u003e overexpression plants [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Similarly, our findings reveal that \u003cem\u003eAtERF103\u003c/em\u003e modulates leaf morphology, suggesting a potential link to auxin-mediated leaf development. The altered phenotypes in \u003cem\u003eAtERF103\u003c/em\u003e-overexpressing and genome-edited lines indicate that \u003cem\u003eAtERF103\u003c/em\u003e is a key regulator of auxin homeostasis, coordinating both vegetative and reproductive development in Arabidopsis.\u003c/p\u003e\u003cp\u003eBeyond its role in auxin-mediated growth regulation, \u003cem\u003eAtERF103\u003c/em\u003e also influences anthocyanin biosynthesis, a crucial secondary metabolic pathway involved in plant stress responses and pigmentation. \u003cem\u003eAtERF103\u003c/em\u003e overexpression significantly enhanced anthocyanin accumulation in Arabidopsis leaf lamella, leaf petiole, nodes, and rosette junctions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This observation suggests that \u003cem\u003eAtERF103\u003c/em\u003e functions as a positive regulator of anthocyanin biosynthesis. Conversely, genome-edited \u003cem\u003eERF103\u003c/em\u003e lines exhibited reduced anthocyanin levels across these tissues, confirming its regulatory role in anthocyanin metabolism. A recent study on \u003cem\u003eERF012\u003c/em\u003e revealed that \u003cem\u003eERF012\u003c/em\u003e-overexpressing plants accumulated more anthocyanin, while \u003cem\u003eerf012\u003c/em\u003e mutants and wild-type Col-0 plants had lower anthocyanin levels, suggesting that \u003cem\u003eERF012\u003c/em\u003e promotes cold adaptability [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, \u003cem\u003eAtERF103\u003c/em\u003e overexpression enhanced anthocyanin accumulation in Arabidopsis, while genome-edited lines exhibited reduced anthocyanin levels. Furthermore, our analysis of key anthocyanin biosynthetic genes (\u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003eC4H\u003c/em\u003e, \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eF3H\u003c/em\u003e, and \u003cem\u003eDFR\u003c/em\u003e) revealed a strong correlation with \u003cem\u003eAtERF103\u003c/em\u003e expression. These genes were significantly upregulated in \u003cem\u003eAtERF103\u003c/em\u003e-overexpressing lines, corresponding with higher anthocyanin levels across various tissues, while their downregulation in genome-edited lines resulted in reduced anthocyanin levels. In pear fruit, \u003cem\u003eERF24\u003c/em\u003e and \u003cem\u003eERF96\u003c/em\u003e increased anthocyanin levels by inducing \u003cem\u003ePAL\u003c/em\u003e, \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eCHI\u003c/em\u003e, and \u003cem\u003eDFR\u003c/em\u003e expression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent studies suggest that \u003cem\u003eAtERF19\u003c/em\u003e regulates plant immunity and environmental stress responses [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Consistent with these findings, our study demonstrated that \u003cem\u003eAtERF103\u003c/em\u003e enhances anthocyanin accumulation, a known stress-mitigating compound. Although we did not directly evaluate the environmental stress response of \u003cem\u003eAtERF103\u003c/em\u003e overexpression lines, previous studies indicate that anthocyanin overaccumulation improves stress tolerance [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Anthocyanin accumulation in vacuoles protects against abiotic stress by regulating reactive oxygen species levels through enzymatic and non-enzymatic antioxidant activity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Based on this, we propose that \u003cem\u003eAtERF103\u003c/em\u003e enhances stress resilience by regulating anthocyanin biosynthesis, highlighting the functional diversity of ERF TFs in plant adaptation. Thus, these findings provide a basis for future research on the role of \u003cem\u003eAtERF103\u003c/em\u003e in environmental stress resilience, its regulatory networks, and its potential application in improving crop tolerance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that the \u003cem\u003eATERF013\u003c/em\u003e transcription factor is a key regulator of vegetative and reproductive development, as well as secondary metabolism, in \u003cem\u003eA. thaliana\u003c/em\u003e. Overexpression of A\u003cem\u003etERF013\u003c/em\u003e markedly enhances auxin levels, root and shoot growth, leaf size, early flowering, silique development, seed production, and anthocyanin biosynthesis, highlighting its positive role in promoting growth and yield related traits. Conversely, loss of its function impairs these processes, leading to reduced growth, delayed flowering, reduced fertility, and compromised seed viability. These findings provide valuable insights into the multifaceted role of \u003cem\u003eAtERF013\u003c/em\u003e in plant development and metabolism, offering a target for genetic improvement of agronomic traits in \u003cem\u003eA. thaliana\u003c/em\u003e and potentially in crop species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Cooperative Research Program for Agriculture Science and Technology Development (Grant No. RS-2024-00348677), Rural Development Administration, Republic of Korea. The authors extend their appreciation to Northern Border University, Saudi Arabia, for supporting this work through project number (NBU-CRP-2025-249).