SiPHO2 orchestrates phosphorus homeostasis and regulates heading date through SiFTc-dependent pathway in foxtail millet (Setaria italica)

SiPHO2 orchestrates phosphorus homeostasis and regulates heading date through SiFTc-dependent pathway in foxtail millet (Setaria italica) | 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 SiPHO2 orchestrates phosphorus homeostasis and regulates heading date through SiFTc-dependent pathway in foxtail millet ( Setaria italica ) Yuqian Li, Hailong Wang, Huamin Fei, Ruifang Qu, Yonghu Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6218826/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Phosphorus (P) is a crucial macronutrient and its deficiency severely limits plant growth and yield. Although multiple inorganic phosphate (Pi) signaling regulators have been identified, the function of them in plant development and flowering time regulatory remains inadequately characterized in C4 model species like Setaria italica . Here, CRISPR/Cas9-generated SiPHO2 knockout lines exhibited disrupted Pi homeostasis, and the lines showed shoot Pi accumulation, leaf tip necrosis, modified root architecture and reduced yield compared with wildtype (Ci846) under Pi deficient conditions. Transcriptome analysis suggested these phenotypic abnormalities might due to expression patterns alteration of Pi starvation-responsive genes. Notably, SiPHO2 knockout lines displayed earlier heading date under Pi deficiency but delayed heading date under normal conditions compared to Ci846 plants. Expression profiling and transgenic functional verification revealed that the heading date reversal correlated with the expression pattern of FLOWERING LOCUS T c ( SiFTc ), rather than SiFTa , which is the closest homolog of Heading date 3a ( OsHd3a ). This study identifies a novel flowering regulator as a potential target for coordinating phosphorus-mediated heading date regulation and yield production. Our findings elucidate genetic mechanisms underlying phosphorus-dependent developmental regulation and propose a strategic approach for improving crop yield under Pi starvation. Setaria italica Pi homeostasis SiPHO2 Heading date Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Phosphorus (P), an essential macronutrient for plant growth and development, plays a pivotal role in energy production, photosynthesis, glycolysis, respiration and the biosynthesis of nucleic acids and membranes system [ 1 ]. Under Pi deficient conditions, crops exhibit distinct deficiency symptoms, including impaired growth, reduced biomass, purple leaf coloration and anthocyanin accumulation [ 2 ]. Plants primarily acquire inorganic phosphate (Pi) from soil solutions [ 3 ], where available Pi concentrations typically average 1 mM, substantially below the 5–20 mM threshold required to sustain normal plant growth and yield [ 4 , 5 ]. Current mitigation strategies involving excessive application of phosphate fertilizers prove ecologically unsustainable, with merely 10–20% fertilizer utilization efficiency, while residual inputs drive aquatic eutrophication and biodiversity loss [ 6 , 7 ]. These realities underscore improving phosphorus use efficiency (PUE) as a critical determinant of sustainable agriculture, demanding mechanistic elucidation of plant Pi homeostasis regulation [ 8 ]. Pi deficiency adversely affects tiller number, panicle formation, setting rate and overall yield. The root system, as the primary organ for Pi sensing and acquisition, plays a decisive role in Pi absorption and yield [ 9 ]. Under Pi deficient conditions, plants often increase the root-to-shoot ratio and modify root architecture to improve Pi uptake [ 10 ]. Root architecture, including root length, root hair (RH) density and root angle are primarily influenced [ 11 ]. To counteract these effects, extensive researches have been conducted on the morphological, physiological and molecular mechanisms underlying Pi homeostasis in plants. The regulation of Pi homeostasis involves a complex network, central to this network is PHOSPHATE STARVATION RESPONSE1 ( PHR1 ), a MYB-CC family transcription factor that senses Pi status and orchestrates Pi deficiency response strategies [ 12 ]. In Arabidopsis, PHR1 overexpression leads to increased Pi concentration in shoot tissues [ 13 ]. There are four PHR1 homologs ( OsPHR1 / 2 / 3 / 4 ) in rice, OsPHR2 overexpression results in Pi accumulation in leaves, increased root length, root-to-shoot ratio and RH density, alongside up-regulation of Pi starvation-induced (PSI) genes [ 14 ]. PHR1 induces the expression of PSI genes, such as PHOSPHATE TRANSPORTER1 ( PHT1 ) and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 ( PHF1 ), to regulate Pi absorption, translocation and remobilization [ 15 ]. Further research indicates that OsPHR2 acts as a central regulator, with higher expression levels than those of OsPHR1 and OsPHR3 [ 16 , 17 ]. Under Pi deficiency, miR399 expression is strongly induced, leading to the down-regulation of PHO2 , which encodes an E2 UBC enzyme. The degradation of PHO2 transcripts by miR399 is regulated by long non-coding RNA (lncRNA) INDUCED BY PHOSPHATE STARVA-TION 1,2 ( IPS1,2 ) [ 18 ], such as homologs of IPS1 in rice ( OsIPS1 and OsIPS2 ) and PI-DEFICIENCY-INDUCED LNCRNA 2 ( PILNCR2 ) of maize exhibit similar functions [ 19 ]. In Arabidopsis and rice, PHO2, NITROGEN LIMITATION ADAPTATION ( NLA1 ) and miR827 maintain Pi homeostasis in a NO 3− dependent manner [ 20 ]. Further researches found that both PHO2 and NLA1 are essential for PHT1 polyubiquitination at the plasma membrane and subsequent degradation via the 26S proteasome system [ 21 ]. In addition, PHO2 negatively regulates PHO1 and PHF1 abundance under Pi sufficient conditions, and it was identified that OsPHO1 and OsPHF1 as the potential substrates of OsPHO2, with yeast two-hybrid and bimolecular fluorescence complementation assays, which confirmed interactions between OsPHO1, OsPHF1 and OsPHO2, but not with OsPHT1;2/6/8 [ 22 , 23 ]. Furthermore, OsSP X1 expression is induced in Ospho2 mutants of rice, indicating its involvement in OsPHO2 -mediated Pi signaling networks [ 24 , 25 ]. Beyond its primary role in regulating Pi homeostasis, PHO2 has been demonstrated to participate in multiple functional pathways in plants, including nitrogen assimilation, iron uptake and flowering regulation [ 26 , 20 , 27 ]. The regulation of flowering time in plants is a complex process that integrates environmental cues with internal signaling pathways [ 28 ]. Among these, FT belonging to the phosphatidyl ethanolamine binding protein (PEBP) family, serves as a central integrator of flowering signals. In Arabidopsis thali ana, the GIGANTEA (GI) protein induces the expression of CONSTANS ( CO ), which subsequently activates the transcription of FT , and the GI-CO-FT pathway is highly conserved in plants [ 29 ]. In rice, the orthologous components of this pathway include OsGI , OsHd1 and OsHd3a. OsHd3a and SiFTa (Setaira) are homologous to AtFT, they are all the florigenic proteins and can induce flowering [ 30 – 32 ]. In rice, OsPHO2 regulates flowering through its interaction with OsGI. Both Ospho2 and Osgi mutants display reduced growth, leaf tip necrosis, delayed flowering and excessive Pi accumulation in leaves. The transcriptome analysis reveals the interaction between OsPHO2 and OsGI links high-level regulators of Pi homeostasis and flowering time in rice [ 33 ]. Moreover, Atpho2 mutants exhibit early flowering at 23 ℃ compared to wild-type (WT) plants, uncovering a novel role for the miR399- PHO2 pathway in mediating ambient temperature-responsive flowering in Arabidopsis [ 27 ]. These findings highlight the multifaceted role of PHO2 in coordinating not only Pi homeostasis but also critical developmental processes such as flowering,which underscore its importance in plant growth and adaptation to environmental conditions. Under Pi deficient conditions, foxtail millet develops an extensive root system with longer LR [ 34 , 35 ]. Different foxtail millet varieties exhibit varying responses to Pi starvation, with up-regulation of Pi transporter genes SiPHT1 ; 1 , SiPHT1 ; 2 and SiPHT1 ; 4 in roots promoting Pi absorption, while down-regulation of SiPHT1;3 leads to Pi accumulation in stems [ 36 ]. Pi deficient conditions also alter SPAD and soluble protein content, balancing the nitrogen-to-phosphorus ratio in plants. PHR1 directly promotes expression of NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 ( NIGT1 ), nitrate-induced nitrogen receptor and transporter, family genes and NIGT1 integrates nitrogen and Pi signals to maintain nutrient balance [ 37 ]. Despite these advances, systematic studies on Pi starvation responses and the other functions of SiPHO2 in foxtail millet remain limited. Setaria italica originates in China, is a model plant for studying nutrient utilization mechanisms due to higher resistance to drought and nutrient-poor soils [ 35 ]. In this study, we identified SiPHO2 was essential not only for Pi homeostasis but also for heading date and plant development regulation in foxtail millet. Our findings also verified SiFTc act as a novel factor of heading date regulation in Setaria under Pi starvation conditions and provided a targeted genetic strategy for breeding Pi-efficient foxtail millet cultivars through optimized heading date regulation. 2. Materials and methods 2.1. Phylogenetic and SiPHO2 promoter analysis For the phylogenetic analysis, protein sequences of UBCs in Setaria italica , Oryza sativa and Arabidopsis thaliana , and FT homology proteins in Setaria italica , Oryza sativa , Zea mays , Sorghum bicolor and Arabidopsis thaliana were downloaded from PHYTOZOME v.13 ( https://phytozome.jgi.doe.gov ). Multiple-sequence alignments of SiPHO2 proteins were carried out using the Clustal W (version2.0) program [ 38 ]. Then, phylogenetic analysis was performed with MEGA7.0 [ 39 ] using theneighbor-joining method (1000 bootstrap replications), and the results were visualized in Itol [ 40 ]. A region approximately 2000 bp upstream of the start codon (ATG) in SiPHO2 was investigated from the reference genome sequence of Yugu1. The online software Plant CARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html ) was used to search for cis-acting regulatory elements in the promoters of SiPHO2. 