The Rice Cysteine Protease OsEP3A Promotes Seedling Growth and Seed Development and Contains a Nitrogen Starvation Responsive Sequence

The Rice Cysteine Protease OsEP3A Promotes Seedling Growth and Seed Development and Contains a Nitrogen Starvation Responsive Sequence | 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 The Rice Cysteine Protease OsEP3A Promotes Seedling Growth and Seed Development and Contains a Nitrogen Starvation Responsive Sequence Shinn Jia Tzeng, Yi Hsuan Hou, Shin Lon Ho This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8421153/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Apr, 2026 Read the published version in Plant Cell Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Nitrogen (N) is a critical macronutrient that influences plant growth, development, and productivity. This study characterizes the rice cysteine protease gene OsEP3A and its promoter to elucidate its role in N-mediated developmental and transcriptional regulation. Transgenic rice lines overexpressing ( OsEP3A-Ox ) or silenced ( OsEP3A-Ri ) for OsEP3A were generated to assess its physiological functions. Overexpression of OsEP3A significantly enhanced shoot and root growth, whereas RNAi -silenced plants exhibited reduced height, shorter roots, and smaller seeds compared to wild type, indicating that OsEP3A positively regulates seedling and seed development. Expression analyses revealed that OsEP3A transcription was strongly induced under N deficient conditions and repressed by both inorganic (NH₄NO₃) and organic (glutamine, asparagine) N sources. Under N-limited hydroponic culture, OsEP3A-RNAi seedlings showed severely impaired growth, underscoring the gene’s essential role in internal N remobilization during deficiency. Promoter-reporter analyses using OsEP3A::GUS lines demonstrated strong activation of the OsEP3A promoter under N starvation and repression upon N resupply, suggesting N dependent transcriptional control. Deletion and insertion analyses of the OsEP3A promoter identified a 43 bp N starvation responsive sequence ( NSRS ; -278 to -236 bp) as necessary and sufficient for starvation-induced transcriptional activation. This NSRS represents a novel cis-regulatory element responsive to N deprivation. Overall, OsEP3A acts as a N starvation-activated cysteine protease that facilitates N recycling and seedling vigor, providing new insight into N responsive regulatory mechanisms in rice and offering a potential molecular target for improving N use efficiency in cereal crops. rice cysteine protease nitrogen nitrogen starvation responsive sequence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nitrogen (N) is an essential macronutrient that frequently limits plant growth, development, and productivity (Miller and Cramer, 2004 ). To cope in N deficient conditions, plants have evolved intricate regulatory networks that modulate N acquisition, assimilation, and remobilization to optimize N use efficiency (NUE). These adaptive mechanisms include transcriptional and post-transcriptional regulation, root architectural adjustments, and coordinated signaling between N and carbon metabolism (Forde, 2014 ; Krapp et al., 2014 ; Kiba and Krapp, 2016 ). Nitrogen acquisition and signaling involve multiple uptake systems. Plants utilize a low-affinity transport system (LATS) when external nitrate (NO₃⁻) concentrations exceed 0.5 mM, and a high-affinity transport system (HATS) under lower concentrations (< 0.5 mM) (Wang et al., 2012 ). Nitrate uptake and assimilation depend on nitrate transporter ( NRT ) genes and associated enzymes. In Arabidopsis thaliana , two major families of nitrate transporters, NRT1 and NRT2 , both play critical roles in nitrate absorption and transport. NRT1 proteins belong to the LATS and function primarily when external nitrate concentrations are high, whereas NRT2 proteins are part of the HATS and mediate nitrate uptake under low-nitrate conditions (Esteban et al., 2016 ; Wang et al., 2018 ). Nitrogen signaling involves intricate regulatory components that orchestrate gene expression and metabolic pathways in response to N availability. Genome-wide transcriptome analyses have identified thousands of nitrate responsive genes, including those encoding hormone related proteins, kinases, transcription factors, transport proteins, and metabolic enzymes (Wang et al., 2000 ; Bi et al., 2007 ; Yang et al., 2015 ; Song et al., 2022 ). Under N deficient conditions, plants exhibit distinct physiological and molecular responses such as reduced photosynthetic activity (Ding et al., 2005 ), enhanced amino acid remobilization from older leaves (Ono et al., 1996 ), accelerated chlorophyll degradation, and increased anthocyanin accumulation (Díaz et al., 2006 ). At the molecular level, N starvation induces HATS components to facilitate nitrate uptake. NRT1.1 acts as a dual-affinity nitrate transceptor that senses and transports nitrate, thereby initiating primary nitrate responses (Liu et al., 1999 ; Sun et al., 2014 ; Wang et al., 2020 ). Moreover, the expression of high-affinity nitrate transporter genes such as NRT2.1 (Filleur et al., 2001 ) and transcription factors like AtNsr1 (Todd et al., 2004 ) promotes efficient nitrate uptake and utilization. In Arabidopsis , AtNRT2.4 is recognized as an N starvation marker gene responsive to N deficiency (Kiba et al., 2012 , 2016). The transcriptional repressor NIGT1/HRS1 binds to the promoter of AtNRT2.4 to repress N deficient responses under high N availability, indicating its key role in balancing N acquisition and utilization under fluctuating N conditions (Kiba et al., 2018 ). Overexpression of AtNRT2.4 significantly enhances plant height, silique number, biomass, and seed yield under normal N conditions, while also promoting anthocyanin accumulation under low nitrate stress. These findings underscore the multifaceted role of AtNRT2.4 in the hierarchical regulation of N signaling and plant adaptive responses. (Zhang et al., 2025 ). In rice, the salt- and drought-tolerant transcription factor OsSNAC1 (Hu et al., 2006 ; You et al., 2016 ) has been shown to bind to and activate the promoters of nitrate responsive genes, including OsNRT2.1 , OsNRT2.2 , OsNRT1.1A , and OsNRT1.1B , thereby enhancing nitrate acquisition. Overexpression of OsSNAC1 enhances nitrate uptake, root development, and grain yield, suggesting a role in coordinating stress responses under N limitation and drought conditions (Qi et al., 2023 ). Similarly, OsHHO3 acts as a transcriptional repressor of AMMONIUM TRANSPORTER1 genes, where reduced OsHHO3 expression improves ammonium uptake under N limitation (Yang et al., 2025 ). Post-transcriptional mechanisms further fine tunes plant responses to N. The expression of microRNAs (miRNAs) is differentially regulated by N deprivation (Paul et al., 2015 ; Kong et al., 2021 ). N deficiency alters the expression of specific miRNAs that regulate root system architecture (RSA) and stress adaptation. In rice, decreased expression of miR164 and miR167 is closely associated with low N tolerance (Nischal et al., 2012 ). In wheat, miR1118 is N responsive and contributes to RSA development under N starvation (Sinha et al., 2015 ; Zhao et al., 2015 ). Similarly, in Arabidopsis , overexpression of miR160 promotes lateral root formation under N-limited conditions (Liang et al., 2012 ). Plant hormones also play a key role in N signaling. In Arabidopsis and maize, limited N availability enhances auxin transport from shoots to roots, promoting lateral root development through auxin-dependent acid growth and the TARGET OF RAPAMYCIN (TOR) kinase signaling pathway (Tian et al., 2008 ; Asim et al., 2020 ; Sun et al., 2020 ; Stitz et al., 2023 ). Nitrate availability also regulates root development; low NO₃⁻ levels promote root elongation, whereas high levels suppress it via abscisic acid (ABA)-mediated signaling (Signora et al., 2001 ). In wheat, increased ABA content and upregulation of zeaxanthin epoxidase under low N stress further support the role of ABA in N deficiency tolerance (Kang et al., 2019 ; Mahmoud et al., 2020 ). Additionally, calcium signaling also contributes to N stress responses as elevated Ca²⁺ levels modulate the expression of N responsive genes (Zhang et al., 2016 ; Tang et al., 2018 ). Calcium-dependent protein kinases (CPKs) phosphorylate NIN-LIKE PROTEIN (NLP) transcription factors, thereby activating downstream nitrate responsive pathways (Liu et al., 2017 ), thus exemplifying the crosstalk between calcium and nitrate signaling in plants. Proteases are ubiquitous that include diverse families (such as cysteine-, serine-, aspartic- and metallo-protease, etc.) and act roles in protein proteolysis to mediate protein turnover, processing, and degradation across all life forms (van der Hoorn and Rivas, 2018 ). Among them, Papain-like cysteine proteases (PLCPs) are widespread enzymes that play crucial roles in plant growth, development, and immunity (Liu et al., 2018 ; Li et al., 2022 ; Xu et al., 2025 ). Characterization by a conserved catalytic triad (Cys-His-Asn), PLCPs are synthesized as inactive precursors and participate protein degradation that functional correlation with plant senescence, seed germination, pollen formation, and responses to biotic and abiotic stresses (Liu et al., 2018 ; Martínez et al., 2012 ). In Arabidopsis , the PLCP RESPONSIVE TO DEHYDRATION 21 (RD21) is essential for defense against necrotrophic pathogens, while in wheat, TaRD21A enhances resistance to wheat yellow mosaic virus through release small peptides (Shindo et al., 2012 ; Liu et al., 2023 ). In rice ( Oryza sativa ), genome-wide analyses have identified multiple PLCP families with diverse expression patterns and regulatory functions, including roles in seed development and stress response (Wang et al., 2018 ; Niño et al., 2020 ). For instance, root specific PLCPs such as OsCP14 , OsCP16 , OsCP19 , and OsCP25 are crucial for maintaining soil microbial colonization (Xu et al., 2025 ). The oryzain alpha chain precursor (OCP), a rice ortholog of Arabidopsis RD21, was shown to negatively regulate resistance to rice blast disease. Loss-of-function of OCP using CRISPR/Cas9 enhanced blast resistance by activating jasmonic acid (JA) and ethylene (ET) signaling pathways while repressing auxin signaling pathways (Li et al., 2022 ). Few studies have explored the role of cysteine proteinases in response to N starvation in plants. Previous research has shown that the abundances of Rubisco greatly decreases in the old leaves of Arabidopsis and Phaseolus vulgaris under N deficient conditions (Crafts-Brandner et al., 1996 ; Izumi et al., 2010 ). The Senescence-Associated Gene 12 ( SAG12 ), which encodes a vacuolar cysteine protease, is highly induced during leaf senescence (Lohman et al., 1994 ; Otegui et al., 2005 ). Therefore, SAG12 is proposed to participate in the degradation of Rubisco proteins in the vacuoles of senescing leaves. In previous studies (Ho et al., 2000 ), we found that expression of the rice cysteine protease gene OsEP3A is induced by N starvation in suspension-cultured cells, germinating seeds, and senescing leaves, suggesting that OsEP3A participates in protein degradation to supply N for plant growth. These findings highlight the pivotal role of N sensing, acquisition, transport, assimilation, and the modulation of N signaling in maintaining N homeostasis throughout the plant life cycle. Despite extensive research on nitrate transporters and transcriptional regulation, the cis-regulatory elements involved in the N starvation responsive expression of proteolytic genes remain poorly characterized. In this study, we conducted a promoter deletion and insertion analysis to identify the N starvation responsive sequence ( NSRS ) within the promoter of the rice OsEP3A gene, aiming to elucidate the molecular mechanisms underlying N-mediated regulation of cysteine protease ( OsEP3A ) expression in rice. Materials and Methods Plant materials The rice cultivar Oryza sativa L. cv. Tainung 67 was used throughout this study. Among the OsEP3A overexpression ( OsEP3A - Ox ) and RNA interference ( OsEP3A - Ri ) transgenic lines, three independent T₃ lines carrying a single-copy transgene were selected (Fig. S1) to evaluate the functional role of OsEP3A in rice development. Callus induction Immature rice seeds collected 10 days after flowering were air-dried for 7 days. Thirty dehulled seeds were sterilized with 2.4% NaOCl for 30 minutes and rinsed thoroughly with sterile water. The sterilized seeds were cultured on callus induction medium containing N6 salts (Chu et al., 1975 ), 10 µM 2, 4-D, and 3% sucrose. Cultures were maintained at 28°C under a 16 h/8 h light/dark photoperiod with a light intensity of 3500 lux (Ho et al., 2000 ). After 30 days, calli were transferred to fresh medium and cultured for an additional 7 days before Agrobacterium -mediated transformation. Primer sequences The nucleotide sequences of all primers used for PCR, RT-PCR, and qRT-PCR are listed in Supplementary Table S1. Construction of OsEP3A-Ox, OsEP3A-Ri and OsEP3A::GUS vectors To construct the OsEP3A overexpression vector ( OsEP3A - Ox ), a 1007-bp fragment containing the full coding sequence of OsEP3A was amplified using primers OsEP3A-F1 and OsEP3A-R1 . The PCR product was digested with Bam HI and ligated into the Bam HI - digested pAHC18 vector under the control of the maize ubiquitin promoter (Bruce et al., 1989 ). The resulting plasmid was linearized with Hin dIII and inserted into the Hin dIII site of the binary vector pSMY1H (Ho et al., 2000 ). For the OsEP3A RNA interference construct ( OsEP3A-Ri ), a 420-bp fragment comprising 100 bp of the 3′ coding region and 320 bp of the 3′ untranslated region of OsEP3A was amplified using primers OsEP3A-Xho I and OsEP3A-Kpn I, and OsEP3A-Xba I and OsEP3A-Hin dIII, respectively. The two DNA fragments were inserted downstream of the maize ubiquitin promoter in both sense and antisense orientations, separated by a truncated GFP spacer (Ho et al., 2013 ). The