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRahmatullah Jan, Sajjad Asaf, Lubna, and Eman R. Elsharkawy: designed the study. Rahmatullah Jan, Saleem Asif, and Zakirullah Khan: performed most of the experiments and analyzed data. Muhammad Farooq and Zakirullah Khan: performed IAA and anthocyanin analysis. Rahmatullah Jan, Sajjad Asaf, and Kyung-Min Kim: wrote the manuscript. Kyung-Min Kim: provided resources and funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work has not received any formal funding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe RNA sequence has been deposited in the National Center for Biotechnology Information (NCBI) under accession number PV929047 (available at: link will be provided soon). All data generated or analyzed during this study are included in this article and its supplementary information file.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest associated with this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRiechmann JL, Meyerowitz EM: \u003cstrong\u003eThe AP2/EREBP family of plant transcription factors\u003c/strong\u003e. \u003cem\u003eBiological chemistry \u003c/em\u003e1998, \u003cstrong\u003e379\u003c/strong\u003e:633-646.\u003c/li\u003e\n\u003cli\u003eNakano T, Suzuki K, Fujimura T, Shinshi H: 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\u003cstrong\u003e143\u003c/strong\u003e:517-530.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"auxin, anthocyanin biosynthesis, ERF013, Arabidopsis thaliana, early flowering, root and shoot growth","lastPublishedDoi":"10.21203/rs.3.rs-7070846/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7070846/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the role of the transcription factor \u003cem\u003eAtERF013\u003c/em\u003e in regulating root and shoot development, flowering time, leaf morphology, anthocyanin biosynthesis, and reproduction in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eAtERF013\u003c/em\u003e overexpression (\u003cem\u003e35s-AtERF013\u003c/em\u003e) enhanced vegetative growth, increasing auxin (IAA) levels in seedlings by 131%, significantly increasing root length, and accelerating lateral root development. In contrast, genome-edited (\u003cem\u003eGE-aterf013\u003c/em\u003e) lines reduced growth, with a 50% decrease in IAA levels and shorter primary and lateral roots. Overexpression also induced early flowering, accelerated stem elongation, and increased silique length by 38% and 33% compared to wild-type (Col-0) plants. In contrast, genome-edited lines delayed flowering and reduced silique length by 32% and 27%. Leaf morphology was significantly altered, with \u003cem\u003e35s-AtERF013\u003c/em\u003e lines showing a 71\u0026ndash;84% increase in leaf length and an 82\u0026ndash;85% increase in total leaf area, while \u003cem\u003eGE-etaref013\u003c/em\u003e line exhibited 17\u0026ndash;18% and 81\u0026ndash;111% reductions in leaf length and area, respectively. \u003cem\u003eAtERF013\u003c/em\u003e overexpression also enhanced anthocyanin biosynthesis, increasing anthocyanin accumulation and upregulating the \u003cem\u003eDFR\u003c/em\u003e gene seven-fold. Regarding reproductive traits, overexpression increased seed count per silique by 72\u0026ndash;111%, while genome-edited lines showed a 50\u0026ndash;77% decrease compared to Col-0 plants. Furthermore, \u003cem\u003eGE-aterf013\u003c/em\u003e lines displayed underdeveloped stigmas and a higher proportion of non-viable seeds. These findings highlight \u003cem\u003eAtERF013\u003c/em\u003e as a crucial regulator of plant growth, development, and metabolism, with significant implications for enhancing agronomic traits in \u003cem\u003eA. thalian\u003c/em\u003e and other crops.\u003c/p\u003e","manuscriptTitle":"Unlocking the Functional Dynamics of ERF103 in Arabidopsis thaliana: A key player in Plant Growth Regulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 11:07:10","doi":"10.21203/rs.3.rs-7070846/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0c80e5b2-497a-42c9-8033-32d54a25892a","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-01T05:53:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-17 11:07:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7070846","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7070846","identity":"rs-7070846","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}