2.2. Subcellular localization assay To explore the subcellular localization of SiPHO2, the coding sequence of SiPHO2 was cloned into the pCAMBIA1305-eGFP vector to generate pUbi::SiPHO2-eGFP, and the plasmid was co-transformed with the ER marker pUbi::AtWAK2-mcherry into foxtail millet leaf protoplasts using PEG-mediated transfection [ 41 ]. TE2000-E inverted confocal microscope (Nikon A1R) was used to detect GFP and mCherry signals. The primers used are shown in Supplemental Table S1 . 2.3. Vector construction and plant transformation For CRISPR/Cas9-mediated knockout of SiPHO2 ( Seita.3G150400 ) in Setaria italica , sgRNAs targeting the first exon were designed using CRISPR-P ( http://crispr.hzau.edu.cn/CRISPR2/ ). The pYLCRISPR-Cas9-MH vector was digested and ligated with sgRNA-containing fragments amplified using primers SiPHO2-target-F/R and U-F/gR-R [ 42 ]. The construct was transformed into Agrobacterium tumefaciens strain EHA105 and introduced into Ci846 using mature seeds as explants [ 43 , 44 ]. T3 transgenic lines were used for further analysis. Primers are listed in Supplementary Table S1 . The full-length coding sequence (CDS) of SiFTc was amplified from Ci846 cDNA and cloned into the pTCK303-Flag vector to generate pUbi::SiFTc-Flag. The construct was transformed into Agrobacterium tumefaciens strain EHA105 and introduced into Zhonghua11 (ZH11) using mature seeds as explants. T2 transgenic lines were used for further experiments. Primers are listed in Supplementary Table S1 . 2.4. Plants growth conditions and Pi treatment Pot experiments were conducted in vermiculite and supplied with basal nutrient solution with or without Pi. The solution contained the following components (mM): 2.0 mM Ca(NO 3 ) 2 , 0.5 mM MgSO 4 , 0.1 mM KCl, 0.3 mM KH 2 PO 4 , 10 mM H 3 BO 3 , 0.5 mM MnCl 2 , 0.5 mM ZnCl 2 , 0.2 mM CuCl 2 , 0.1 mM Na 2 MoO 4 and 0.1 mM Fe-EDTA. For the treatment of Pi deficiency, KH 2 PO 4 was substituted with KCl. The pH of all culture solutions was adjusted to 5.8, and the medium was changed every three days [ 45 ]. All foxtail millet and rice plants were grown in a greenhouse in Beijing (116.34′E, 39.97′N). Root architecture analysis experiments were conducted in illumination incubator and supplied with Murashige & Skoog Basal Medium with vitamins (Phyto Technology # M519).The seedlings with similar growth to continue be cultured for one week on MS medium used for root architecture analysis. Agronomic traits analysis was according to methods described in the previous study [ 46 ], such as plant height, leaf length, leaf width, PR length, LR length, LR number, SPAD, panicle weight, panicle length, grains number per panicle and 1000-grains weight. Percentage of necrotic area of the second leaf from the top was used for leaf Pi toxic analysis [ 47 ]. Heading date was measured as the number of days from seed germination to the fully emergency of the panicle. At least six plants in three different pots were used for phenotypic statistics [ 33 ]. Three biological replicates were used in each treatment. Data and statistics were analyzed by GraphPad Prism8 (GraphPad Software, USA). 2.5. Pi concentration assay Pi concentration analysis was quantified in various tissues of SiPHO2-CR and Ci846 lines grown under Pi normal (0.3 mM) and Pi deficient (0.01 mM) conditions. Samples were collected from shoots and roots of two-week-old seedlings, leaves at the heading stage, and roots, stems, leaves and spikes at the mature stage. For seedling root Pi concentration analysis, one-week-old seedlings grown on MS medium were used for Pi uptake assays. All samples were homogenized in a 1:5 (w/v) extraction buffer, centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant liquid was collected. The supernatant liquid, distilled water and color-developing reagent were mixed in a 1:9:10 ratio, incubated at 40°C for 10 min, and then cooled to room temperature. Absorbance was measured at 660 nm using a spectrophotometer. Pi concentration was calculated as follows: Tissue Pi concentration (mmol/g) = \(\:\frac{\text{C}\text{s}\times\:\varDelta\:\text{A}\text{t}\times\:\text{V}\text{t}}{\text{W}\times\:\varDelta\:\text{A}\text{s}}\) = \(\:\frac{0.002\times\:\varDelta\:\text{A}\text{t}}{\text{W}\text{t}\times\:\varDelta\:\text{A}\text{s}}\) where: ΔAt = At-Ab (OD660 of the tested sample minus the blank), ΔAs = As-Ab (OD660 of the standard sample minus the blank), Cs = concentration of the standard solution (2 mmol/L), Vt = total volume of the tested sample (1 mL = 1×10⁻³ L), Wt = weight of the tested sample (g). 2.6. β-glucuronidase (GUS) staining assay A 3600-bp genomic fragment upstream of SiPHO2 was cloned as the promoter to drive the expression of the GUS reporter gene in the pCAMBIA1301 vector to generate pSiPHO2::GUS transgenic plants. Histochemical localization of GUS activity was performed by incubating plant tissues in a staining solution containing 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) at 37°C for three hours. 2.7. RNA extraction, gene expression and transcriptome sequencing analysis Total RNA was extracted from leaves of Ci846 and SiPHO2-CR plants at the heading stage using the RNAprep Pure Plant Plus Kit (Tiangen, Cat. # DP441). RNA (5 µg) was reverse-transcribed into cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Cat. #6210A). Quantitative PCR (qPCR) was performed using 2x Realtime PCR Super mix (Mei5bio, Cat. # MF013-05) with gene-specific primers. SiCullin was used as the control. Primers are listed in Supplementary Table S1 . For transcriptome sequencing, 16 RNA-seq libraries (four biological replicates per treatment) were constructed and sequenced on an Illumina HiSeq 2500 platform in 150-bp paired-end mode. Clean reads were mapped and analyzed as previously described [ 48 ]. 3. Results 3.1. SiPHO2 is a homologous gene of OsPHO2 and responds to a variety of abiotic stresses. The PHO2 encodes a UBC E2 enzyme critical for phosphate homeostasis, characterized by a conserved C-terminal ubiquitin-binding domain. Phylogenetic analysis of UBC family members by conserved UBC domains in Arabidopsis, rice and foxtail millet showed SiPHO2 ( Seita.3G150400 ) is an ortholog of OsPHO2 ( LOC Os05g48390 ), and shares 79.04% amino acid sequence identity with OsPHO2 (Figure S1 A). The genome sequence of SiPHO2 is 2,625 base pair nucleotides, contains eight exons and seven introns, encoding 875 amino acids. SiPHO2 belongs to Group 3 together with AtPHO2 (AT2G33770, UBC24) and OsPHO2 (Figure S1 A).These findings indicated that PHO2 -mediated regulatory mechanisms might be functionally conserved in Pi signaling across monocot and dicot plants. The SiPHO2 promoter region contains several cis-elements associated with diverse biological processes, including photoresponse, MeJA response, SA response, stress response and meristem development (Figure S1 B). To further characterize its stress-responsive properties, we analyzed the expression pattern of SiPHO2 under various abiotic stress conditions. Our results revealed distinct expression patterns in response to different treatments. Low temperature (4 ℃), drought stress and gibberellic acid (GA) treatment generally suppressed SiPHO2 expression within 24 hours (h). In contrast, MeJA and PEG 6000 treatment induced SiPHO2 expression after 24 h. In addition, we observed significant changes in SiPHO2 expression levels under short-term treatments, ABA (1 h), SA (1 h and 3 h) and NaCl (1 h to 6 h) treatments all elicited rapid responses compared to the 0 h control (Fig. 1 C). These findings indicated that SiPHO2 was involved in dual-phase stress response mechanisms, mediating both rapid transcriptional regulation for immediate stress signaling and sustained induction for long-term adaptive responses. 3.2. SiPHO2 is widely expressed in different tissues and localized in the endoplasmic reticulum (ER) of cells. To investigate the tissue-specific expression pattern of SiPHO2 , we generated transgenic plants carrying the GUS reporter gene driven by SiPHO2 promoter (pSiPHO2::GUS). In foxtail millet, histochemical analysis revealed GUS activity in whole seedlings, and in stems, nodes, leaves and panicle of the palnts at reproductive stage (Figure. 1D-I). Similarly, in transgenic rice lines, GUS signals were also observed in seedlings, and roots, stem, leaves, florets and grains during the reproductive stage (Figure S2 ). The expression pattern of SiPHO2 was analyzed in tissues at different developmental stages, such as shoot elongation stage, booting stage, panicle developmental stage, flowering stage and grain filling stage. According to the results, SiPHO2 was widely expressed throughout plant development and was highly expressed in seedlings and panicles (Figure. 1J). The Ubi::GFP (control) or Ubi::SiPHO2-eGFP vector was constructed and co-transformed with an ER marker, AtWAK2-mCherry [ 49 ], in foxtail millet protoplasts to analyze the subcellular location of SiPHO2. The SiPHO2-eGFP fusion protein predominantly colocalized with ER marker, it indicated that SiPHO2 was primarily localized at the ER (Fig. 1 A). To elucidate the response of SiPHO2 to Pi availability, we performed time-course expression analysis of both shoots and roots under Pi normal and Pi deficient conditions, with sampling intervals at 0, 2, 6, 12, 24 and 72 hours post-treatment [ 45 ]. The results indicated that the expression of SiPHO2 in the shoot remained relatively stable, and it exhibited a downward trend in roots in six hours under different Pi conditions. The results implied that the expression of SiPHO2 might response to Pi deficiency during the short term in roots (Fig. 1 B, C). 