construct was linearized with Pst I and inserted into the Pst I site of pSMY1H binary vector. To generate the OsEP3A::GUS reporter construct, a 2015-bp promoter fragment including the 5′ untranslated region (upstream of the ATG start codon) was amplified using primers OsEP3A-5P and OsEP3A-3B . The PCR product was digested with Pst I and Bam HI and ligated upstream of the gusA reporter gene in the pBX-2 vector (Ho et al., 2000 ). The resulting plasmid was linearized with Pst I and inserted into the Pst I site of the pSMY1H binary vector. Construction of serial deletion OsEP3A::GUS vectors To identify the NSRS within the OsEP3A promoter, a 2000 bp upstream regulatory sequence ( URS ) from the OsEP3A promoter region (Fig. 6 A) was subjected to a series of 5′-end serial deletions, internal deletions, and insertions using PCR-based methods. The 3′ end of all fragments was fixed at the first nucleotide upstream of the TATA box (-35) (Fig. 6 A). The resulting URS fragments of varying lengths were digested with Hin dIII and Bam HI and cloned upstream of the CaMV 35S minimal promoter (Amack and Antunes, 2020 ) to drive the gusA reporter gene using the same restriction sites (Fig. 6 B). The constructed vectors were used to assess N starvation responsive activity. In total, seven URS serial deletion constructs were generated: 2000::GUS (-2000 to -35), 837::GUS , 531::GUS , 278::GUS , 236::GUS , 183::GUS , and 113::GUS . Three internal deletion constructs were also produced by removing fragments from − 236 to -278, -183 to -236, or -113 to -183 of the 837::GUS construct, and designated as D278/236::GUS , D236/183::GUS , and D183/113::GUS , respectively. In addition, two insertion constructs containing fragments from − 236 to -837 or -236 to -278 were fused upstream of the CaMV 35S minimal promoter and named A837-236::GUS and A278-236::GUS . The CaMV 35S minimal promoter fused only to the gusA reporter gene served as a control and was designated 35mp::GUS . Each expression construct was cloned into a binary vector and introduced into rice cells via Agrobacterium -mediated transformation. Three independent T₂ lines from each transgenic construct were used for callus induction, followed by the establishment of suspension-cultured cell lines. These lines were cultured in media containing N (+ N) or lacking N (-N) for 10 days and subjected to qRT-PCR analysis of gusA expression and GUS staining to identify the N responsive sequence within the OsEP3A promoter. Plant transformation Transgenic rice plants were generated via Agrobacterium -mediated transformation. Recombinant plasmids were introduced into Agrobacterium tumefaciens strain EHA101 by electroporation, and the transformed bacteria were co-cultivated with rice calli to transfer the T-DNA, following established protocols (Ho et al., 2000 ). Phenotypic analysis Dehulled rice seeds were surface-sterilized with 2.4% NaOCl for 30 min, rinsed thoroughly with sterile water, and sown on half-strength MS (Murashige and Skoog, 1962 ) solid medium supplemented with 1.5% sucrose. Cultures were maintained at 28°C under a 16 h photoperiod with a light intensity of 3500 lux. After 4 days, seedlings were transferred to soil-filled pots and grown for either 8 days (12-day-old plants; Fig. 1 C) or 5 days (9-day-old plants; Fig. S2B). Phenotypic traits including shoot length and root length were measured. Statistical analyses were performed using one-way ANOVA. Histochemical GUS staining Rice suspension-cultured cells were grown in MS medium with or without N for 5 days and then collected for GUS staining. For GUS staining of developing seeds, spikelets were collected before flowering and at 1, 3, 6, and 20 days after flowering, followed by GUS staining. Samples were incubated at 37°C in the dark for 4 hours in a staining buffer containing 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.1% Triton X-100, and 1 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc). After staining, samples were stored in 70% ethanol, rinsed with water, and imaged. Quantitative RT-PCR Thirty dehulled and sterilized rice seeds were sown on half-strength MS medium containing 1.5% sucrose and cultured under a 16 h/8 h light/dark photoperiod at 28°C with a light intensity of 3500 lux. Five-day-old seedlings (Fig. 1 B) of WT, OsEP3A - Ri , and OsEP3A - Ox lines, as well as calli treated with or without N for 5 days (Fig. 5 and Fig. 6 ), were collected. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and treated with the TURBO DNA-free kit (Ambion, USA). First-strand cDNA was synthesized from 5 µg of RNA using M-MuLV reverse transcriptase (New England Biolabs, USA) and oligo (dT) primers. Quantitative RT-PCR was performed on an Eco Real-Time PCR System (Illumina, USA) following the manufacturer’s instructions. Gene-specific primers targeting the 3′ untranslated region of OsEP3A and the GUS coding sequence were used (Table S1). Expression levels were normalized to OsActin1 , with WT set to 1.0. All reactions were performed in triplicate. Southern blot analysis Genomic DNA was extracted from 5-d-old seedlings using the standard phenol-chloroform method. A total of 10 µg of genomic DNA was digested with Hin dIII at 37°C for 2 h. The digested DNA was separated on agarose gel in TBE buffer. The DNA was then depurinated by incubating the gel in 0.2 N HCl for 10 min, followed by denaturation in denaturation solution (1.5M NaCl, 0.5M NaOH) for 45 min and neutralization in neutralization solution (1M Tris pH 7.4, 1.5M NaCl) for 30 min. The DNA was transferred to a nylon membrane using capillary transfer with 10 X SSC buffer overnight. After transfer, the membrane was UV-crosslinked to fix the DNA. The membrane was then hybridized with the DIG-labeled Hpt probe using the DIG-High Prime DNA Labeling and Detection Starter Kit according to the supplier’s instructions (Roche). Northern blot analysis Total RNA was extracted from rice suspension cells using TRIzol reagent according to the manufacturer’s instructions. Northern blot was performed with the DIG-labeled 420 bp of OsEP3A specific region (containing 120 bp downstream coding sequence followed by 300 bp 3’-untranslated region) as a probe using the DIG-High Prime DNA Labeling and Detection Starter Kit according to the supplier’s instructions (Roche). Statistical analysis All experiments were performed with at least three biological replicates. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test using SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA). Statistical significance was set at P < 0.01 (Fig. 2 ) or P < 0.05 (Figs. 1 , 4 – 6 ). Results Phenotypic changes in transgenic rice plants overexpressing or silenced for OsEP3A The expression of a rice cysteine proteinase gene, OsEP3A , is induced by gibberellic acid (GA) in germinating seeds, suggesting that it plays a role in degrading storage proteins in the endosperm to provide N sources required for seedling growth (Ho et al., 2000 ). The expression of OsEP3A is also induced under N starvation in suspension-cultured cells and is subjected to feedback repression by supplemental N (Ho et al., 2000 ). In this study, both gene overexpression and RNA interference ( RNAi )-mediated gene silencing approaches were used to dissect the role of OsEP3A in rice seedling growth and to identify the promoter sequences responsive to N starvation in rice suspension-cultured cells. The expression vectors (Fig. 1 A) for overexpression ( OsEP3A - Ox , referred to as Ox ) and RNA interference ( OsEP3A - Ri , referred to as Ri ) were constructed under the control of the maize ubiquitin ( Ubi ) promoter (Christensen et al. 1992 ). After Agrobacterium -mediated transformation of rice calli and regeneration of the transgenic plants, five overexpression lines (a total of 11 independent lines) and three RNAi -silenced lines (a total of 8 independent lines) carrying single-copy transgenes were confirmed by Southern blot hybridization using the hygromycin phosphotransferase gene ( Hpt ) as a probe (Fig. S1). Among these, three independent single-copy lines were selected from each construct and designated as Ox-1 , Ox-2 , and Ox-3 , and Ri-1, Ri-2 , and Ri-3 , respectively (Fig. S1). To validate the expression levels of OsEP3A mRNA in the different transgenic lines, thirty T 3 transgenic seeds from each line were dehulled, sterilized, and germinated on half-strength MS medium for 5 days. Embryos along with shoots were collected for total RNA extraction, and OsEP3A mRNA accumulation was analyzed by qRT-PCR. The results (Fig. 1 B) showed that OsEP3A mRNA was highly expressed in the three OsEP3A -overexpressing lines, whereas endogenous OsEP3A expression was either completely absent or very low in the RNAi -silenced lines. To examine the physiological function effect of OsEP3A on rice seedling development, seedling phenotypes were analyzed. As shown in Fig. 1 C, significant differences in plant height and root length were observed at 10 days after germination (DAG) among the seedlings of wild type (WT), Ox-1, Ox-2, Ox-3, Ri-1, Ri-2 , and Ri-3. Compared with the WT, the plant height of Ox lines was 14.5% taller, whereas that of Ri lines was 28.6% shorter (Figs. 1 C, D). Similarly, crown root length in Ox and Ri lines was 13.8% longer and 31.0% shorter, respectively, than in the WT (Figs. 1 C, E). These results indicate that OsEP3A plays a positive role in rice seedling development. As consistent phenotypes were observed among the OsEP3A -overexpressing and RNAi -silenced plants, the Ox-1 and Ri-1 lines were selected for further study. Therefore, the expression levels of OsEP3A mRNA in the Ox-1 and Ri-1 lines were reconfirmed by northern blot hybridization using the OsEP3A 3′-UTR as a probe. Compared with the wild type, the results showed that the accumulation of OsEP3A mRNA was much higher and lower in the Ox-1 and Ri-1 lines, respectively (Fig. S2A). Consistent with the results shown in Fig. 1 , the 7-day-old seedlings of both shoots and roots were longer in Ox-1 and shorter in Ri-1 , respectively, compared with those of the WT (Fig. S2, B-D). Silencing of OsEP3A decreased seed size To determine whether OsEP3A is involved in other plant developmental processes, seed morphology was further analyzed. The results indicated no significant differences in grain length (Fig. 2 A, C) or width (Fig. 2 B, D) between the WT (0.70 ± 0.05 cm for length and 0.36 ± 0.02 cm for width) and the overexpression line Ox-1 (0.72 ± 0.04 cm for length and 0.37 ± 0.01 cm for width). By contrast, the RNA interference line Ri-1 (0.64 ± 0.05 cm for length and 0.32 ± 0.02 cm for width) showed significant reductions of 9.0% and 11.1% in length and width, respectively, compared with the WT. These results indicate that OsEP3A plays a positive role in rice seed development. Expression profiles of OsEP3A in developing rice seeds Our results demonstrated that OsEP3A regulates rice seed development. Therefore, the spatial and temporal expression pattern of OsEP3A was examined in developing rice seeds. The OsEP3A::GUS transgenic line was constructed using a β-glucuronidase ( GUS ) reporter gene driven by the OsEP3A promoter (a 2,015 bp region upstream of the translational start site) containing the 5′ untranslated region (121 bp) of OsEP3A mRNA (Fig. 3 A). As shown in Fig. 3 B, strong GUS staining was observed in the anthers and ovaries before flowering (BF), whereas much weaker or almost no GUS activity was detected in these tissues at 1 day after flowering (1 DAF). Similarly, little or no GUS staining was observed in developing seeds at 3, 6, and 20 DAF. These results indicate that OsEP3A is expressed in a tissue-specific and developmental stage–dependent manner, showing high expression in anthers and ovaries before flowering but very low expression after flowering during seed development. OsEP3A expression is induced by N starvation and required for normal growth in rice seedlings Previous studies (Ho et al., 2000 ) showed that the expression of OsEP3A was induced in N-starved (-N) suspension-cultured rice cells. As shown in Fig. 4 A of this study, OsEP3A expression was moderately induced after 5 days and highly induced after 10 days of -N treatment. In contrast, its expression was repressed by both the organic N source glutamine and the inorganic N source NH₄NO₃. These results suggest that OsEP3A expression is activated under N starvation to degrade target proteins for N supplementation and is subsequently downregulated through feedback repression by available N sources. To examine the effect of N on plant growth in WT, Ox-1 , and Ri-1 , twenty seeds from each line were germinated in Kimura hydroponic solution (Ehara et al., 2001 ) either containing N sources ((NH₄)₂SO₄ and KNO₃) or lacking N for 10 days. When seeds were germinated and grown in medium containing N, the results (Fig. 4 B and 4 C) were consistent with those shown in Fig. 1 C and 1 D. When compared with WT, the shoot length was longer in Ox-1 and shorter in Ri-1 . Under N deficient (-N) hydroponic conditions, the shoot length of all three lines was shorter than that under + N conditions, and no significant difference was observed between WT and Ox-1 . Interestingly, Ri-1 seedlings exhibited severely impaired growth under -N conditions and appeared unable to grow normally (Fig. 4 B and 4 C). These results indicate that OsEP3A is functionally correlated with N and is essential for rice seed germination and seedling growth. The OsEP3A promoter is activated under N-limited conditions To verify the effect of N on OsEP3A expression, OsEP3A::GUS reporter lines were used. Calli derived from three independent OsEP3A::GUS transgenic lines were induced from immature seeds and subsequently cultured as suspension cells. These suspension cells were then grown in MS medium with or without N for 10 days, followed by GUS staining or qRT-PCR analysis. As shown in Figs. 5 A, all three lines exhibited stronger GUS staining in -N medium than in + N medium. Consistent with these results, qRT-PCR analysis revealed that gusA mRNA levels were approximately nine-fold higher in -N cells than in + N cells (Fig. 5 B). These results demonstrate that the transcriptional activity of the OsEP3A promoter is activated under N-starved conditions. Furthermore, to determine whether the transcriptional activity of the OsEP3A promoter is repressed by N, calli derived from OsEP3A::GUS transgenic line were cultured in -N MS medium for 5 days (Fig. 5 C, lane 1), followed by subculture in MS medium without N or in medium supplemented with inorganic N (NH₄NO₃) or organic N sources (glutamine or asparagine) for an additional 5 days (Fig. 5 C, lanes 2–5). Total RNA was isolated and analyzed by northern blot hybridization using the gusA coding sequence as a probe. As shown in Fig. 5 C, gusA mRNA accumulated abundantly in cells starved of N for 5 days (lane 1). When the cells were subsequently cultured in medium containing either inorganic or organic N, gusA mRNA accumulation was significantly repressed, and the hybridization signal became almost undetectable (lanes 2–4). In contrast, continued culture in -N medium further enhanced gusA expression (lane 5). These results demonstrate that the transcriptional activity of the OsEP3A promoter is repressed by metabolizable N sources and activated by N starvation in rice cells. Identification of a N responsive sequence in the OsEP3A promoter The results (Fig. 4 , 5 ) showed that expression of the OsEP3A gene was induced in cells grown in medium lacking N sources, and repressed in medium containing N. To investigate the mechanism underlying N-dependent regulation of OsEP3A expression, we analyzed the N starvation responsive sequence ( NSRS ) within the OsEP3A promoter. A 2000 bp upstream regulatory sequence ( URS ) from the OsEP3A promoter region (Fig. 6 A) was subjected to a series of 5′-end serial deletions, internal deletions, and insertions. The 3′ end of all deletion fragments was fixed at the first nucleotide upstream of the TATA box (-35) (Fig. 6 A). The resulting URS fragments of varying lengths were fused upstream of the CaMV 35S minimal promoter and ligated to the gusA reporter gene (Fig. 6 B) to assess promoter activity. Each expression construct was cloned into a binary vector and introduced into rice cells via Agrobacterium -mediated transformation. Three independent T₂ lines from each transgenic construct were used for callus induction, followed by the establishment of suspension-cultured cell lines. These lines were cultured in media + N or -N for 10 days and subjected to qRT-PCR analysis of gusA expression and GUS staining to identify the NSRS within the OsEP3A promoter. Results (Fig. 6 C, D) found that deletions from − 2000 (2000::GUS) up to -837, -531, and − 278 did not affect GUS staining or the -N/+N expression ratio, which remained approximately 4.3- to 6.2-fold. This indicates that gusA expression in these cell lines was still induced under N-starved conditions and repressed when N was available. Further deletions up to -236, -183, and − 113 abolished N responsiveness, as GUS staining and gusA expression were no longer induced under -N conditions and remained at low, similar levels under both conditions, showing nearly 1.0-fold -N/+N ratios. The internal deletion construct D278/236::GUS also lost -N inducibility, whereas D236/183::GUS and D183/113::GUS retained -N induction of gusA expression. Moreover, the insertion constructs A837-236::GUS and A278-236::GUS conferred N starvation inducibility to the minimal CaMV 35S promoter, showing 3.8- and 3.4-fold -N/+N expression ratios, respectively. These results identify the − 278 to -236 bp region of the OsEP3A promoter as essential for N starvation responsiveness, defining it as a putative NSRS . Discussion Nitrogen is an essential macronutrient that regulates multiple aspects of plant growth, development, and metabolism. Plants have evolved complex signaling and regulatory networks to sense, acquire, and modified the RSA for efficiently utilize N (Forde, 2014 ; Krapp et al., 2014 ; Kiba and Krapp, 2016 ). These systems coordinate transcriptional, post-transcriptional, and hormonal responses to maintain N homeostasis. In this study, we characterized the rice cysteine protease gene OsEP3A and its promoter to elucidate its role in N-mediated developmental regulation and transcriptional control under N-limited conditions. OsEP3A positively regulates seedling growth and seed development Our results demonstrate that OsEP3A promotes early seedling growth and seed development. The shoot and crown root lengths were slightly longer in OsEP3A - overexpressing ( Ox ) lines and significantly shorter in RNAi-silenced ( Ri ) lines (Fig. 1 ), suggesting that OsEP3A -mediated proteolysis facilitates N mobilization from storage proteins during germination, thereby playing a positive role in supporting seedling growth. These findings are consistent with our previous study, which showed strong GUS staining localized in the germinating endosperm of OsEP3A::GUS transgenic rice seeds and suggested a functional role for OsEP3A in providing N sources during seed germination (Ho et al., 2000 ). Moreover, GUS staining was also abundant in the anther before flowering but scarcely detected after flowering in florets or spikelets (Fig. 3 ), suggesting a functional role in anther development prior to flowering. A similar expression pattern was observed for OsCP1 , a rice papain-like cysteine protease and ortholog of OsEP3A , whose promoter fused with the GUS reporter gene showed strong expression in immature anthers (Lee et al., 2004 ). The T-DNA-tagged oscp1 mutant exhibited defective pollen development, resulting in reduced seed set, suggesting that OsCP1 plays a positive role in rice pollen development. However, our results (Fig. 2 ) showed that silencing OsEP3A ( Ri lines) produced smaller seeds, while no significant differences in seed size were observed between Ox and wild-type (WT) plants. No differences in seed setting were detected among the WT, Ox, and Ri lines. This indicates that OsEP3A influences seed development but not pollen development, which may explain the lack of changes in seed setting. These differences may reflect functional specificity between the orthologous genes OsEP3A and OsCP1 , which may cooperate to regulate stamen development in rice. This study shows that silencing OsEP3A results in smaller grains, whereas its overexpression has no significant effect, suggesting that a basal level of OsEP3A protease activity is required for normal seed development. These findings indicate that OsEP3A acts as a positive regulator of seedling growth and seed development in rice. OsEP3A expression is induced by N starvation and repressed by metabolizable N To maintain N homeostasis under fluctuating environmental conditions, plants through sophisticated regulatory networks that integrate transcriptional, post-transcriptional, and hormonal control mechanisms to coordinate N uptake, assimilation, and signaling (Forde, 2014 ; Krapp et al., 2014 ; Kiba & Krapp, 2016 ). Under N starvation, several response genes are induced, including high-affinity nitrate transporters (NRT2 family), transcriptional activators such as SNAC1, and signaling factors such as NIN-like proteins (NLPs ) , while repressors such as NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) are repressed, cooperatively fine-tuning N acquisition and utilization (Konishi and Yanagisawa, 2013 ; Sawaki et al., 2013 ; Qi et al., 2023 ; Yang et al., 2025 ). In addition to these well-known regulatory pathways controlling the NRT families involved in N uptake, other mechanisms also contribute to the regulation of N homeostasis. The proteolytic enzymes, particularly papain-like cysteine proteases (PLCPs), play crucial roles in N remobilization, senescence, and stress adaptation (van der Hoorn et al., 2004 ; Liu et al., 2018 ; Li et al., 2022 ; Xu et al., 2025 ). Despite their broad physiological importance, the role of PLCPs in N signaling remains poorly understood. In this study, our results (Fig. 4 ) demonstrated that OsEP3A transcript accumulation was markedly enhanced under N starvation and rapidly repressed by metabolizable N sources, including both inorganic (NH₄NO₃) and organic (glutamine, asparagine) forms (Figs. 4 and 5 ). This dual responsiveness suggests a feedback mechanism in which N sufficiency represses OsEP3A transcription to modulate proteolytic N recycling. Such regulation mirrors the transcriptional behavior of other N starvation inducible genes, including nitrate transporter NRT2.1 , NRT2.4 and NRT2.5 (Ma et al., 2015 ; Kiba et al., 2012 , 2018 ), nitrate reductase ( NIA1 and NIA2 ) and the nitrite reductase ( NIR ) (Medici and Krouk, 2014 ) in Arabidopsis , whose expression is tightly controlled by N availability. Moreover, under N-limited hydroponic conditions, OsEP3A-RNAi seedlings exhibited severe stunting growth, while wild-type and overexpression lines maintained near-normal growth (Fig. 4 ). These phenotypic differences were minimized under N sufficient conditions, indicating that OsEP3A is important during N scarcity. The failure of OsEP3A - RNAi lines to sustain growth under N limitation highlights the importance of cysteine protease OsEP3A-mediated proteolysis for internal N remobilization for providing amino acid substrates for metabolism when external N sources are restricted. Moreover, the OsEP3A::GUS analyses confirmed that the OsEP3A promoter is transcriptionally activated under N starvation and repressed upon N supply (Fig. 5 ). Strong GUS activity and elevated gusA expression were observed under N deficient conditions in OsEP3A::GUS transgenic lines, whereas supplementation with either inorganic or organic N suppressed promoter activity. These results indicate that OsEP3A regulation depends on N metabolizability rather than N form, consistent with the broader principle of metabolic feedback control observed in other N responsive genes (Yang et al., 2015 ; kong et al., 2021 ; Zhang et al., 2025 ). Identification of an N starvation responsive sequence ( NSRS ) in the OsEP3A promoter Although numerous studies have identified genes whose expression is activated by N starvation, such as NRT2.1 , NRT2.4 , and NRT2.5 (Wang et al., 2018 ; Zhang et al., 2025 ), there remains limited information regarding the identification of N starvation responsive cis-regulatory sequences. In Arabidopsis , the NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) protein functions as a transcriptional repressor that binds to one GAATATTC motif and three GAATC motifs within the promoter region of the high affinity nitrate transporter NRT2.4 (254–389 bp upstream of the translation start site) under high N availability, thereby repressing the expression of N starvation responsive genes (Kiba et al., 2018 ). In contrast, our study identified a 43 bp region (-236 to -278) upstream of the OsEP3A transcription start site as essential for activating N starvation responsiveness (Fig. 6 ). Deletion of this 43 bp DNA fragment abolished GUS induction under N starvation, whereas insertion of the fragment upstream of a minimal CaMV 35S promoter conferred N starvation inducible expression. These results define the − 236 to -278 region as a N starvation responsive sequence ( NSRS ), which likely contains a novel cis-element recognized by transcriptional activators involved in N starvation signaling. In soybean ( Glycine max L.), the NAC transcription factor GmNAC039 functions as a positive regulator of nodule senescence (Yu et al., 2023 ). Electrophoretic mobility shift assay (EMSA) analysis revealed that GmNAC039 directly binds to a CACAAA motif located in the GmCYP37 promoter region (93–121 bp upstream of the translation start codon), thereby activating GmCYP37 expression during nodule senescence. Overexpression or knockout of GmCYP37 in nodules accelerated or delayed senescence, respectively, indicating that GmNAC039 promotes nodule senescence by directly activating GmCYP37 through binding to the CACAAA motif. Our study defines a 43 bp NSRS within the OsEP3A promoter that mediates transcriptional activation under N starvation. As described above, the transcriptional repressor NIGT1 binds to GAATATTC and GAATC cis-elements in the NRT2.4 promoter under N replete conditions to repress N starvation responsive genes, while the CACAAA motif in the GmCYP37 promoter serves as a binding site for the transcriptional activator GmNAC039 during nodule senescence. To date, no cis-element has been characterized as an N starvation activating motif. In this study, the 43 bp NSRS sequence in the OsEP3A promoter appears to function as a positive regulatory element in response to N starvation. Notably, no cis-element similarity was found between the NSRS and previously characterized motifs such as GAATATTC or CACAAA. We therefore propose that the NSRS may interact with previously unidentified transcriptional regulators that activate OsEP3A expression, thereby enhancing its proteolytic activity to supply N under N-limited conditions. Further protein-DNA interaction studies will clarify how OsEP3A transcription is modulated in response to N availability. In conclusion, this study identifies OsEP3A as an N starvation activated cysteine protease that enhances seedling growth and seed development in rice. We further define a 43 bp N starvation responsive sequence within the OsEP3A promoter that mediates transcriptional activation under N deficient conditions. These findings uncover a previously uncharacterized regulatory mechanism in rice N signaling and suggest that OsEP3A could serve as a molecular target for improving N utilization during cereal crop development. Declarations Data availability The data supporting the findings of this study are available within the article and its supplementary materials from the date of publication, and may also be obtained from the corresponding author upon reasonable request. Author contributions Shin Lon Ho conceived and designed the experiments. Shinn Jia Tzeng performed agronomic trait measurements and statistical analyses. Yi Hsuan Hou constructed the expression vectors and carried out Agrobacterium-mediated gene transformation. Shin Lon Ho analyzed the data and wrote the manuscript. Funding This work was supported by grants from the National Science and Technology Council of Taiwan (Grant No. NSTC 114-2313-B-415-003- and MOST 111-2313-B-415-004-). Competing interests The authors declare no competing interests. References Amack SC, Antunes MS (2020) CaMV35S promoter – A plant biology and biotechnology workhorse in the era of synthetic biology. 