3.3. SiPHO2 was involved in the Pi homeostasis and development of Setaria italica . The CRISPR/Cas9 system was used to create SiPHO2 loss-of-function mutants in the background of the Ci846 genotype. From ten independent knockout transgenic lines, we identified three distinct editing types: SiPHO2-CR#2 (T insertion), SiPHO2-CR # 3 (C deletion) and SiPHO2-CR # 4 (C insertion) in the coding sequence compared to the Ci846. These frame-shift mutations resulted in premature termination codons, as confirmed by sequence analysis (Fig. 2 A and Figure S3 ). Consistent with the Pi toxicity phenotype observed in rice [ 14 , 50 , 51 ], the SiPHO2-CR plants exhibited leaf tip necrotic phenotype during the heading date (Fig. 2 F). The percentage of leaf tip necrosis area relative to the whole leaf was significantly higher in SiPHO2-CR plants than that in the Ci846 (Fig. 2 G). In addition, the Pi concentration in the leaves of SiPHO2-CR was two to three times higher than that in Ci846 (Fig. 2 H). These findings suggested that excessive Pi accumulation may lead to leaf damage in SiPHO2-CR plants. Comprehensive phenotype analysis demonstrated that SiPHO2-CR plants exhibited significant growth impairments compared to Ci846, including reductions in plant height, leaf length and width, panicle length and width and SPAD (Fig. 2 B-E and 2 K-Q). At the seedling stage, the whole plant Pi concentration was higher in SiPHO2-CR lines compared to Ci846 (Fig. 2 C). Tissue-specific analysis revealed that the shoot to root ratio of Pi concentration was increased in SiPHO2-CR plants (Fig. 2 D). During maturity, while root and stem Pi concentrations remained comparable between SiPHO2-CR and Ci846, the mutants accumulated significantly higher Pi levels in leaves, particularly in apical young leaves (Figure. 2I). This altered spatial distribution of Pi was associated with the abnormal phenotype, such as, pronounced Pi toxicity symptoms in apical leaves, enhanced basal-to-apical (stem to leaf) Pi translocation and reduced Pi transfer efficiency from apical leaves to panicles (Fig. 2 J). Notably, SiPHO2-CR plants showed 43.60% reduction in panicle weight and 50.54% reduction in grain weight per panicle (Figs. 2 R-T). The broken of phosphorus homeostasis may be the cause of panicle development defects and yield reduction, suggesting that SiPHO2 plays a crucial role in coordinating both Pi homeostasis and agronomic traits in Setaria italica through the regulation of interorgan Pi partitioning. 3.4. Some Pi transporters and SPX proteins were responsible for root architecture changes of SiPHO2 plants Root architecture plays a crucial role in ion absorption and plant development. Under sufficient Pi condition, SiPHO2-CR plants exhibited significant alterations in root morphology compared to Ci846, including reduced PR length and LR number, but increased LR length (Fig. 3 A, E-G). These architectural changes were associated with Pi distribution patterns. While root Pi concentration was significantly lower in SiPHO2-CR plants, the higher shoot Pi concentration resulted in an increase shoot to root Pi ratio relative to Ci846 (Fig. 3 B-D). These findings demonstrated that SiPHO2 played an integrative role in coordinating root development with Pi acquisition and distribution in Setaria italica . To investigate the molecular mechanisms underling root architecture alterations and Pi signal regulation, we analyzed the expression patterns of key genes involved in Pi homeostasis, including high-affinity Pi transporters and SPX domain-containing proteins using qRT-PCR. The OsPHO1 homolog SiPHO1;H1 was up-regulated in shoots, while several Pi transporters ( SiPHO1 , SiPHT1 and SiPHF1 ) and SPX domain-containing proteins ( SiSPX1 and SiMSF ) showed significant up-regulation in roots (Fig. 3 H). The different tissue-specific expression patterns are consistent with the established functional specialization of these genes, where PHO1;H1 primarily regulates shoot Pi homeostasis and PHT1 family members maintain root Pi homeostasis [ 52 ]. These findings suggested that SiPHO2 modulated Pi uptake and translocation by regulating the expression of key Pi homeostasis-related genes, thereby influencing both root architecture and systemic Pi distribution. 3.5. SiPHO2-CR plants exhibited developmental inhibition during the heading date. The flowering stage is an important period which needs more water and nutrient resources to maintain the crops development, where Pi homeostasis plays pivotal regulatory roles. The phenotype comparison between Ci846 and SiPHO2-CR mutants revealed the mutant lines exhibited a mean 5.81 day delay in heading date under normal Pi condition (Fig. 4 A, 4 C). It demonstrated the functional involvement of SiPHO2 in reproductive phase regulation. Furthermore, SiPHO2-CR plants displayed significant alterations in key agronomic traits, including 38.84% reduction in mean plant height (Figs. 4 B), 17.33% decrease in leaf length (Figs. 4 D) and 25.50% reduction in leaf width (Figs. 4 E). To elucidate the SiPHO2 -mediated heading date regulatory mechanisms, we performed transcriptomic sequencing between Ci846 and SiPHO2-CR mutants. Differential expression analysis (|log 2 FC| ≥ 1; FDR ≤ 0.001) identified 1,437 differential expressed genes (DEGs) (813 up-regulated and 624 down-regulated) in SiPHO-CR mutants (Fig. 4 F). Gene Ontology (GO) enrichment analysis revealed predominant enrichment of these DEGs were mainly enriched in carbohydrate / polysaccharide metabolism, cell wall organization and cellular amide metabolic process (Fig. 4 G). In addition, through functional annotation screening of DEGs, 17 heading date related genes exhibiting significant expression alterations in SiPHO2-CR mutants compared to Ci846 (Fig. 4 H) and six key flowering genes were verified by qRT-PCR. The results showed a complete suppression of SiFTc (paralogous of SiFTa , 0.15 fold of Ci846), significant down regulation of SiMADS15 and SiMADS3 (MADS-box transcription factor, 0.15-fold and 0.37-fold of Ci846 respectively) and SiNFYA10 (CCAAT-box binding factor, 0.29-fold of Ci846), while up regulation of SiHY5 (photomorphogenesis regulator, 1.65-fold of Ci846) under Pi normal conditions in SiPHO2-CR mutants. However, expression trends of these genes were reversed under Pi deficient conditions, the up-regulation of SiFTc , SiCOL9 , SiMADS15 , SiMADS3 and SiNFYA10 , while the significant down-regulation of SiHY5 in SiPHO2-CR mutants compared to Ci846 (Fig. 4 I). These results indicate that SiPHO2 modulates heading date of foxtail millet through transcriptional regulation of conserved flowering pathway factors, particularly via its significant impact on SiFTc and SiMADS3/15 . Phylogenetic analysis of the FT gene family showed SiFTc was a paralogous gene of SiFTa (Figure S4 ), and SiFTa protein showed significantly higher conservation with OsHd3a (87.71% identity), SiFTc exhibited relatively lower sequence conservation with OsHd3a (75.42% identity). To investigate the functional role of SiFTc , eight SiFTc overexpression transgenic lines obtained in ZH11 background, seven of them were transgenic positive lines and showed overexpression (Figure S5 A-B). The three lines with the highest SiFTc expression levels for further analysis. The SiFTc-OE lines exhibited an accelerated floral transition, with heading dates advanced by 28 days compared to ZH11 (22 ± 1.8 days vs 50 ± 2.1 days). Concurrently, these transgenic plants displayed pleiotropic morphological changes, including obvious dwarfism and reduced leaf width and length (Fig. 4 J, K). It was confirmed that SiFTc was function as a flowering inducer and integrated into the PHO2 -mediated heading date regulatory network in foxtail millet. 3.6. Pi deficient conditions influenced the whole plant development of SiPHO2-CR plants. Under Pi deficient conditions, the plant height of SiPHO2-CR mutants was significantly reduced compared to that of Ci846, both at the seedling stage (47.9% reduction) and at the flowering stage (41.4% reduction) (Fig. 5 A, B and Figure S6 ). Moreover, the Pi deficiency response was obvious in the leaf architecture, with SiPHO2-CR mutants exhibiting decreases in both leaf length and width relative to Ci846 (Fig. 5 C). These findings underscored the necessity of functional SiPHO2 for the maintaining of plant development under Pi limited conditions. To dissect the molecular mechanisms underlying SiPHO2 -mediated growth regulation under Pi limitation, we conducted comparative transcriptome analysis of SiPHO2-CR mutants and Ci846 at flowering stage under Pi deficient conditions. Differential expression analysis identified 1,146 significantly DEGs (549 up-regulated and 597 down-regulated) in the mutants. GO analysis results showed that the most significant enrichment functional terms was the canonical Pi response pathways, including cell response to phosphate starvation, phospholipid catabolic process and cell response to nutrient level. These genes included some nutrition-related genes, such as phosphorus-related genes ( PHT1s , purple acid phosphatases ( PAPs ), sulfoquinovosyl diacylglycerol ( SQD ), monogalactosyldiacylglycerol ( MGDG ) and SPXs (Fig. 5 D). These results suggested that the SiPHO2 mutation might mimic the natural Pi starvation signal and activate a deficiency Pi response in the SiPHO2 - CR mutants. In addition, the pathways involved in auxin biosynhetic process regulation, cell communication, cell periphery and ion transmembrane transport activities were enriched (Fig. 5 E-H). Among them, auxin bioanabolic process and cell periphery are closely related to plant growth and development. In the samples of Ci846 and SiPHO2 - CR plants cultivated under the Pi deficient condition, the expression of three development-associated DEGs, containing ALTERED SEED GERMINATION 3 ( SiASG3 ), ABNORMAL LEAF SHAPE 2 ( SiALE2 ) and QUANTITATIVE RESISTANCE TO PLECTOSPHAERELLA 1 ( SiQRP1 ), were selected for qRT-PCR. Consistent with RNA-seq data, SiASG3 and SiALE2 showed suppressed expression in SiPHO2-CR mutants, while SiQRP1 was induced (Fig. 5 I). In Arabidopsis, AtASG3 is associate with cell cycle regulation during early leaf and root growth [ 53 ], AtALE2 positively regulates protoderm-specific gene expression and for the formation of leaf [ 54 ] and AtQRP1 negatively regulates epidermal and mesophyll development [ 55 ]. These results collectively indicated that SiPHO2 played a role in plant growth and development by modulating the activity of numerous genes. 4. Discussion 4.1. SiPHO2 modulates root architecture and leaf development through Pi homeostasis. Pi acquisition and distribution in plants are governed by complex spatial regulation involving root cytoplasm and vascular tissues, a process mediated by the coordinated xylem loading and phloem redistribution [ 56 ]. In poaceae species, adaptive responses to Pi deficiency often include enhanced PR elongation and increased LR formation, as observed in rice and maize [ 57 , 58 ]. In addition, the sbpho2 mutant showed reduced PR growth under Pi sufficient conditions [ 47 ]. Pi deficiency tolerant genotype showed a lower expression of PvPHO2 , while the Pi content and root biomass increased compared to the sensitive genotype under Pi starvation in bean [ 59 ]. However, the genetic mechanisms underlying these adaptive responses in Setaria italica , a model C4 model, remain poorly understood. We investigated the modifications in root system architecture under Pi sufficient conditions and found that SiPHO2-CR mutants exhibited significant alterations in root morphology compared to Ci846, including shortened PR, reduced LR density and elongated individual LR (Fig. 3 A, E-G). These architectural variations suggested a crucial role for SiPHO2 in modulating root plasticity and Pi sensitivity, consistent with conserved functions observed in Ospho2 and Atpho2 mutants, where PHO2 regulates root elongation, and this regulation is enhanced in the ltn1 mutant under Pi starvation [ 26 ]. Building on these observations, we systematically characterized tissue-specific Pi redistribution patterns across different developmental stages. The SiPHO2-CR mutants showed root Pi reduced concomitant with shoot hyper-accumulation, particularly in leaves during both vegetative and reproductive phases (Fig. 2 D, H-J). Subsequent analysis revealed an enhanced vascular translocation capacity from stems to leaves, along with preferential Pi transporting to apical leaf regions. Notably, this altered redistribution pattern correlated with progressive chlorosis and necrotic lesion development at leaf tip during maturation, which can be attributed to Pi toxicity resulting from compartmentalization defects. Collectively, these findings underscore the pivotal role of SiPHO2 in maintaining Pi spatial homeostasis through the coordinated regulation of source-to-sink translocation dynamics. Previous research has shown that the knockout of TaPHO2 -A1 in wheat led to a significantly enhancement in Pi uptake and grain yield, with no discernible adverse effects under high Pi conditions [ 60 ]. In contrast, our study observed that SiPHO2-CR mutants displayed impaired panicle development, marked by substantial reductions in panicle length, width and weight (Fig. 2 P-T). Notably, our analysis of Pi absorption and transport dynamics revealed a significant decrease in Pi translocation efficiency from leaves to panicles (Fig. 2 J), which may be responsible for the observed abnormalities in panicle development. This finding highlights the importance of SiPHO2 in maintaining the balance of Pi distribution and the crucial role in panicle development of Setaita italica . 4.2. SiPHO2 plays a crucial role in plant growth and development through both Pi signaling pathways and other gene regulatory mechanisms. The PHR1 -miR399- PHO2 signaling pathway, a well-established regulatory network for Pi homestasis, has been extensively characterized in rice and Arabidopsis [ 61 , 18 ]. However, this pathway remains to be elucidated in foxtail millet. Our study has uncovered distinct molecular responses in foxtail millet under Pi sufficient conditions. While the expression profiles of key Pi-starvation-responsive genes were found to be relatively stable in the shoots, a significant up-regulation of numerous genes associated with Pi starvation was observed in the roots. Notably, we identified two pivotal transporters showed a increased expression in roots of SiPHO2-CR lines: SiPHT1 ( Seita.9G238300 ), a homolog of the rice Pi transporter OsPT2 , which is implicated in root-to-shoot translocation of Pi [ 62 ], SiPHO1 ( Seita.1G360400 ), the homolog of OsPHO1 , which is known to facilitate Pi transport from root to shoot in rice [ 63 ] (Fig. 3 H). Our research indicated that SiPHT1 and SiPHO1 might act in concert to regulate the translocation of Pi from the root to the shoot in foxtail millet, potentially accounting for the higher Pi levels observed in the leaves of SiPHO2-CR mutants compared to Ci846 (Fig. 2 H, I). This finding aligns with the role of these transporters in other species, such as rice, where they are involved in Pi translocation. Furthermore, we have identified SiPHF1 ( Seita.2G065200 ), a homolog of OsPHF1 in rice, which is known to regulate the plasma membrane localization of both high and low affinity Pi transporters, thereby controlling Pi uptake and transport [ 64 ], was higher expressed in SiPHO2-CR lines (Fig. 3 H). This suggests that SiPHF1 may play a similar role in foxtail millet with that of rice, facilitating Pi trafficking from the endoplasmic reticulum to the plasma membrane. Under Pi deficient conditions, we observed significant differential expression of Pi starvation responsive genes. GO analysis revealed a prominent clustering of DEGs within the Pi starvation response module (Fig. 5 E), which is consistent with previous observations of PHO2 -mediated gene expression changes in rice and Arabidopsis [ 26 ]. These findings collectively demonstrated that SiPHO2 mediated conserved regulatory functions in plant growth and development through Pi signaling pathways in foxtail millet. GO enrichment analysis of DEGs in SiPHO2-CR plants under Pi deficient conditions also revealed significant enrichment of terms associated with auxin biosynthesis and metabolism, cell periphery organization and nutrient homeostasis (Fig. 5 F-H). Considering the key role of hormonal regulation in root development [ 65 ], we conducted a detail investigation into auxin signaling dynamics in the SiPHO2-CR mutant. Notably, key auxin biosynthesis genes, such as those in the YUC family, which are responsive to Pi starvation, exhibited differently regulation. In rice, auxin derived from YUC activates the transcription of OsWOX11 , thereby promoting crown root development [ 66 ]. Similarly, the up regulated of SiPGP (Fig. 5 G), encoding an auxin efflux carrier involved in auxin polar transport [ 67 ], suggests an enhanced redistribution of auxin in SiPHO2-CR . Collectively, these findings indicate that SiPHO2 modulates auxin pathway activity to influence growth architecture. Mechanistic investigations uncovered multiple developmental regulators responsive to Pi starvation. For instance, ASG3 , which regulates leaf and root growth early in the cell cycle [ 53 ], QRP1 , which affects epidermal and mesophyll development [ 55 ], ALE2 , involved in the regulation of the formation of leafy organs [ 54 ], BRL2 , which controls cell division and elongation to regulate organ development [ 68 , 69 ], the homolog of these genes showed increased expression in SiPHO2-CR mutants under Pi starvation, suggesting that they might be regulated by SiPHO2. This SiPHO2 -mediated transcriptional network likely drives the observed phenotypic reductions in plant height and leaf width in SiPHO2-CR mutants. These findings demonstrate that SiPHO2 orchestrates plant growth through dual mechanisms: the first being canonical Pi signaling and the second involving a multi-layered regulatory encompassing auxin signaling, cell cycle control and tissue morphogenesis. This highlights the integrative role of SiPHO2 in coordinating developmental plasticity under Pi stress. 4.3. SiPHO2 regulates heading date through modulation of SiFTc Flowering time in plants is regulated by a variety of abiotic stresses, including cold and nutrient deficiencies, such as nitrogen, phosphorus and potassium [ 70 , 71 ]. Flowering integration genes expressed predominantly in the companion cells of the leaf phloem, particularly in the distal secondary vein of leaves [ 72 ]. The FT protein is transported from the leaf to the shoot apical meristem through the phloem to execute its biological functions [ 72 ]. It is well established that the GI-CO-FT module constitutes a classic flowering pathway across different species [ 73 ]. Several regulatory factors can modulate flowering by influencing the expression of FT . For instance, CO , a B-box zinc finger transcription factor, promotes FT expression in sieve tube companion cell under long-day conditions in Arabidopsis leaves, leading to the formation of an FT protein complex that activates downstream genes such as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 ( SOC1 ) and APETALA1 ( AP1 ), thereby promoting flowering [ 72 ]. Overexpression of FT in Arabidopsis results in extremely early flowering, while the ft mutant exhibits a delayed-flowering phenotype [ 73 ]. In Arabidopsis, both miR399-OE plants and pho2 mutants exhibited an early flowering phenotype under normal temperature conditions (23°C) [ 27 ]. However, the early flowering phenotype is abolished in pho2 mutant plants when grown under low Pi conditions, with delayed flowering even being observed [ 74 ]. In contrast, our study demonstrated that SiPHO2-CR mutants displayed a delayed heading date under Pi sufficient conditions, whereas under Pi deficiency cultivation, the heading date of SiPHO2-CR mutants was significantly earlier compared to that of Ci846. Notably, under Pi sufficient conditions, the expression of SiFTc in SiPHO2-CR mutants was lower than that of Ci846, but the expression pattern was reversed under Pi deficiency cultivation (Fig. 4 I). These findings