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17:51:15","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185671,"visible":true,"origin":"","legend":"","description":"","filename":"9419ec9d8e5749519e7cccf45be4a4221structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/a525778cc6b149b2868d3079.xml"},{"id":99249125,"identity":"a960f16c-f567-45b0-af01-c74bfcd921d2","added_by":"auto","created_at":"2025-12-30 17:51:15","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":204678,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/9a1be371bcf1d1bcbf6059c7.html"},{"id":99249101,"identity":"4f702327-cdec-49ae-a424-1feadabbb4dc","added_by":"auto","created_at":"2025-12-30 17:51:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":403843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEctopic expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsEP3A-Ox\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsEP3A-Ri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e constructs in transgenic rice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic maps of the overexpression of \u003cem\u003eOsEP3A-Ox\u003c/em\u003e and the double strand RNA gene silencing \u003cem\u003eOsEP3A-Ri \u003c/em\u003econstructs. (B) qRT-PCR analysis of \u003cem\u003eOsEP3A\u003c/em\u003e in 5-day-old of WT and T\u003csub\u003e3 \u003c/sub\u003etransgenic rice seedlings. (C) Phenotypes of WT, \u003cem\u003eOsEP3A-Ox\u003c/em\u003e, and \u003cem\u003eOsEP3A-Ri\u003c/em\u003e seedlings 10 days after germination. (D) Quantitative analysis of shoot length as shown in (C). (E) Quantitative analysis of root length as shown in (C). Each value is the mean ± \u003cem\u003eSD\u003c/em\u003e of three independent measurements. Different letters above the bars indicate significant differences, identified by performing ANOVA, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 for (B), and P\u0026lt;0.01 for (D) and (E).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/3ea0149550c91fe828f2fc36.png"},{"id":99249104,"identity":"bea54f56-516a-4e26-b0d1-ef011230d22b","added_by":"auto","created_at":"2025-12-30 17:51:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265124,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe grain length and width decrease in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsEP3A-Ri\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Phenotypic analysis of WT, \u003cem\u003eOsEP3A-Ox\u003c/em\u003e, and \u003cem\u003eOsEP3A-Ri\u003c/em\u003e grains. (C) Quantitative analysis of grain length as shown in (A). (D) Quantitative analysis of grain width as shown in (B). Fifteen seeds per line were positioned in a row. Columns represent means ± \u003cem\u003eSD\u003c/em\u003e (n = 45). Different letters above the bars indicate significant differences, identified by performing ANOVA (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/eff9c4706d16aa260b3fce0a.png"},{"id":99249102,"identity":"c44382c0-89ce-48f4-b487-cc548763f1c7","added_by":"auto","created_at":"2025-12-30 17:51:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":277851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistochemical staining showing GUS activity in anthers before flowering.\u003c/strong\u003e (A) Schematic diagram of the \u003cem\u003eOsEP3A::GUS\u003c/em\u003e expression construct. The black block represents the 5’-untranslated region (5’-UTR) in \u003cem\u003eOsEP3A\u003c/em\u003e mRNA. (B) GUS staining of spikelets before flowering (BF) and 1, 3, 6, 20 days after flowering of the developing seeds.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/c4b9229353d6d9a1e2c91a46.png"},{"id":99249105,"identity":"169434cf-d25f-46a4-90c2-62e0c0d821c8","added_by":"auto","created_at":"2025-12-30 17:51:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":278323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of N availability on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsEP3A \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression and seedling growth in rice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Rice suspension cells were cultured in MS medium without N (-N) or in medium containing 10 mM NH₄NO₃ or 10 mM KNO₃ as the sole N source for 5 or 10 days. Total RNA was isolated and analyzed by Northern blot hybridization using a DIG-labeled \u003cem\u003eOsEP3A\u003c/em\u003e \u003cem\u003e3′-UTR \u003c/em\u003eprobe. (B) Rice seeds from WT, \u003cem\u003eOx-1\u003c/em\u003e, and \u003cem\u003eRi-1\u003c/em\u003e were germinated in Kimura hydroponic solution containing N sources (0.37 mM (NH₄)₂SO₄ and 0.23 mM KNO₃) (+N) or lacking N (-N) for 10 days, and then photographed. (C) Quantitative analysis of shoot length as shown in (B).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/c8f808102a5b077ec0b763b9.png"},{"id":99319296,"identity":"2e2c9773-00ee-4a00-9c22-d69c6fa69bff","added_by":"auto","created_at":"2025-12-31 16:36:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":189838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe OsEP3A promoter is activated under N starvation conditions\u003c/strong\u003e\u003cbr\u003e\nRice calli derived from three independent \u003cem\u003eOsEP3A::GUS \u003c/em\u003etransgenic lines were induced from immature seeds and subsequently cultured as suspension cells. These suspension cells were then grown in MS medium with or without N for 10 days, followed by (A) GUS staining or (B) qRT-PCR analysis. (C) Calli derived from an \u003cem\u003eOsEP3A::GUS \u003c/em\u003etransgenic line were cultured in N deficient MS medium for 5 days (lane 1), and then subcultured for an additional 5 days in MS medium without N (lane 5) or in medium supplemented with inorganic N (NH₄NO₃) or organic N sources (glutamine or asparagine) (lanes 2-4). Total RNA was isolated and analyzed by Northern blot hybridization using a DIG-labeled \u003cem\u003egusA \u003c/em\u003ecoding sequence as a probe\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/20c4ebe6f4f2d25679a5d46b.png"},{"id":99318153,"identity":"5853bca5-6e7a-421e-9284-cd70029c478a","added_by":"auto","created_at":"2025-12-31 16:31:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":254616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of a N responsive sequence in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsEP3A\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter. \u003c/strong\u003e(A) Upper panel: Schematic representation of the 2000 bp \u003cem\u003eOsEP3A::GUS\u003c/em\u003eexpression vector construct. Lower panel: Schematic representation of the TATA-less 2000 bp promoter region fused to the\u003cem\u003e CaMV 35S\u003c/em\u003e minimal promoter, followed by the \u003cem\u003egusA\u003c/em\u003e reporter gene. (B) Schematic representation of a series of 5′-end serial deletions, internal deletions, and insertions generated using PCR-based methods. The 3′ end of all fragments was fixed at the first nucleotide upstream of the TATA box (-35). The resulting DNA fragments of varying lengths were cloned upstream of the \u003cem\u003eCaMV 35S\u003c/em\u003e minimal promoter to drive \u003cem\u003egusA\u003c/em\u003e expression. (C) and (D) Analysis of \u003cem\u003eOsEP3A::GUS\u003c/em\u003e gene expression and GUS activity staining. Immature seeds derived from three independent T₂ lines for each transgenic construct were used for callus induction, followed by the establishment of suspension-cultured cell lines. These lines were cultured in MS medium containing N (+N) or lacking N (-N) for 10 days, then subjected to qRT-PCR analysis of \u003cem\u003egusA\u003c/em\u003e expression (C) and GUS activity staining\u003cstrong\u003e \u003c/strong\u003e(D) to identify the N starvation responsive sequences within the \u003cem\u003eOsEP3A\u003c/em\u003e promoter. The relative expression level of the \u003cem\u003eGUS\u003c/em\u003e gene was normalized to that of the internal control \u003cem\u003eOsActin\u003c/em\u003e. The expression level of \u003cem\u003eGUS\u003c/em\u003e in the N containing (+N) 35mp::GUS line was set to 1.0. Data represent means ± SD from three independent experiments (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/ff379b47d844586d638f4b16.png"},{"id":107928060,"identity":"210021a2-957f-40ed-bb4d-5d34af6f6859","added_by":"auto","created_at":"2026-04-27 16:06:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2201771,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/de6f3701-08ae-4463-a8f5-420db055154f.pdf"},{"id":99249103,"identity":"6acd77bd-c870-4313-8001-4045455440fe","added_by":"auto","created_at":"2025-12-30 17:51:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":816273,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8421153/v1/928ec23fc702613ff607911d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Rice Cysteine Protease OsEP3A Promotes Seedling Growth and Seed Development and Contains a Nitrogen Starvation Responsive Sequence","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNitrogen (N) is an essential macronutrient that frequently limits plant growth, development, and productivity (Miller and Cramer, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). To cope in N deficient conditions, plants have evolved intricate regulatory networks that modulate N acquisition, assimilation, and remobilization to optimize N use efficiency (NUE). These adaptive mechanisms include transcriptional and post-transcriptional regulation, root architectural adjustments, and coordinated signaling between N and carbon metabolism (Forde, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Krapp et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kiba and Krapp, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNitrogen acquisition and signaling involve multiple uptake systems. Plants utilize a low-affinity transport system (LATS) when external nitrate (NO₃⁻) concentrations exceed 0.5 mM, and a high-affinity transport system (HATS) under lower concentrations (\u0026lt;\u0026thinsp;0.5 mM) (Wang et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Nitrate uptake and assimilation depend on nitrate transporter (\u003cem\u003eNRT\u003c/em\u003e) genes and associated enzymes. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, two major families of nitrate transporters, \u003cem\u003eNRT1\u003c/em\u003e and \u003cem\u003eNRT2\u003c/em\u003e, both play critical roles in nitrate absorption and transport. NRT1 proteins belong to the LATS and function primarily when external nitrate concentrations are high, whereas NRT2 proteins are part of the HATS and mediate nitrate uptake under low-nitrate conditions (Esteban et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNitrogen signaling involves intricate regulatory components that orchestrate gene expression and metabolic pathways in response to N availability. Genome-wide transcriptome analyses have identified thousands of nitrate responsive genes, including those encoding hormone related proteins, kinases, transcription factors, transport proteins, and metabolic enzymes (Wang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Bi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Under N deficient conditions, plants exhibit distinct physiological and molecular responses such as reduced photosynthetic activity (Ding et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), enhanced amino acid remobilization from older leaves (Ono et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), accelerated chlorophyll degradation, and increased anthocyanin accumulation (D\u0026iacute;az et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). At the molecular level, N starvation induces HATS components to facilitate nitrate uptake. NRT1.1 acts as a dual-affinity nitrate transceptor that senses and transports nitrate, thereby initiating primary nitrate responses (Liu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, the expression of high-affinity nitrate transporter genes such as NRT2.1 (Filleur et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and transcription factors like AtNsr1 (Todd et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) promotes efficient nitrate uptake and utilization. In \u003cem\u003eArabidopsis\u003c/em\u003e, AtNRT2.4 is recognized as an N starvation marker gene responsive to N deficiency (Kiba et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, 2016). The transcriptional repressor NIGT1/HRS1 binds to the promoter of \u003cem\u003eAtNRT2.4\u003c/em\u003e to repress N deficient responses under high N availability, indicating its key role in balancing N acquisition and utilization under fluctuating N conditions (Kiba et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Overexpression of \u003cem\u003eAtNRT2.4\u003c/em\u003e significantly enhances plant height, silique number, biomass, and seed yield under normal N conditions, while also promoting anthocyanin accumulation under low nitrate stress. These findings underscore the multifaceted role of AtNRT2.4 in the hierarchical regulation of N signaling and plant adaptive responses. (Zhang et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn rice, the salt- and drought-tolerant transcription factor OsSNAC1 (Hu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; You et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) has been shown to bind to and activate the promoters of nitrate responsive genes, including \u003cem\u003eOsNRT2.1\u003c/em\u003e, \u003cem\u003eOsNRT2.2\u003c/em\u003e, \u003cem\u003eOsNRT1.1A\u003c/em\u003e, and \u003cem\u003eOsNRT1.1B\u003c/em\u003e, thereby enhancing nitrate acquisition. Overexpression of \u003cem\u003eOsSNAC1\u003c/em\u003e enhances nitrate uptake, root development, and grain yield, suggesting a role in coordinating stress responses under N limitation and drought conditions (Qi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, OsHHO3 acts as a transcriptional repressor of \u003cem\u003eAMMONIUM TRANSPORTER1\u003c/em\u003e genes, where reduced \u003cem\u003eOsHHO3\u003c/em\u003e expression improves ammonium uptake under N limitation (Yang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePost-transcriptional mechanisms further fine tunes plant responses to N. The expression of microRNAs (miRNAs) is differentially regulated by N deprivation (Paul et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kong et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). N deficiency alters the expression of specific miRNAs that regulate root system architecture (RSA) and stress adaptation. In rice, decreased expression of miR164 and miR167 is closely associated with low N tolerance (Nischal et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In wheat, miR1118 is N responsive and contributes to RSA development under N starvation (Sinha et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similarly, in \u003cem\u003eArabidopsis\u003c/em\u003e, overexpression of miR160 promotes lateral root formation under N-limited conditions (Liang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlant hormones also play a key role in N signaling. In \u003cem\u003eArabidopsis\u003c/em\u003e and maize, limited N availability enhances auxin transport from shoots to roots, promoting lateral root development through auxin-dependent acid growth and the TARGET OF RAPAMYCIN (TOR) kinase signaling pathway (Tian et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Asim et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Stitz et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nitrate availability also regulates root development; low NO₃⁻ levels promote root elongation, whereas high levels suppress it via abscisic acid (ABA)-mediated signaling (Signora et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). In wheat, increased ABA content and upregulation of \u003cem\u003ezeaxanthin epoxidase\u003c/em\u003e under low N stress further support the role of ABA in N deficiency tolerance (Kang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mahmoud et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additionally, calcium signaling also contributes to N stress responses as elevated Ca\u0026sup2;⁺ levels modulate the expression of N responsive genes (Zhang et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Calcium-dependent protein kinases (CPKs) phosphorylate NIN-LIKE PROTEIN (NLP) transcription factors, thereby activating downstream nitrate responsive pathways (Liu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), thus exemplifying the crosstalk between calcium and nitrate signaling in plants.\u003c/p\u003e \u003cp\u003eProteases are ubiquitous that include diverse families (such as cysteine-, serine-, aspartic- and metallo-protease, etc.) and act roles in protein proteolysis to mediate protein turnover, processing, and degradation across all life forms (van der Hoorn and Rivas, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Among them, Papain-like cysteine proteases (PLCPs) are widespread enzymes that play crucial roles in plant growth, development, and immunity (Liu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Characterization by a conserved catalytic triad (Cys-His-Asn), PLCPs are synthesized as inactive precursors and participate protein degradation that functional correlation with plant senescence, seed germination, pollen formation, and responses to biotic and abiotic stresses (Liu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mart\u0026iacute;nez et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, the PLCP \u003cem\u003eRESPONSIVE TO DEHYDRATION 21 (RD21)\u003c/em\u003e is essential for defense against necrotrophic pathogens, while in wheat, \u003cem\u003eTaRD21A\u003c/em\u003e enhances resistance to wheat yellow mosaic virus through release small peptides (Shindo et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In rice (\u003cem\u003eOryza sativa\u003c/em\u003e), genome-wide analyses have identified multiple PLCP families with diverse expression patterns and regulatory functions, including roles in seed development and stress response (Wang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ni\u0026ntilde;o et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For instance, root specific PLCPs such as \u003cem\u003eOsCP14\u003c/em\u003e, \u003cem\u003eOsCP16\u003c/em\u003e, \u003cem\u003eOsCP19\u003c/em\u003e, and \u003cem\u003eOsCP25\u003c/em\u003e are crucial for maintaining soil microbial colonization (Xu et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The oryzain alpha chain precursor (OCP), a rice ortholog of \u003cem\u003eArabidopsis\u003c/em\u003e RD21, was shown to negatively regulate resistance to rice blast disease. Loss-of-function of OCP using CRISPR/Cas9 enhanced blast resistance by activating jasmonic acid (JA) and ethylene (ET) signaling pathways while repressing auxin signaling pathways (Li et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFew studies have explored the role of cysteine proteinases in response to N starvation in plants. Previous research has shown that the abundances of Rubisco greatly decreases in the old leaves of \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e under N deficient conditions (Crafts-Brandner et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Izumi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The \u003cem\u003eSenescence-Associated Gene 12\u003c/em\u003e (\u003cem\u003eSAG12\u003c/em\u003e), which encodes a vacuolar cysteine protease, is highly induced during leaf senescence (Lohman et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Otegui et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Therefore, \u003cem\u003eSAG12\u003c/em\u003e is proposed to participate in the degradation of Rubisco proteins in the vacuoles of senescing leaves. In previous studies (Ho et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), we found that expression of the rice cysteine protease gene \u003cem\u003eOsEP3A\u003c/em\u003e is induced by N starvation in suspension-cultured cells, germinating seeds, and senescing leaves, suggesting that \u003cem\u003eOsEP3A\u003c/em\u003e participates in protein degradation to supply N for plant growth.\u003c/p\u003e \u003cp\u003eThese findings highlight the pivotal role of N sensing, acquisition, transport, assimilation, and the modulation of N signaling in maintaining N homeostasis throughout the plant life cycle. Despite extensive research on nitrate transporters and transcriptional regulation, the cis-regulatory elements involved in the N starvation responsive expression of proteolytic genes remain poorly characterized. In this study, we conducted a promoter deletion and insertion analysis to identify the N starvation responsive sequence (\u003cem\u003eNSRS\u003c/em\u003e) within the promoter of the rice \u003cem\u003eOsEP3A\u003c/em\u003e gene, aiming to elucidate the molecular mechanisms underlying N-mediated regulation of \u003cem\u003ecysteine protease\u003c/em\u003e (\u003cem\u003eOsEP3A\u003c/em\u003e) expression in rice.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eThe rice cultivar \u003cem\u003eOryza sativa\u003c/em\u003e L. cv. Tainung 67 was used throughout this study. Among the \u003cem\u003eOsEP3A overexpression\u003c/em\u003e (\u003cem\u003eOsEP3A\u003c/em\u003e-\u003cem\u003eOx\u003c/em\u003e) and \u003cem\u003eRNA interference\u003c/em\u003e (\u003cem\u003eOsEP3A\u003c/em\u003e-\u003cem\u003eRi\u003c/em\u003e) transgenic lines, three independent T₃ lines carrying a single-copy transgene were selected (Fig. S1) to evaluate the functional role of \u003cem\u003eOsEP3A\u003c/em\u003e in rice development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCallus induction\u003c/h3\u003e\n\u003cp\u003eImmature rice seeds collected 10 days after flowering were air-dried for 7 days. Thirty dehulled seeds were sterilized with 2.4% NaOCl for 30 minutes and rinsed thoroughly with sterile water. The sterilized seeds were cultured on callus induction medium containing N6 salts (Chu et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1975\u003c/span\u003e), 10 \u0026micro;M 2, 4-D, and 3% sucrose. Cultures were maintained at 28\u0026deg;C under a 16 h/8 h light/dark photoperiod with a light intensity of 3500 lux (Ho et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). After 30 days, calli were transferred to fresh medium and cultured for an additional 7 days before \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation.\u003c/p\u003e\n\u003ch3\u003ePrimer sequences\u003c/h3\u003e\n\u003cp\u003eThe nucleotide sequences of all primers used for PCR, RT-PCR, and qRT-PCR are listed in Supplementary Table S1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of\u003c/b\u003e \u003cb\u003eOsEP3A-Ox, OsEP3A-Ri\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eOsEP3A::GUS\u003c/b\u003e \u003cb\u003evectors\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo construct the \u003cem\u003eOsEP3A\u003c/em\u003e overexpression vector (\u003cem\u003eOsEP3A\u003c/em\u003e-\u003cem\u003eOx\u003c/em\u003e), a 1007-bp fragment containing the full coding sequence of \u003cem\u003eOsEP3A\u003c/em\u003e was amplified using primers \u003cem\u003eOsEP3A-F1\u003c/em\u003e and \u003cem\u003eOsEP3A-R1\u003c/em\u003e. The PCR product was digested with \u003cem\u003eBam\u003c/em\u003eHI and ligated into the \u003cem\u003eBam\u003c/em\u003eHI\u003cem\u003e-\u003c/em\u003edigested \u003cem\u003epAHC18\u003c/em\u003e vector under the control of the maize ubiquitin promoter (Bruce et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). The resulting plasmid was linearized with \u003cem\u003eHin\u003c/em\u003edIII and inserted into the \u003cem\u003eHin\u003c/em\u003edIII site of the binary vector pSMY1H (Ho et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003eOsEP3A\u003c/em\u003e RNA interference construct (\u003cem\u003eOsEP3A-Ri\u003c/em\u003e), a 420-bp fragment comprising 100 bp of the 3\u0026prime; coding region and 320 bp of the 3\u0026prime; untranslated region of \u003cem\u003eOsEP3A\u003c/em\u003e was amplified using primers \u003cem\u003eOsEP3A-Xho\u003c/em\u003eI and \u003cem\u003eOsEP3A-Kpn\u003c/em\u003eI, and \u003cem\u003eOsEP3A-Xba\u003c/em\u003eI and \u003cem\u003eOsEP3A-Hin\u003c/em\u003edIII, respectively. The two DNA fragments were inserted downstream of the maize ubiquitin promoter in both sense and antisense orientations, separated by a truncated \u003cem\u003eGFP\u003c/em\u003e spacer (Ho et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The construct was linearized with \u003cem\u003ePst\u003c/em\u003eI and inserted into the \u003cem\u003ePst\u003c/em\u003eI site of \u003cem\u003epSMY1H\u003c/em\u003e binary vector.\u003c/p\u003e \u003cp\u003eTo generate the \u003cem\u003eOsEP3A::GUS\u003c/em\u003e reporter construct, a 2015-bp promoter fragment including the 5\u0026prime; untranslated region (upstream of the ATG start codon) was amplified using primers \u003cem\u003eOsEP3A-5P\u003c/em\u003e and \u003cem\u003eOsEP3A-3B\u003c/em\u003e. The PCR product was digested with \u003cem\u003ePst\u003c/em\u003eI and \u003cem\u003eBam\u003c/em\u003eHI and ligated upstream of the \u003cem\u003egusA\u003c/em\u003e reporter gene in the \u003cem\u003epBX-2\u003c/em\u003e vector (Ho et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The resulting plasmid was linearized with \u003cem\u003ePst\u003c/em\u003eI and inserted into the \u003cem\u003ePst\u003c/em\u003eI site of the \u003cem\u003epSMY1H\u003c/em\u003e binary vector.