suggest that SiPHO2 plays a critical role in the crosstalk between Pi homeostasis and the regulation of flowering time. The loss of OsPHO2 function resulted in a delayed flowering phenotype under Pi normal conditions in rice, it is similar with our results [ 33 ]. These results further supporting the hypothesis that the regulatory mechanisms governing flowering time in response to Pi availability differ between monocots and dicots. The regulatory network between Pi signaling and flowering time involves complex molecular crosstalk across plant species. In Arabidopsis, PHO1 modulates the floral transition through jasmonic acid (JA) dependent suppression of FT and TWIN SISTER OF FT ( TSF ), a paralogue of FT [ 74 ], while OsPHO2 coordinates Pi homeostasis and flowering time via interaction with GI , a core flowering regulator [ 33 ]. Building on these models, our transcriptome analysis in foxtail millet uncovered a distinct mechanism: SiPHO2 deficiency specifically disrupts the expression of SiFTc (an ortholog of OsHd3a ), while was not FTa , which is the most ortholog of OsHd3a and is a flowering inducer [ 32 ]. This reveals evolutionary divergence in Pi responsive flowering regulation and establishes SiFTc as a novel Pi-stress-responsive flowering determinant. This finding contrasts with the conserved GI-CO-FT module[ 73 ]. Our results thus delineated a SiPHO2-SiFTc regulatory module that reconfigures heading date pathways under Pi limitation, providing mechanistic insights into nutrient-environment coordination of heading date in Setaria italica . Pi deficiency is indeed a major yield-limiting factor in crop production, as it influences crop yield and development [ 75 – 78 ]. The excessive reliance on Pi fertilizers to address Pi deficiency is unsustainable, and while the molecular mechanisms of enhancing yield stability under Pi starvation remain poorly understood. Our results showed that SiFTc overexpression accelerated the heading date, providing critical insights into Pi-responsive developmental regulation. Therefore, we speculate that SiFTc suppression or loss-of-function under Pi limitation will delay the heading date while enhancing yield potential. These findings establish SiFTc as a key regulator of Pi starvation adaptation and provide a targeted genetic strategy to develop more resilient crop varieties in the face of Pi scarcity through optimized heading date regulation. Declarations Conflict of interest The authors declare that they have no conflict of interest. Author contributions Xing GF and Zhang JW supervised the study; Li YQ, Wang HL and Fei HM performed experiments; Jia DD generated transgenetic plants; Qu RF and Wei R organized the vectors of transgenetic, Zhang YH, Liao HZ, Wei JH and Zhao XW provided technical assistance; Wang HL and Li YQ analysed the whole data and writing the manuscript; Xing GF revised the manuscript. Acknowledgments This work was funded by Beijing Natural Science Foundation (6234044), the National Natural Science Foundation of China (32372062, 32241041), the Youth Scientific Research Foundation of Beijing Academy of Agriculture and Forestry Sciences (QNJJ202414), the National Key R&D Program of China (No. 2023YFD1200704, 2023YFD1200700), the Shanxi Province Postgraduate Innovation Project (2023KY328, 2021Y335). References De Bang TC, Husted S, Laursen KH, Persson DP, Schjoerring JK (2021) The molecular-physiological functions of mineral macronutrients and their consequences for deficiency symptoms in plants. New Phytol 229:2446–2469 Cong WF, Suriyagoda LDB, Lambers H (2020) Tightening the phosphorus cycle through phosphorus-efficient crop genotypeS. Trends Plant Sci 25:967–975 Jiang M, Caldararu S, Zaehle S, Ellsworth DS, Medlyn BE (2019) Towards a more physiological representation of vegetation phosphorus processes in land surface models. 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The SiPHO2 proteins sequence alignment between Ci846 and SiPHO2-CR mutants. FigureS4.tif Figure S4. Phylogenetic and protein sequence analysis of FT homologys . FigureS5.tif Figure S5. SiFTc-OE lines identified in ZH11 background. FigureS6.tif Figure S6. Phenotypic analysis of Ci846 and SiPHO2-CR plants at two-week-old seedlings under Pi normal (A) and Pi deficient (B) conditions. FigureS7.tif Figure S7. Tissue-specific Pi concentration analysis between Ci846 and SiPHO2-CR plants at maturity under Pi deficient conditions. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6218826","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431814698,"identity":"61ba34e8-df19-48f6-82af-25e1c1045bd6","order_by":0,"name":"Yuqian Li","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuqian","middleName":"","lastName":"Li","suffix":""},{"id":431814699,"identity":"844d2cce-909b-434b-af0e-2b0dfb128288","order_by":1,"name":"Hailong Wang","email":"","orcid":"","institution":"Beijing Academy of Agriculture and Forestry 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University","correspondingAuthor":false,"prefix":"","firstName":"Yonghu","middleName":"","lastName":"Zhang","suffix":""},{"id":431814703,"identity":"1a3edbfb-9206-4c9c-9ade-871ef1a85b98","order_by":5,"name":"Hongze Liao","email":"","orcid":"","institution":"Guangxi Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Hongze","middleName":"","lastName":"Liao","suffix":""},{"id":431814704,"identity":"8bbf599c-1ed1-449c-9d19-28161c0658dd","order_by":6,"name":"Rui Wen","email":"","orcid":"","institution":"Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Wen","suffix":""},{"id":431814705,"identity":"f22615b4-db1a-4ecf-bb87-90a88591bf21","order_by":7,"name":"Xiongwei Zhao","email":"","orcid":"","institution":"Shanxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiongwei","middleName":"","lastName":"Zhao","suffix":""},{"id":431814706,"identity":"f6779aa2-f811-4289-b6c9-5a5e07118e5b","order_by":8,"name":"Jianhua Wei","email":"","orcid":"","institution":"Beijing Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jianhua","middleName":"","lastName":"Wei","suffix":""},{"id":431814707,"identity":"879cd87d-e7c6-4094-8c8a-7cf6c6d920e5","order_by":9,"name":"Jiewei Zhang","email":"","orcid":"","institution":"Beijing Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jiewei","middleName":"","lastName":"Zhang","suffix":""},{"id":431814708,"identity":"2b938a41-97c4-4ffc-b530-3b97371b57fb","order_by":10,"name":"Guofang Xing","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDAC5gMMBxgYbKA8NmK0sCWAtKSRqAUIDpOgRd6Nx/Bwwa/z9gbHzxgwfCg7zMA/uwG/FsNjbAmHZ/bdTpzZk2PAOOPcYQaJOwcIaJnffOAwb8/tBH6GHANm3rbDDAYSCQS0tDE2ALWcs2fjf2PA/JcYLfJszAcO8/w4wNgvAbSFkRgtBmxAv/A2JCfOnPGs4GDPuXQeiRuEbGnjMf7M88fO3uB88sYHP8qs5fhnELLlAJBgbINwQGwe/OpBtjSAyD8E1Y2CUTAKRsFIBgB1DEOdocLxdgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4834-6209","institution":"Shanxi Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Guofang","middleName":"","lastName":"Xing","suffix":""}],"badges":[],"createdAt":"2025-03-13 10:07:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6218826/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6218826/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79073876,"identity":"f1cb9660-3087-4cf8-b4c2-044c24d0fd59","added_by":"auto","created_at":"2025-03-24 06:44:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":472557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization and expression patterns analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSiPHO2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in \u003c/strong\u003e\u003cem\u003eSetaria italica. \u003c/em\u003e(A) Subcellular localization analysis of \u003cem\u003eSiPHO2\u003c/em\u003e in \u003cem\u003eSetaria italica\u003c/em\u003e protoplasts. Scale bar = 10 µm. (B-C) Expression pattern analysis of \u003cem\u003eSiPHO2 \u003c/em\u003eunder Pi deficient (B) and Pi normal (C) conditions. (D–I) Different tissues expression pattern of \u003cem\u003eSiPHO2 \u003c/em\u003eanalysis by GUS staining in Setaria italica.seedling (D), stem (E), node (F), leaf (G), milking panicle (H) and leaf sheath (I). Scale bar = 1 cm. (J) Tissue-specific expression analysis of \u003cem\u003eSiPHO2\u003c/em\u003e at different developmental stages. Data represent mean ± SD (n = three biological replicates). Values labeled with the same lowercase letters are not significantly different, while the different letters represent significant differences as determined by Duncan’s multiple range test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/eefcde53b25b245c2943580c.png"},{"id":79073176,"identity":"f8227bb1-67e3-4000-83a8-e7bac7ac2b0a","added_by":"auto","created_at":"2025-03-24 06:36:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":272785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe agriculture traits and Pi concentration analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSiPHO2-CR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants and Ci846 under normal conditions. \u003c/strong\u003e(A) Diagram showing the location of the CRISPR-Cas9 edited site within the \u003cem\u003eSiPHO2\u003c/em\u003e sequence, sequence and protein traces of this region in Ci846 and SiPHO2knockout lines. (B) Phenotype comparison of two-week-old seedlings of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants under Pi normal conditions. Scale bar = 2 cm. (C) Whole plant Pi concentration analysis of two-week-old seedlings. (D) Shoot-to-root Pi ratio analysis of two-week-old seedlings. (E) Phenotype comparison of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at maturity. Scale bar = 10 cm. (F) The leaf tip necrosis phenotype of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at maturity. Scale bar = 1 cm. (G) Necrotic area ratio of the second fully expanded leaves in Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants. (H) Pi concentration comparison of the second fully expanded leaves between Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants. (I) Tissue-specific Pi concentration analysis of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at maturity. (J) Pi uptake and transport ratio analysis of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at maturity. (K) Panicle phenotype of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at maturity. Scale bar = 1 cm. (L–T) Agriculture traits analysis of plant height (L), leaf length (M), leaf width (N), SPAD (O), panicle length (P), panicle width (Q), panicle weight (R), 1000-grains weight (S) and grains number per panicle (T) of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at maturity. Data represent mean ± SD (n = three biological replicates). Values labeled with the same lowercase letters are not significantly different, while the different letters represent significant differences as determined by Duncan’s multiple range test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Significant differences between two groups were assessed using unpaired two-sided Student’s t-tests (*, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/6187a1d48997dce510867974.png"},{"id":79073193,"identity":"8c52696f-e5cb-4c45-a016-71b53560dacd","added_by":"auto","created_at":"2025-03-24 06:36:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":869831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRoot architecture analysis of Ci846 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSiPHO2-CR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants under Pi-normal conditions\u003c/strong\u003e. Phenotype comparison of one-week-old seedlings of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants under normal conditions. Scale bar = 1 cm. (B–D) Pi concentration analysis of shoots (B), roots (C) and shoot-to-root ratio (D) of one-week-old seedlings. (E–G) Phenotype analysis of PR length (E), LR number (F) and LR length (G) of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at one-week-old seedlings. (H) The transcript level analysis of six phosphorus-related genes in shoots and roots between Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at one-week-old seedlings. Data represent mean ± SD (n = three biological replicates). Values labeled with the same lowercase letters are not significantly different, while the different letters represent significant differences as determined by Duncan’s multiple range test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Significant differences between two groups were assessed using unpaired two-sided Student’s t-tests (*, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/99b99f7d7b9a4bba9b98662f.png"},{"id":79073183,"identity":"977a6296-905d-4001-a8b3-304f3bfabe5d","added_by":"auto","created_at":"2025-03-24 06:36:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":316525,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq analysis of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants and functional verification of \u003cem\u003eSiFTc \u003c/em\u003ein rice. \u003cstrong\u003e(A)\u003c/strong\u003e Phenotype comparison of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003eplants at the heading stage. Scale bar = 10 cm. \u003cstrong\u003e(B–E)\u003c/strong\u003e Phenotypic analysis of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003eplants in plant height \u003cstrong\u003e(B)\u003c/strong\u003e, heading date \u003cstrong\u003e(C)\u003c/strong\u003e, leaf length \u003cstrong\u003e(D)\u003c/strong\u003e and leaf width \u003cstrong\u003e(E)\u003c/strong\u003e at the heading stage. \u003cstrong\u003e(F)\u003c/strong\u003e Volcano plot analysis of DEGs betweenCi846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at the heading stage. \u003cstrong\u003e(G)\u003c/strong\u003e GOenrichment analysis of DEGs in Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at the heading stage.\u003cstrong\u003e (H)\u003c/strong\u003e Heat map analysis of key flowering-related genes in DEGs between Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at the heading stage. \u003cstrong\u003e(I)\u003c/strong\u003e Transcript levelanalysis of six flowering-related genes between Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003eplants. \u003cstrong\u003e(J)\u003c/strong\u003e Phenotype comparison of ZH11 and \u003cem\u003eSiFTc-OE\u003c/em\u003e in rice background. \u003cstrong\u003e(K)\u003c/strong\u003e Heading date analysis between ZH11 and \u003cem\u003eSiFTc-OE \u003c/em\u003etransgenicplants. Data represent mean ± SD (n = three biological replicates). Values labeled with the same lowercase letters are not significantly different, while the different letters represent significant differences as determined by Duncan’s multiple range test (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/485b443d8ebb8a34d0b6a02f.png"},{"id":79073875,"identity":"549d40bd-464e-4e33-b449-c774540317a1","added_by":"auto","created_at":"2025-03-24 06:44:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":563969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA-seq analysis of Ci846 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSiPHO2-CR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants under Pi deficient conditions.\u003c/strong\u003e (A) Phenotype comparison of two-week-old seedlings between Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants under Pi deficient conditions. Scale bar = 1 cm. (B) Phenotype comparison of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at the heading stage under Pi deficient conditions. Scale bar = 10 cm. (C) Agriculture traits analysis of heading date, plant height, leaf length and leaf width in Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at the heading stage under Pi deficient conditions. (D) GO analysis between Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants under Pi deficient conditions. (E–H) Expression profiles of key genes involved in phosphate starvation response (E), cell periphery (F), auxin biosynthesis and metabolism (G) and nutrient response pathways (H) in Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants under Pi deficient conditions. (I) The expression analysis of leaf development-related genes in Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants under Pi deficient conditions. Data represent mean ± SD (n = three biological replicates). Values labeled with the same lowercase letters are not significantly different, while the different letters represent significant differences as determined by Duncan’s multiple range test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/835c1134bb5ee263aeca58c5.png"},{"id":89062885,"identity":"3a087b7e-3c5f-4eb4-938e-7bcac4944911","added_by":"auto","created_at":"2025-08-14 09:55:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3827748,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/bed75403-edf1-40c6-9868-1e27160fa631.pdf"},{"id":79073179,"identity":"c42f2bb1-44c7-4aa3-812c-8fa2c020113c","added_by":"auto","created_at":"2025-03-24 06:36:25","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11520,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. \u003cstrong\u003ePrimers used in this study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Supplementarytable1.Primersusedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/098982eeb8178127e3a6dc54.xlsx"},{"id":79073191,"identity":"6ecc6d59-348e-4844-8907-9c63094e08cc","added_by":"auto","created_at":"2025-03-24 06:36:26","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20605976,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1.\u003cstrong\u003e Phylogenetic analysis of UBCs family and abiotic stresses response of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSiPHO2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/235210d3031482089903f074.tif"},{"id":79073190,"identity":"bf4138c8-f900-4e4e-8950-00c9f89a4f26","added_by":"auto","created_at":"2025-03-24 06:36:26","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23228472,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2. Different tissues expression pattern of \u003cem\u003eSiPHO2 \u003c/em\u003ein \u003cem\u003eOryza sativa \u003c/em\u003erevealed by GUS staining.\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/d54e5ad68dbebf9a0fb157e8.tif"},{"id":79073877,"identity":"9228a2a6-f4ce-403c-9446-52efdf5c9770","added_by":"auto","created_at":"2025-03-24 06:44:26","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":33295696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3. The SiPHO2 proteins sequence alignment between Ci846 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSiPHO2-CR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutants.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/cf820e997b1afeb04f2b69f3.tif"},{"id":79073881,"identity":"2ecaad07-59d2-46c1-8684-334f2549f099","added_by":"auto","created_at":"2025-03-24 06:44:26","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":73691456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S4. Phylogenetic and protein sequence analysis of FT\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ehomologys\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/ec3f65b351c8ced00615c6bd.tif"},{"id":79073231,"identity":"2be46755-02c8-4278-949a-79c239e6424d","added_by":"auto","created_at":"2025-03-24 06:36:27","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":74722360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S5. \u003c/strong\u003e\u003cem\u003eSiFTc-OE\u003c/em\u003e\u003cstrong\u003e lines identified in ZH11 background.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"FigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/aba33755f5dafb6b6a553565.tif"},{"id":79073180,"identity":"5e5a2b45-1a57-4c8a-9912-a76881f859b0","added_by":"auto","created_at":"2025-03-24 06:36:25","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1495032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S6. \u003c/strong\u003ePhenotypic analysis of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at two-week-old seedlings under Pi normal \u003cstrong\u003e(A) \u003c/strong\u003eand Pi deficient \u003cstrong\u003e(B)\u003c/strong\u003e conditions.