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of serial deletion\u003c/b\u003e \u003cb\u003eOsEP3A::GUS\u003c/b\u003e \u003cb\u003evectors\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify the \u003cem\u003eNSRS\u003c/em\u003e within the \u003cem\u003eOsEP3A\u003c/em\u003e promoter, a 2000 bp upstream regulatory sequence (\u003cem\u003eURS\u003c/em\u003e) from the \u003cem\u003eOsEP3A\u003c/em\u003e promoter region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) was subjected to a series of 5\u0026prime;-end serial deletions, internal deletions, and insertions using PCR-based methods. The 3\u0026prime; end of all fragments was fixed at the first nucleotide upstream of the TATA box (-35) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The resulting \u003cem\u003eURS\u003c/em\u003e fragments of varying lengths were digested with \u003cem\u003eHin\u003c/em\u003edIII and \u003cem\u003eBam\u003c/em\u003eHI and cloned upstream of the \u003cem\u003eCaMV 35S\u003c/em\u003e minimal promoter (Amack and Antunes, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) to drive the \u003cem\u003egusA\u003c/em\u003e reporter gene using the same restriction sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The constructed vectors were used to assess N starvation responsive activity. In total, seven \u003cem\u003eURS\u003c/em\u003e serial deletion constructs were generated: \u003cem\u003e2000::GUS\u003c/em\u003e (-2000 to -35), \u003cem\u003e837::GUS\u003c/em\u003e, \u003cem\u003e531::GUS\u003c/em\u003e, \u003cem\u003e278::GUS\u003c/em\u003e, \u003cem\u003e236::GUS\u003c/em\u003e, \u003cem\u003e183::GUS\u003c/em\u003e, and \u003cem\u003e113::GUS\u003c/em\u003e. Three internal deletion constructs were also produced by removing fragments from \u0026minus;\u0026thinsp;236 to -278, -183 to -236, or -113 to -183 of the \u003cem\u003e837::GUS\u003c/em\u003e construct, and designated as \u003cem\u003eD278/236::GUS\u003c/em\u003e, \u003cem\u003eD236/183::GUS\u003c/em\u003e, and \u003cem\u003eD183/113::GUS\u003c/em\u003e, respectively. In addition, two insertion constructs containing fragments from \u0026minus;\u0026thinsp;236 to -837 or -236 to -278 were fused upstream of the \u003cem\u003eCaMV 35S\u003c/em\u003e minimal promoter and named \u003cem\u003eA837-236::GUS\u003c/em\u003e and \u003cem\u003eA278-236::GUS\u003c/em\u003e. The \u003cem\u003eCaMV 35S\u003c/em\u003e minimal promoter fused only to the \u003cem\u003egusA\u003c/em\u003e reporter gene served as a control and was designated \u003cem\u003e35mp::GUS\u003c/em\u003e. Each expression construct was cloned into a binary vector and introduced into rice cells via \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. Three independent T₂ lines from each transgenic construct were used for callus induction, followed by the establishment of suspension-cultured cell lines. These lines were cultured in media containing N (+\u0026thinsp;N) or lacking N (-N) for 10 days and subjected to qRT-PCR analysis of \u003cem\u003egusA\u003c/em\u003e expression and GUS staining to identify the N responsive sequence within the \u003cem\u003eOsEP3A\u003c/em\u003e promoter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePlant transformation\u003c/h3\u003e\n\u003cp\u003eTransgenic rice plants were generated via \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. Recombinant plasmids were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA101 by electroporation, and the transformed bacteria were co-cultivated with rice calli to transfer the T-DNA, following established protocols (Ho et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhenotypic analysis\u003c/h3\u003e\n\u003cp\u003eDehulled rice seeds were surface-sterilized with 2.4% NaOCl for 30 min, rinsed thoroughly with sterile water, and sown on half-strength MS (Murashige and Skoog, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) solid medium supplemented with 1.5% sucrose. Cultures were maintained at 28\u0026deg;C under a 16 h photoperiod with a light intensity of 3500 lux. After 4 days, seedlings were transferred to soil-filled pots and grown for either 8 days (12-day-old plants; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) or 5 days (9-day-old plants; Fig. S2B). Phenotypic traits including shoot length and root length were measured. Statistical analyses were performed using one-way ANOVA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistochemical GUS staining\u003c/h2\u003e \u003cp\u003eRice suspension-cultured cells were grown in MS medium with or without N for 5 days and then collected for GUS staining. For GUS staining of developing seeds, spikelets were collected before flowering and at 1, 3, 6, and 20 days after flowering, followed by GUS staining. Samples were incubated at 37\u0026deg;C in the dark for 4 hours in a staining buffer containing 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.1% Triton X-100, and 1 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc). After staining, samples were stored in 70% ethanol, rinsed with water, and imaged.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative RT-PCR\u003c/h3\u003e\n\u003cp\u003eThirty dehulled and sterilized rice seeds were sown on half-strength MS medium containing 1.5% sucrose and cultured under a 16 h/8 h light/dark photoperiod at 28\u0026deg;C with a light intensity of 3500 lux. Five-day-old seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) of WT, \u003cem\u003eOsEP3A\u003c/em\u003e-\u003cem\u003eRi\u003c/em\u003e, and \u003cem\u003eOsEP3A\u003c/em\u003e-\u003cem\u003eOx\u003c/em\u003e lines, as well as calli treated with or without N for 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e), were collected. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and treated with the TURBO DNA-free kit (Ambion, USA). First-strand cDNA was synthesized from 5 \u0026micro;g of RNA using M-MuLV reverse transcriptase (New England Biolabs, USA) and oligo (dT) primers. Quantitative RT-PCR was performed on an Eco Real-Time PCR System (Illumina, USA) following the manufacturer\u0026rsquo;s instructions. Gene-specific primers targeting the 3\u0026prime; untranslated region of \u003cem\u003eOsEP3A\u003c/em\u003e and the GUS coding sequence were used (Table S1). Expression levels were normalized to \u003cem\u003eOsActin1\u003c/em\u003e, with WT set to 1.0. All reactions were performed in triplicate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSouthern blot analysis\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from 5-d-old seedlings using the standard phenol-chloroform method. A total of 10 \u0026micro;g of genomic DNA was digested with \u003cem\u003eHin\u003c/em\u003edIII at 37\u0026deg;C for 2 h. The digested DNA was separated on agarose gel in TBE buffer. The DNA was then depurinated by incubating the gel in 0.2 N HCl for 10 min, followed by denaturation in denaturation solution (1.5M NaCl, 0.5M NaOH) for 45 min and neutralization in neutralization solution (1M Tris pH 7.4, 1.5M NaCl) for 30 min. The DNA was transferred to a nylon membrane using capillary transfer with 10 X SSC buffer overnight. After transfer, the membrane was UV-crosslinked to fix the DNA. The membrane was then hybridized with the DIG-labeled \u003cem\u003eHpt\u003c/em\u003e probe using the DIG-High Prime DNA Labeling and Detection Starter Kit according to the supplier\u0026rsquo;s instructions (Roche).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eNorthern blot analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from rice suspension cells using TRIzol reagent according to the manufacturer\u0026rsquo;s instructions. Northern blot was performed with the DIG-labeled 420 bp of \u003cem\u003eOsEP3A\u003c/em\u003e specific region (containing 120 bp downstream coding sequence followed by 300 bp 3\u0026rsquo;-untranslated region) as a probe using the DIG-High Prime DNA Labeling and Detection Starter Kit according to the supplier\u0026rsquo;s instructions (Roche).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed with at least three biological replicates. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s test using SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA). Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e) or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePhenotypic changes in transgenic rice plants overexpressing or silenced for\u003c/b\u003e \u003cb\u003eOsEP3A\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe expression of a rice cysteine proteinase gene, \u003cem\u003eOsEP3A\u003c/em\u003e, is induced by gibberellic acid (GA) in germinating seeds, suggesting that it plays a role in degrading storage proteins in the endosperm to provide N sources required for seedling growth (Ho et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The expression of \u003cem\u003eOsEP3A\u003c/em\u003e is also induced under N starvation in suspension-cultured cells and is subjected to feedback repression by supplemental N (Ho et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In this study, both gene overexpression and RNA interference (\u003cem\u003eRNAi\u003c/em\u003e)-mediated gene silencing approaches were used to dissect the role of \u003cem\u003eOsEP3A\u003c/em\u003e in rice seedling growth and to identify the promoter sequences responsive to N starvation in rice suspension-cultured cells. The expression vectors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) for overexpression (\u003cem\u003eOsEP3A\u003c/em\u003e-\u003cem\u003eOx\u003c/em\u003e, referred to as \u003cem\u003eOx\u003c/em\u003e) and RNA interference (\u003cem\u003eOsEP3A\u003c/em\u003e-\u003cem\u003eRi\u003c/em\u003e, referred to as \u003cem\u003eRi\u003c/em\u003e) were constructed under the control of the maize ubiquitin (\u003cem\u003eUbi\u003c/em\u003e) promoter (Christensen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1992\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAfter \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of rice calli and regeneration of the transgenic plants, five overexpression lines (a total of 11 independent lines) and three \u003cem\u003eRNAi\u003c/em\u003e-silenced lines (a total of 8 independent lines) carrying single-copy transgenes were confirmed by Southern blot hybridization using the hygromycin phosphotransferase gene (\u003cem\u003eHpt\u003c/em\u003e) as a probe (Fig. S1). Among these, three independent single-copy lines were selected from each construct and designated as \u003cem\u003eOx-1\u003c/em\u003e, \u003cem\u003eOx-2\u003c/em\u003e, and \u003cem\u003eOx-3\u003c/em\u003e, and \u003cem\u003eRi-1, Ri-2\u003c/em\u003e, and \u003cem\u003eRi-3\u003c/em\u003e, respectively (Fig. S1).\u003c/p\u003e \u003cp\u003eTo validate the expression levels of \u003cem\u003eOsEP3A\u003c/em\u003e mRNA in the different transgenic lines, thirty T\u003csub\u003e3\u003c/sub\u003e transgenic seeds from each line were dehulled, sterilized, and germinated on half-strength MS medium for 5 days. Embryos along with shoots were collected for total RNA extraction, and \u003cem\u003eOsEP3A\u003c/em\u003e mRNA accumulation was analyzed by qRT-PCR. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) showed that \u003cem\u003eOsEP3A\u003c/em\u003e mRNA was highly expressed in the three \u003cem\u003eOsEP3A\u003c/em\u003e-overexpressing lines, whereas endogenous \u003cem\u003eOsEP3A\u003c/em\u003e expression was either completely absent or very low in the \u003cem\u003eRNAi\u003c/em\u003e-silenced lines.\u003c/p\u003e \u003cp\u003eTo examine the physiological function effect of \u003cem\u003eOsEP3A\u003c/em\u003e on rice seedling development, seedling phenotypes were analyzed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, significant differences in plant height and root length were observed at 10 days after germination (DAG) among the seedlings of wild type (WT), \u003cem\u003eOx-1, Ox-2, Ox-3, Ri-1, Ri-2\u003c/em\u003e, and \u003cem\u003eRi-3.\u003c/em\u003e Compared with the WT, the plant height of \u003cem\u003eOx\u003c/em\u003e lines was 14.5% taller, whereas that of \u003cem\u003eRi\u003c/em\u003e lines was 28.6% shorter (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Similarly, crown root length in \u003cem\u003eOx\u003c/em\u003e and \u003cem\u003eRi\u003c/em\u003e lines was 13.8% longer and 31.0% shorter, respectively, than in the WT (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, E).\u003c/p\u003e \u003cp\u003eThese results indicate that \u003cem\u003eOsEP3A\u003c/em\u003e plays a positive role in rice seedling development. As consistent phenotypes were observed among the \u003cem\u003eOsEP3A\u003c/em\u003e-overexpressing and \u003cem\u003eRNAi\u003c/em\u003e-silenced plants, the \u003cem\u003eOx-1\u003c/em\u003e and \u003cem\u003eRi-1\u003c/em\u003e lines were selected for further study. Therefore, the expression levels of \u003cem\u003eOsEP3A\u003c/em\u003e mRNA in the \u003cem\u003eOx-1\u003c/em\u003e and \u003cem\u003eRi-1\u003c/em\u003e lines were reconfirmed by northern blot hybridization using the \u003cem\u003eOsEP3A 3\u0026prime;-UTR\u003c/em\u003e as a probe. Compared with the wild type, the results showed that the accumulation of \u003cem\u003eOsEP3A\u003c/em\u003e mRNA was much higher and lower in the \u003cem\u003eOx-1\u003c/em\u003e and \u003cem\u003eRi-1\u003c/em\u003e lines, respectively (Fig. S2A). Consistent with the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the 7-day-old seedlings of both shoots and roots were longer in \u003cem\u003eOx-1\u003c/em\u003e and shorter in \u003cem\u003eRi-1\u003c/em\u003e, respectively, compared with those of the WT (Fig. S2, B-D).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing of\u003c/b\u003e \u003cb\u003eOsEP3A\u003c/b\u003e \u003cb\u003edecreased seed size\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether \u003cem\u003eOsEP3A\u003c/em\u003e is involved in other plant developmental processes, seed morphology was further analyzed. The results indicated no significant differences in grain length (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C) or width (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D) between the WT (0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 cm for length and 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 cm for width) and the overexpression line \u003cem\u003eOx-1\u003c/em\u003e (0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 cm for length and 0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 cm for width). By contrast, the RNA interference line \u003cem\u003eRi-1\u003c/em\u003e (0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 cm for length and 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 cm for width) showed significant reductions of 9.0% and 11.1% in length and width, respectively, compared with the WT. These results indicate that \u003cem\u003eOsEP3A\u003c/em\u003e plays a positive role in rice seed development.