\u003c/p\u003e","description":"","filename":"FigureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/8fff3cb8800714f6e62247dd.tif"},{"id":79073882,"identity":"9ed24844-b1fe-40a0-825f-37b49f302a06","added_by":"auto","created_at":"2025-03-24 06:44:27","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":78469896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S7. \u003c/strong\u003eTissue-specific Pi concentration analysis between Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at maturity under Pi deficient conditions.\u003c/p\u003e","description":"","filename":"FigureS7.tif","url":"https://assets-eu.researchsquare.com/files/rs-6218826/v1/0a55cb6e9a56443933d8638f.tif"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003eSiPHO2\u003c/em\u003e orchestrates phosphorus homeostasis and regulates heading date through SiFTc-dependent pathway in foxtail millet (\u003cem\u003eSetaria italica\u003c/em\u003e)\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePhosphorus (P), an essential macronutrient for plant growth and development, plays a pivotal role in energy production, photosynthesis, glycolysis, respiration and the biosynthesis of nucleic acids and membranes system [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Under Pi deficient conditions, crops exhibit distinct deficiency symptoms, including impaired growth, reduced biomass, purple leaf coloration and anthocyanin accumulation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Plants primarily acquire inorganic phosphate (Pi) from soil solutions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], where available Pi concentrations typically average 1 mM, substantially below the 5\u0026ndash;20 mM threshold required to sustain normal plant growth and yield [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Current mitigation strategies involving excessive application of phosphate fertilizers prove ecologically unsustainable, with merely 10\u0026ndash;20% fertilizer utilization efficiency, while residual inputs drive aquatic eutrophication and biodiversity loss [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These realities underscore improving phosphorus use efficiency (PUE) as a critical determinant of sustainable agriculture, demanding mechanistic elucidation of plant Pi homeostasis regulation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePi deficiency adversely affects tiller number, panicle formation, setting rate and overall yield. The root system, as the primary organ for Pi sensing and acquisition, plays a decisive role in Pi absorption and yield [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Under Pi deficient conditions, plants often increase the root-to-shoot ratio and modify root architecture to improve Pi uptake [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Root architecture, including root length, root hair (RH) density and root angle are primarily influenced [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To counteract these effects, extensive researches have been conducted on the morphological, physiological and molecular mechanisms underlying Pi homeostasis in plants.\u003c/p\u003e \u003cp\u003eThe regulation of Pi homeostasis involves a complex network, central to this network is \u003cem\u003ePHOSPHATE STARVATION RESPONSE1\u003c/em\u003e (\u003cem\u003ePHR1\u003c/em\u003e), a MYB-CC family transcription factor that senses Pi status and orchestrates Pi deficiency response strategies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In Arabidopsis, \u003cem\u003ePHR1\u003c/em\u003e overexpression leads to increased Pi concentration in shoot tissues [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. There are four \u003cem\u003ePHR1\u003c/em\u003e homologs (\u003cem\u003eOsPHR1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e) in rice, \u003cem\u003eOsPHR2\u003c/em\u003e overexpression results in Pi accumulation in leaves, increased root length, root-to-shoot ratio and RH density, alongside up-regulation of Pi starvation-induced (PSI) genes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003ePHR1\u003c/em\u003e induces the expression of PSI genes, such as \u003cem\u003ePHOSPHATE TRANSPORTER1\u003c/em\u003e (\u003cem\u003ePHT1\u003c/em\u003e) and \u003cem\u003ePHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1\u003c/em\u003e (\u003cem\u003ePHF1\u003c/em\u003e), to regulate Pi absorption, translocation and remobilization [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Further research indicates that \u003cem\u003eOsPHR2\u003c/em\u003e acts as a central regulator, with higher expression levels than those of \u003cem\u003eOsPHR1\u003c/em\u003e and \u003cem\u003eOsPHR3\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder Pi deficiency, miR399 expression is strongly induced, leading to the down-regulation of \u003cem\u003ePHO2\u003c/em\u003e, which encodes an E2 UBC enzyme. The degradation of \u003cem\u003ePHO2\u003c/em\u003e transcripts by miR399 is regulated by long non-coding RNA (lncRNA) \u003cem\u003eINDUCED BY PHOSPHATE STARVA-TION 1,2\u003c/em\u003e (\u003cem\u003eIPS1,2\u003c/em\u003e) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], such as homologs of \u003cem\u003eIPS1\u003c/em\u003e in rice (\u003cem\u003eOsIPS1\u003c/em\u003e and \u003cem\u003eOsIPS2\u003c/em\u003e) and \u003cem\u003ePI-DEFICIENCY-INDUCED LNCRNA 2\u003c/em\u003e (\u003cem\u003ePILNCR2\u003c/em\u003e) of maize exhibit similar functions [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In Arabidopsis and rice, \u003cem\u003ePHO2, NITROGEN LIMITATION ADAPTATION\u003c/em\u003e (\u003cem\u003eNLA1\u003c/em\u003e) and miR827 maintain Pi homeostasis in a NO\u003csup\u003e3\u0026minus;\u003c/sup\u003e dependent manner [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Further researches found that both PHO2 and NLA1 are essential for PHT1 polyubiquitination at the plasma membrane and subsequent degradation via the 26S proteasome system [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, PHO2 negatively regulates PHO1 and PHF1 abundance under Pi sufficient conditions, and it was identified that OsPHO1 and OsPHF1 as the potential substrates of OsPHO2, with yeast two-hybrid and bimolecular fluorescence complementation assays, which confirmed interactions between OsPHO1, OsPHF1 and OsPHO2, but not with OsPHT1;2/6/8 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, OsSP\u003cem\u003eX1\u003c/em\u003e expression is induced in \u003cem\u003eOspho2\u003c/em\u003e mutants of rice, indicating its involvement in \u003cem\u003eOsPHO2\u003c/em\u003e-mediated Pi signaling networks [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond its primary role in regulating Pi homeostasis, \u003cem\u003ePHO2\u003c/em\u003e has been demonstrated to participate in multiple functional pathways in plants, including nitrogen assimilation, iron uptake and flowering regulation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The regulation of flowering time in plants is a complex process that integrates environmental cues with internal signaling pathways [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Among these, FT belonging to the phosphatidyl ethanolamine binding protein (PEBP) family, serves as a central integrator of flowering signals. In \u003cem\u003eArabidopsis thali\u003c/em\u003eana, the GIGANTEA (GI) protein induces the expression of \u003cem\u003eCONSTANS\u003c/em\u003e (\u003cem\u003eCO\u003c/em\u003e), which subsequently activates the transcription of \u003cem\u003eFT\u003c/em\u003e, and the \u003cem\u003eGI-CO-FT\u003c/em\u003e pathway is highly conserved in plants [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In rice, the orthologous components of this pathway include \u003cem\u003eOsGI\u003c/em\u003e, \u003cem\u003eOsHd1\u003c/em\u003e and \u003cem\u003eOsHd3a.\u003c/em\u003e OsHd3a and SiFTa (Setaira) are homologous to AtFT, they are all the florigenic proteins and can induce flowering [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In rice, OsPHO2 regulates flowering through its interaction with OsGI. Both \u003cem\u003eOspho2\u003c/em\u003e and \u003cem\u003eOsgi\u003c/em\u003e mutants display reduced growth, leaf tip necrosis, delayed flowering and excessive Pi accumulation in leaves. The transcriptome analysis reveals the interaction between \u003cem\u003eOsPHO2\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e links high-level regulators of Pi homeostasis and flowering time in rice [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, \u003cem\u003eAtpho2\u003c/em\u003e mutants exhibit early flowering at 23 ℃ compared to wild-type (WT) plants, uncovering a novel role for the miR399-\u003cem\u003ePHO2\u003c/em\u003e pathway in mediating ambient temperature-responsive flowering in Arabidopsis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These findings highlight the multifaceted role of \u003cem\u003ePHO2\u003c/em\u003e in coordinating not only Pi homeostasis but also critical developmental processes such as flowering,which underscore its importance in plant growth and adaptation to environmental conditions.\u003c/p\u003e \u003cp\u003eUnder Pi deficient conditions, foxtail millet develops an extensive root system with longer LR [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Different foxtail millet varieties exhibit varying responses to Pi starvation, with up-regulation of Pi transporter genes \u003cem\u003eSiPHT1\u003c/em\u003e;\u003cem\u003e1\u003c/em\u003e, \u003cem\u003eSiPHT1\u003c/em\u003e;\u003cem\u003e2\u003c/em\u003e and \u003cem\u003eSiPHT1\u003c/em\u003e;\u003cem\u003e4\u003c/em\u003e in roots promoting Pi absorption, while down-regulation of \u003cem\u003eSiPHT1;3\u003c/em\u003e leads to Pi accumulation in stems [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Pi deficient conditions also alter SPAD and soluble protein content, balancing the nitrogen-to-phosphorus ratio in plants. \u003cem\u003ePHR1\u003c/em\u003e directly promotes expression of \u003cem\u003eNITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1\u003c/em\u003e (\u003cem\u003eNIGT1\u003c/em\u003e), nitrate-induced nitrogen receptor and transporter, family genes and \u003cem\u003eNIGT1\u003c/em\u003e integrates nitrogen and Pi signals to maintain nutrient balance [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Despite these advances, systematic studies on Pi starvation responses and the other functions of \u003cem\u003eSiPHO2\u003c/em\u003e in foxtail millet remain limited.