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression profiles of\u003c/b\u003e \u003cb\u003eOsEP3A\u003c/b\u003e \u003cb\u003ein developing rice seeds\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results demonstrated that \u003cem\u003eOsEP3A\u003c/em\u003e regulates rice seed development. Therefore, the spatial and temporal expression pattern of \u003cem\u003eOsEP3A\u003c/em\u003e was examined in developing rice seeds. The \u003cem\u003eOsEP3A::GUS\u003c/em\u003e transgenic line was constructed using a \u003cem\u003eβ-glucuronidase\u003c/em\u003e (\u003cem\u003eGUS\u003c/em\u003e) reporter gene driven by the \u003cem\u003eOsEP3A\u003c/em\u003e promoter (a 2,015 bp region upstream of the translational start site) containing the 5\u0026prime; untranslated region (121 bp) of \u003cem\u003eOsEP3A\u003c/em\u003e mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, strong GUS staining was observed in the anthers and ovaries before flowering (BF), whereas much weaker or almost no GUS activity was detected in these tissues at 1 day after flowering (1 DAF). Similarly, little or no GUS staining was observed in developing seeds at 3, 6, and 20 DAF. These results indicate that \u003cem\u003eOsEP3A\u003c/em\u003e is expressed in a tissue-specific and developmental stage\u0026ndash;dependent manner, showing high expression in anthers and ovaries before flowering but very low expression after flowering during seed development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOsEP3A\u003c/b\u003e \u003cb\u003eexpression is induced by N starvation and required for normal growth in rice seedlings\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious studies (Ho et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) showed that the expression of \u003cem\u003eOsEP3A\u003c/em\u003e was induced in N-starved (-N) suspension-cultured rice cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA of this study, \u003cem\u003eOsEP3A\u003c/em\u003e expression was moderately induced after 5 days and highly induced after 10 days of -N treatment. In contrast, its expression was repressed by both the organic N source glutamine and the inorganic N source NH₄NO₃. These results suggest that \u003cem\u003eOsEP3A\u003c/em\u003e expression is activated under N starvation to degrade target proteins for N supplementation and is subsequently downregulated through feedback repression by available N sources.\u003c/p\u003e \u003cp\u003eTo examine the effect of N on plant growth in WT, \u003cem\u003eOx-1\u003c/em\u003e, and \u003cem\u003eRi-1\u003c/em\u003e, twenty seeds from each line were germinated in Kimura hydroponic solution (Ehara et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) either containing N sources ((NH₄)₂SO₄ and KNO₃) or lacking N for 10 days. When seeds were germinated and grown in medium containing N, the results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) were consistent with those shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eD. When compared with WT, the shoot length was longer in \u003cem\u003eOx-1\u003c/em\u003e and shorter in \u003cem\u003eRi-1\u003c/em\u003e. Under N deficient (-N) hydroponic conditions, the shoot length of all three lines was shorter than that under +\u0026thinsp;N conditions, and no significant difference was observed between WT and \u003cem\u003eOx-1\u003c/em\u003e. Interestingly, \u003cem\u003eRi-1\u003c/em\u003e seedlings exhibited severely impaired growth under -N conditions and appeared unable to grow normally (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results indicate that \u003cem\u003eOsEP3A\u003c/em\u003e is functionally correlated with N and is essential for rice seed germination and seedling growth.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003eOsEP3A\u003c/b\u003e \u003cb\u003epromoter is activated under N-limited conditions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo verify the effect of N on \u003cem\u003eOsEP3A\u003c/em\u003e expression, \u003cem\u003eOsEP3A::GUS\u003c/em\u003e reporter lines were used. Calli derived from three independent \u003cem\u003eOsEP3A::GUS\u003c/em\u003e transgenic lines were induced from immature seeds and subsequently cultured as suspension cells. These suspension cells were then grown in MS medium with or without N for 10 days, followed by GUS staining or qRT-PCR analysis. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, all three lines exhibited stronger GUS staining in -N medium than in +\u0026thinsp;N medium. Consistent with these results, qRT-PCR analysis revealed that \u003cem\u003egusA\u003c/em\u003e mRNA levels were approximately nine-fold higher in -N cells than in +\u0026thinsp;N cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results demonstrate that the transcriptional activity of the \u003cem\u003eOsEP3A\u003c/em\u003e promoter is activated under N-starved conditions.\u003c/p\u003e \u003cp\u003eFurthermore, to determine whether the transcriptional activity of the \u003cem\u003eOsEP3A\u003c/em\u003e promoter is repressed by N, calli derived from \u003cem\u003eOsEP3A::GUS\u003c/em\u003e transgenic line were cultured in -N MS medium for 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, lane 1), followed by subculture in MS medium without N or in medium supplemented with inorganic N (NH₄NO₃) or organic N sources (glutamine or asparagine) for an additional 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, lanes 2\u0026ndash;5). Total RNA was isolated and analyzed by northern blot hybridization using the \u003cem\u003egusA\u003c/em\u003e coding sequence as a probe. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cem\u003egusA\u003c/em\u003e mRNA accumulated abundantly in cells starved of N for 5 days (lane 1). When the cells were subsequently cultured in medium containing either inorganic or organic N, \u003cem\u003egusA\u003c/em\u003e mRNA accumulation was significantly repressed, and the hybridization signal became almost undetectable (lanes 2\u0026ndash;4). In contrast, continued culture in -N medium further enhanced \u003cem\u003egusA\u003c/em\u003e expression (lane 5). These results demonstrate that the transcriptional activity of the \u003cem\u003eOsEP3A\u003c/em\u003e promoter is repressed by metabolizable N sources and activated by N starvation in rice cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of a N responsive sequence in the\u003c/b\u003e \u003cb\u003eOsEP3A\u003c/b\u003e \u003cb\u003epromoter\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) showed that expression of the \u003cem\u003eOsEP3A\u003c/em\u003e gene was induced in cells grown in medium lacking N sources, and repressed in medium containing N. To investigate the mechanism underlying N-dependent regulation of \u003cem\u003eOsEP3A\u003c/em\u003e expression, we analyzed the N starvation responsive sequence (\u003cem\u003eNSRS\u003c/em\u003e) within the \u003cem\u003eOsEP3A\u003c/em\u003e promoter. A 2000 bp upstream regulatory sequence (\u003cem\u003eURS\u003c/em\u003e) from the \u003cem\u003eOsEP3A\u003c/em\u003e promoter region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) was subjected to a series of 5\u0026prime;-end serial deletions, internal deletions, and insertions. The 3\u0026prime; end of all deletion fragments was fixed at the first nucleotide upstream of the TATA box (-35) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The resulting \u003cem\u003eURS\u003c/em\u003e fragments of varying lengths were fused upstream of the \u003cem\u003eCaMV 35S\u003c/em\u003e minimal promoter and ligated to the \u003cem\u003egusA\u003c/em\u003e reporter gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) to assess promoter activity. Each expression construct was cloned into a binary vector and introduced into rice cells via \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. Three independent T₂ lines from each transgenic construct were used for callus induction, followed by the establishment of suspension-cultured cell lines. These lines were cultured in media\u0026thinsp;+\u0026thinsp;N or -N for 10 days and subjected to qRT-PCR analysis of \u003cem\u003egusA\u003c/em\u003e expression and GUS staining to identify the \u003cem\u003eNSRS\u003c/em\u003e within the \u003cem\u003eOsEP3A\u003c/em\u003e promoter.\u003c/p\u003e \u003cp\u003eResults (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D) found that deletions from \u0026minus;\u0026thinsp;2000 (2000::GUS) up to -837, -531, and \u0026minus;\u0026thinsp;278 did not affect GUS staining or the -N/+N expression ratio, which remained approximately 4.3- to 6.2-fold. This indicates that \u003cem\u003egusA\u003c/em\u003e expression in these cell lines was still induced under N-starved conditions and repressed when N was available.\u003c/p\u003e \u003cp\u003eFurther deletions up to -236, -183, and \u0026minus;\u0026thinsp;113 abolished N responsiveness, as GUS staining and \u003cem\u003egusA\u003c/em\u003e expression were no longer induced under -N conditions and remained at low, similar levels under both conditions, showing nearly 1.0-fold -N/+N ratios. The internal deletion construct \u003cem\u003eD278/236::GUS\u003c/em\u003e also lost -N inducibility, whereas \u003cem\u003eD236/183::GUS\u003c/em\u003e and \u003cem\u003eD183/113::GUS\u003c/em\u003e retained -N induction of \u003cem\u003egusA\u003c/em\u003e expression. Moreover, the insertion constructs \u003cem\u003eA837-236::GUS\u003c/em\u003e and \u003cem\u003eA278-236::GUS\u003c/em\u003e conferred N starvation inducibility to the minimal \u003cem\u003eCaMV 35S\u003c/em\u003e promoter, showing 3.8- and 3.4-fold -N/+N expression ratios, respectively. These results identify the \u0026minus;\u0026thinsp;278 to -236 bp region of the \u003cem\u003eOsEP3A\u003c/em\u003e promoter as essential for N starvation responsiveness, defining it as a putative \u003cem\u003eNSRS\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNitrogen is an essential macronutrient that regulates multiple aspects of plant growth, development, and metabolism. Plants have evolved complex signaling and regulatory networks to sense, acquire, and modified the RSA for efficiently utilize N (Forde, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Krapp et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kiba and Krapp, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These systems coordinate transcriptional, post-transcriptional, and hormonal responses to maintain N homeostasis. In this study, we characterized the rice cysteine protease gene \u003cem\u003eOsEP3A\u003c/em\u003e and its promoter to elucidate its role in N-mediated developmental regulation and transcriptional control under N-limited conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOsEP3A\u003c/b\u003e \u003cb\u003epositively regulates seedling growth and seed development\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur results demonstrate that \u003cem\u003eOsEP3A\u003c/em\u003e promotes early seedling growth and seed development. The shoot and crown root lengths were slightly longer in \u003cem\u003eOsEP3A\u003c/em\u003e-\u003cem\u003eoverexpressing\u003c/em\u003e (\u003cem\u003eOx\u003c/em\u003e) lines and significantly shorter in \u003cem\u003eRNAi-silenced\u003c/em\u003e (\u003cem\u003eRi\u003c/em\u003e) lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting that \u003cem\u003eOsEP3A\u003c/em\u003e-mediated proteolysis facilitates N mobilization from storage proteins during germination, thereby playing a positive role in supporting seedling growth. These findings are consistent with our previous study, which showed strong GUS staining localized in the germinating endosperm of \u003cem\u003eOsEP3A::GUS\u003c/em\u003e transgenic rice seeds and suggested a functional role for \u003cem\u003eOsEP3A\u003c/em\u003e in providing N sources during seed germination (Ho et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, GUS staining was also abundant in the anther before flowering but scarcely detected after flowering in florets or spikelets (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting a functional role in anther development prior to flowering. A similar expression pattern was observed for \u003cem\u003eOsCP1\u003c/em\u003e, a rice papain-like cysteine protease and ortholog of \u003cem\u003eOsEP3A\u003c/em\u003e, whose promoter fused with the GUS reporter gene showed strong expression in immature anthers (Lee et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The T-DNA-tagged \u003cem\u003eoscp1\u003c/em\u003e mutant exhibited defective pollen development, resulting in reduced seed set, suggesting that \u003cem\u003eOsCP1\u003c/em\u003e plays a positive role in rice pollen development. However, our results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e) showed that silencing \u003cem\u003eOsEP3A\u003c/em\u003e (\u003cem\u003eRi\u003c/em\u003e lines) produced smaller seeds, while no significant differences in seed size were observed between \u003cem\u003eOx\u003c/em\u003e and wild-type (WT) plants. No differences in seed setting were detected among the WT, Ox, and Ri lines. This indicates that \u003cem\u003eOsEP3A\u003c/em\u003e influences seed development but not pollen development, which may explain the lack of changes in seed setting. These differences may reflect functional specificity between the orthologous genes \u003cem\u003eOsEP3A\u003c/em\u003e and \u003cem\u003eOsCP1\u003c/em\u003e, which may cooperate to regulate stamen development in rice. This study shows that silencing \u003cem\u003eOsEP3A\u003c/em\u003e results in smaller grains, whereas its overexpression has no significant effect, suggesting that a basal level of OsEP3A protease activity is required for normal seed development. These findings indicate that \u003cem\u003eOsEP3A\u003c/em\u003e acts as a positive regulator of seedling growth and seed development in rice.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOsEP3A expression is induced by N starvation and repressed by metabolizable N\u003c/h2\u003e \u003cp\u003eTo maintain N homeostasis under fluctuating environmental conditions, plants through sophisticated regulatory networks that integrate transcriptional, post-transcriptional, and hormonal control mechanisms to coordinate N uptake, assimilation, and signaling (Forde, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Krapp et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kiba \u0026amp; Krapp, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Under N starvation, several response genes are induced, including high-affinity nitrate transporters (NRT2 family), transcriptional activators such as SNAC1, and signaling factors such as NIN-like proteins (NLPs\u003cb\u003e)\u003c/b\u003e, while repressors such as NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) are repressed, cooperatively fine-tuning N acquisition and utilization (Konishi and Yanagisawa, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sawaki et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Qi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In addition to these well-known regulatory pathways controlling the NRT families involved in N uptake, other mechanisms also contribute to the regulation of N homeostasis. The proteolytic enzymes, particularly papain-like cysteine proteases (PLCPs), play crucial roles in N remobilization, senescence, and stress adaptation (van der Hoorn et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Despite their broad physiological importance, the role of PLCPs in N signaling remains poorly understood. In this study, our results (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e) demonstrated that \u003cem\u003eOsEP3A\u003c/em\u003e transcript accumulation was markedly enhanced under N starvation and rapidly repressed by metabolizable N sources, including both inorganic (NH₄NO₃) and organic (glutamine, asparagine) forms (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This dual responsiveness suggests a feedback mechanism in which N sufficiency represses \u003cem\u003eOsEP3A\u003c/em\u003e transcription to modulate proteolytic N recycling. Such regulation mirrors the transcriptional behavior of other N starvation inducible genes, including \u003cem\u003enitrate transporter NRT2.1\u003c/em\u003e, \u003cem\u003eNRT2.4\u003c/em\u003e and \u003cem\u003eNRT2.5\u003c/em\u003e (Ma et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kiba et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), \u003cem\u003enitrate reductase\u003c/em\u003e (\u003cem\u003eNIA1\u003c/em\u003e and \u003cem\u003eNIA2\u003c/em\u003e) and the \u003cem\u003enitrite reductase\u003c/em\u003e (\u003cem\u003eNIR\u003c/em\u003e) (Medici and Krouk, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) in \u003cem\u003eArabidopsis\u003c/em\u003e, whose expression is tightly controlled by N availability. Moreover, under N-limited hydroponic conditions, \u003cem\u003eOsEP3A-RNAi\u003c/em\u003e seedlings exhibited severe stunting growth, while wild-type and overexpression lines maintained near-normal growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These phenotypic differences were minimized under N sufficient conditions, indicating that \u003cem\u003eOsEP3A\u003c/em\u003e is important during N scarcity. The failure of \u003cem\u003eOsEP3A\u003c/em\u003e-\u003cem\u003eRNAi\u003c/em\u003e lines to sustain growth under N limitation highlights the importance of cysteine protease OsEP3A-mediated proteolysis for internal N remobilization for providing amino acid substrates for metabolism when external N sources are restricted. Moreover, the \u003cem\u003eOsEP3A::GUS\u003c/em\u003e analyses confirmed that the \u003cem\u003eOsEP3A\u003c/em\u003e promoter is transcriptionally activated under N starvation and repressed upon N supply (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Strong GUS activity and elevated \u003cem\u003egusA\u003c/em\u003e expression were observed under N deficient conditions in \u003cem\u003eOsEP3A::GUS\u003c/em\u003e transgenic lines, whereas supplementation with either inorganic or organic N suppressed promoter activity. These results indicate that \u003cem\u003eOsEP3A\u003c/em\u003e regulation depends on N metabolizability rather than N form, consistent with the broader principle of metabolic feedback control observed in other N responsive genes (Yang et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; kong et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of an N starvation responsive sequence (\u003c/b\u003e \u003cb\u003eNSRS\u003c/b\u003e \u003cb\u003e) in the\u003c/b\u003e \u003cb\u003eOsEP3A\u003c/b\u003e \u003cb\u003epromoter\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAlthough numerous studies have identified genes whose expression is activated by N starvation, such as \u003cem\u003eNRT2.1\u003c/em\u003e, \u003cem\u003eNRT2.4\u003c/em\u003e, and \u003cem\u003eNRT2.5\u003c/em\u003e (Wang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), there remains limited information regarding the identification of N starvation responsive cis-regulatory sequences. In \u003cem\u003eArabidopsis\u003c/em\u003e, the NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) protein functions as a transcriptional repressor that binds to one GAATATTC motif and three GAATC motifs within the promoter region of the high affinity nitrate transporter \u003cem\u003eNRT2.4\u003c/em\u003e (254\u0026ndash;389 bp upstream of the translation start site) under high N availability, thereby repressing the expression of N starvation responsive genes (Kiba et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In contrast, our study identified a 43 bp region (-236 to -278) upstream of the \u003cem\u003eOsEP3A\u003c/em\u003e transcription start site as essential for activating N starvation responsiveness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Deletion of this 43 bp DNA fragment abolished GUS induction under N starvation, whereas insertion of the fragment upstream of a minimal \u003cem\u003eCaMV 35S\u003c/em\u003e promoter conferred N starvation inducible expression. These results define the \u0026minus;\u0026thinsp;236 to -278 region as a N starvation responsive sequence (\u003cem\u003eNSRS\u003c/em\u003e), which likely contains a novel cis-element recognized by transcriptional activators involved in N starvation signaling.\u003c/p\u003e \u003cp\u003eIn soybean (\u003cem\u003eGlycine max\u003c/em\u003e L.), the NAC transcription factor GmNAC039 functions as a positive regulator of nodule senescence (Yu et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Electrophoretic mobility shift assay (EMSA) analysis revealed that GmNAC039 directly binds to a CACAAA motif located in the \u003cem\u003eGmCYP37\u003c/em\u003e promoter region (93\u0026ndash;121 bp upstream of the translation start codon), thereby activating \u003cem\u003eGmCYP37\u003c/em\u003e expression during nodule senescence. Overexpression or knockout of \u003cem\u003eGmCYP37\u003c/em\u003e in nodules accelerated or delayed senescence, respectively, indicating that GmNAC039 promotes nodule senescence by directly activating \u003cem\u003eGmCYP37\u003c/em\u003e through binding to the CACAAA motif.\u003c/p\u003e \u003cp\u003eOur study defines a 43 bp \u003cem\u003eNSRS\u003c/em\u003e within the \u003cem\u003eOsEP3A\u003c/em\u003e promoter that mediates transcriptional activation under N starvation. As described above, the transcriptional repressor NIGT1 binds to GAATATTC and GAATC cis-elements in the \u003cem\u003eNRT2.4\u003c/em\u003e promoter under N replete conditions to repress N starvation responsive genes, while the CACAAA motif in the \u003cem\u003eGmCYP37\u003c/em\u003e promoter serves as a binding site for the transcriptional activator GmNAC039 during nodule senescence. To date, no cis-element has been characterized as an N starvation activating motif. In this study, the 43 bp \u003cem\u003eNSRS\u003c/em\u003e sequence in the \u003cem\u003eOsEP3A\u003c/em\u003e promoter appears to function as a positive regulatory element in response to N starvation. Notably, no \u003cem\u003ecis-element\u003c/em\u003e similarity was found between the \u003cem\u003eNSRS\u003c/em\u003e and previously characterized motifs such as GAATATTC or CACAAA. We therefore propose that the \u003cem\u003eNSRS\u003c/em\u003e may interact with previously unidentified transcriptional regulators that activate \u003cem\u003eOsEP3A\u003c/em\u003e expression, thereby enhancing its proteolytic activity to supply N under N-limited conditions. Further protein-DNA interaction studies will clarify how \u003cem\u003eOsEP3A\u003c/em\u003e transcription is modulated in response to N availability.\u003c/p\u003e \u003cp\u003eIn conclusion, this study identifies \u003cem\u003eOsEP3A\u003c/em\u003e as an N starvation activated cysteine protease that enhances seedling growth and seed development in rice. We further define a 43 bp N starvation responsive sequence within the \u003cem\u003eOsEP3A\u003c/em\u003e promoter that mediates transcriptional activation under N deficient conditions. These findings uncover a previously uncharacterized regulatory mechanism in rice N signaling and suggest that \u003cem\u003eOsEP3A\u003c/em\u003e could serve as a molecular target for improving N utilization during cereal crop development.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available within the article and its supplementary materials from the date of publication, and may also be obtained from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShin Lon Ho\u0026nbsp;conceived and designed the experiments. Shinn Jia Tzeng performed agronomic trait measurements and statistical analyses. Yi Hsuan Hou constructed the expression vectors and carried out Agrobacterium-mediated gene transformation. Shin Lon Ho analyzed the data and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Science and Technology Council of Taiwan (Grant No. NSTC 114-2313-B-415-003- and MOST 111-2313-B-415-004-).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmack SC, Antunes MS (2020) CaMV35S promoter \u0026ndash; A plant biology and biotechnology workhorse in the era of synthetic biology. 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Plant Sci 250:89-96. https://doi.org/10.1016/j.plantsci.2016.05.008\u003c/li\u003e\n\u003cli\u003eZhang H, Huang J, Li Y, Zhao J, Mai W, Khan L, Zhang M, Wang W, Zeng C, and Chen X (2025) Beyond nitrate transport: AtNRT2.4 responds to local and systemic nitrogen signaling in Arabidopsis. BMC Plant Biol 25:655. https://doi.org/10.1186/s12870-025-06695-4\u003c/li\u003e\n\u003cli\u003eZhao Y, Guo L, Lu W, Li X, Chen H, and Guo C (2015) Expression pattern analysis of microRNAs in root tissue of wheat (Triticum aestivum L.) under normal nitrogen and low nitrogen conditions. J Plant Biochem Biotechnol 24:143-153. https://doi.org/10.1007/s13562-013-0246-2\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"rice, cysteine protease, nitrogen, nitrogen starvation responsive sequence","lastPublishedDoi":"10.21203/rs.3.rs-8421153/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8421153/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNitrogen (N) is a critical macronutrient that influences plant growth, development, and productivity. This study characterizes the rice cysteine protease gene \u003cem\u003eOsEP3A\u003c/em\u003e and its promoter to elucidate its role in N-mediated developmental and transcriptional regulation. Transgenic rice lines overexpressing (\u003cem\u003eOsEP3A-Ox\u003c/em\u003e) or silenced (\u003cem\u003eOsEP3A-Ri\u003c/em\u003e) for \u003cem\u003eOsEP3A\u003c/em\u003e were generated to assess its physiological functions. Overexpression of \u003cem\u003eOsEP3A\u003c/em\u003e significantly enhanced shoot and root growth, whereas \u003cem\u003eRNAi\u003c/em\u003e-silenced plants exhibited reduced height, shorter roots, and smaller seeds compared to wild type, indicating that \u003cem\u003eOsEP3A\u003c/em\u003e positively regulates seedling and seed development. Expression analyses revealed that \u003cem\u003eOsEP3A\u003c/em\u003e transcription was strongly induced under N deficient conditions and repressed by both inorganic (NH₄NO₃) and organic (glutamine, asparagine) N sources. Under N-limited hydroponic culture, \u003cem\u003eOsEP3A-RNAi\u003c/em\u003e seedlings showed severely impaired growth, underscoring the gene\u0026rsquo;s essential role in internal N remobilization during deficiency. Promoter-reporter analyses using \u003cem\u003eOsEP3A::GUS\u003c/em\u003e lines demonstrated strong activation of the \u003cem\u003eOsEP3A\u003c/em\u003e promoter under N starvation and repression upon N resupply, suggesting N dependent transcriptional control. Deletion and insertion analyses of the \u003cem\u003eOsEP3A\u003c/em\u003e promoter identified a 43 bp N starvation responsive sequence (\u003cem\u003eNSRS\u003c/em\u003e; -278 to -236 bp) as necessary and sufficient for starvation-induced transcriptional activation. This \u003cem\u003eNSRS\u003c/em\u003e represents a novel cis-regulatory element responsive to N deprivation. Overall, \u003cem\u003eOsEP3A\u003c/em\u003e acts as a N starvation-activated cysteine protease that facilitates N recycling and seedling vigor, providing new insight into N responsive regulatory mechanisms in rice and offering a potential molecular target for improving N use efficiency in cereal crops.\u003c/p\u003e","manuscriptTitle":"The Rice Cysteine Protease OsEP3A Promotes Seedling Growth and Seed Development and Contains a Nitrogen Starvation Responsive Sequence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 17:51:09","doi":"10.21203/rs.3.rs-8421153/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-07T09:22:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-07T05:45:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T04:56:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319161944028575475807895233849593545002","date":"2025-12-29T07:28:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297044199117401958845611536072854671265","date":"2025-12-28T09:00:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130892138539816573509676275591174889385","date":"2025-12-27T06:44:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-26T08:46:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-26T06:31:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-26T06:30:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-12-22T05:25:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"92728b3c-85d3-47f6-b2b8-70b7af0e2377","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:04:41+00:00","versionOfRecord":{"articleIdentity":"rs-8421153","link":"https://doi.org/10.1007/s00299-026-03826-5","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2026-04-25 15:57:38","publishedOnDateReadable":"April 25th, 2026"},"versionCreatedAt":"2025-12-30 17:51:09","video":"","vorDoi":"10.1007/s00299-026-03826-5","vorDoiUrl":"https://doi.org/10.1007/s00299-026-03826-5","workflowStages":[]},"version":"v1","identity":"rs-8421153","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8421153","identity":"rs-8421153","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}