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSetaria italica\u003c/em\u003e originates in China, is a model plant for studying nutrient utilization mechanisms due to higher resistance to drought and nutrient-poor soils [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, we identified \u003cem\u003eSiPHO2\u003c/em\u003e was essential not only for Pi homeostasis but also for heading date and plant development regulation in foxtail millet. Our findings also verified \u003cem\u003eSiFTc\u003c/em\u003e act as a novel factor of heading date regulation in Setaria under Pi starvation conditions and provided a targeted genetic strategy for breeding Pi-efficient foxtail millet cultivars through optimized heading date regulation.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Phylogenetic and \u003cem\u003eSiPHO2\u003c/em\u003e promoter analysis\u003c/h2\u003e \u003cp\u003eFor the phylogenetic analysis, protein sequences of UBCs in \u003cem\u003eSetaria italica\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and FT homology proteins in \u003cem\u003eSetaria italica\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003eSorghum bicolor\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were downloaded from PHYTOZOME v.13 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome.jgi.doe.gov\u003c/span\u003e\u003cspan address=\"https://phytozome.jgi.doe.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple-sequence alignments of SiPHO2 proteins were carried out using the Clustal W (version2.0) program [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Then, phylogenetic analysis was performed with MEGA7.0 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] using theneighbor-joining method (1000 bootstrap replications), and the results were visualized in Itol [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA region approximately 2000 bp upstream of the start codon (ATG) in \u003cem\u003eSiPHO2\u003c/em\u003e was investigated from the reference genome sequence of Yugu1. The online software Plant CARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to search for cis-acting regulatory elements in the promoters of \u003cem\u003eSiPHO2.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Subcellular localization assay\u003c/h2\u003e \u003cp\u003eTo explore the subcellular localization of SiPHO2, the coding sequence of SiPHO2 was cloned into the pCAMBIA1305-eGFP vector to generate pUbi::SiPHO2-eGFP, and the plasmid was co-transformed with the ER marker pUbi::AtWAK2-mcherry into foxtail millet leaf protoplasts using PEG-mediated transfection [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. TE2000-E inverted confocal microscope (Nikon A1R) was used to detect GFP and mCherry signals. The primers used are shown in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Vector construction and plant transformation\u003c/h2\u003e \u003cp\u003eFor CRISPR/Cas9-mediated knockout of \u003cem\u003eSiPHO2\u003c/em\u003e (\u003cem\u003eSeita.3G150400\u003c/em\u003e) in \u003cem\u003eSetaria italica\u003c/em\u003e, sgRNAs targeting the first exon were designed using CRISPR-P (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.hzau.edu.cn/CRISPR2/\u003c/span\u003e\u003cspan address=\"http://crispr.hzau.edu.cn/CRISPR2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The pYLCRISPR-Cas9-MH vector was digested and ligated with sgRNA-containing fragments amplified using primers SiPHO2-target-F/R and U-F/gR-R [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The construct was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 and introduced into Ci846 using mature seeds as explants [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. T3 transgenic lines were used for further analysis. Primers are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe full-length coding sequence (CDS) of \u003cem\u003eSiFTc\u003c/em\u003e was amplified from Ci846 cDNA and cloned into the pTCK303-Flag vector to generate pUbi::SiFTc-Flag. The construct was transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain EHA105 and introduced into Zhonghua11 (ZH11) using mature seeds as explants. T2 transgenic lines were used for further experiments. Primers are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Plants growth conditions and Pi treatment\u003c/h2\u003e \u003cp\u003ePot experiments were conducted in vermiculite and supplied with basal nutrient solution with or without Pi. The solution contained the following components (mM): 2.0 mM Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csup\u003e2\u003c/sup\u003e, 0.5 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.1 mM KCl, 0.3 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 10 mM H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 0.5 mM MnCl\u003csub\u003e2\u003c/sub\u003e, 0.5 mM ZnCl\u003csub\u003e2\u003c/sub\u003e, 0.2 mM CuCl\u003csub\u003e2\u003c/sub\u003e, 0.1 mM Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e and 0.1 mM Fe-EDTA. For the treatment of Pi deficiency, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e was substituted with KCl. The pH of all culture solutions was adjusted to 5.8, and the medium was changed every three days [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. All foxtail millet and rice plants were grown in a greenhouse in Beijing (116.34\u0026prime;E, 39.97\u0026prime;N).\u003c/p\u003e \u003cp\u003eRoot architecture analysis experiments were conducted in illumination incubator and supplied with Murashige \u0026amp; Skoog Basal Medium with vitamins (Phyto Technology # M519).The seedlings with similar growth to continue be cultured for one week on MS medium used for root architecture analysis.\u003c/p\u003e \u003cp\u003eAgronomic traits analysis was according to methods described in the previous study [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], such as plant height, leaf length, leaf width, PR length, LR length, LR number, SPAD, panicle weight, panicle length, grains number per panicle and 1000-grains weight. Percentage of necrotic area of the second leaf from the top was used for leaf Pi toxic analysis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Heading date was measured as the number of days from seed germination to the fully emergency of the panicle. At least six plants in three different pots were used for phenotypic statistics [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Three biological replicates were used in each treatment. Data and statistics were analyzed by GraphPad Prism8 (GraphPad Software, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Pi concentration assay\u003c/h2\u003e \u003cp\u003ePi concentration analysis was quantified in various tissues of \u003cem\u003eSiPHO2-CR\u003c/em\u003e and Ci846 lines grown under Pi normal (0.3 mM) and Pi deficient (0.01 mM) conditions. Samples were collected from shoots and roots of two-week-old seedlings, leaves at the heading stage, and roots, stems, leaves and spikes at the mature stage. For seedling root Pi concentration analysis, one-week-old seedlings grown on MS medium were used for Pi uptake assays. All samples were homogenized in a 1:5 (w/v) extraction buffer, centrifuged at 10,000 \u0026times; g for 10 min at 4\u0026deg;C, and the supernatant liquid was collected. The supernatant liquid, distilled water and color-developing reagent were mixed in a 1:9:10 ratio, incubated at 40\u0026deg;C for 10 min, and then cooled to room temperature. Absorbance was measured at 660 nm using a spectrophotometer.\u003c/p\u003e \u003cp\u003ePi concentration was calculated as follows:\u003c/p\u003e \u003cp\u003eTissue Pi concentration (mmol/g) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{C}\\text{s}\\times\\:\\varDelta\\:\\text{A}\\text{t}\\times\\:\\text{V}\\text{t}}{\\text{W}\\times\\:\\varDelta\\:\\text{A}\\text{s}}\\)\u003c/span\u003e\u003c/span\u003e =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{0.002\\times\\:\\varDelta\\:\\text{A}\\text{t}}{\\text{W}\\text{t}\\times\\:\\varDelta\\:\\text{A}\\text{s}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003ewhere:\u003c/p\u003e \u003cp\u003eΔAt\u0026thinsp;=\u0026thinsp;At-Ab (OD660 of the tested sample minus the blank),\u003c/p\u003e \u003cp\u003eΔAs\u0026thinsp;=\u0026thinsp;As-Ab (OD660 of the standard sample minus the blank),\u003c/p\u003e \u003cp\u003eCs\u0026thinsp;=\u0026thinsp;concentration of the standard solution (2 mmol/L),\u003c/p\u003e \u003cp\u003eVt\u0026thinsp;=\u0026thinsp;total volume of the tested sample (1 mL\u0026thinsp;=\u0026thinsp;1\u0026times;10⁻\u0026sup3; L),\u003c/p\u003e \u003cp\u003eWt\u0026thinsp;=\u0026thinsp;weight of the tested sample (g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. β-glucuronidase (GUS) staining assay\u003c/h2\u003e \u003cp\u003eA 3600-bp genomic fragment upstream of SiPHO2 was cloned as the promoter to drive the expression of the GUS reporter gene in the pCAMBIA1301 vector to generate pSiPHO2::GUS transgenic plants. Histochemical localization of GUS activity was performed by incubating plant tissues in a staining solution containing 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) at 37\u0026deg;C for three hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. RNA extraction, gene expression and transcriptome sequencing analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from leaves of Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants at the heading stage using the RNAprep Pure Plant Plus Kit (Tiangen, Cat. # DP441). RNA (5 \u0026micro;g) was reverse-transcribed into cDNA using the PrimeScript\u0026trade; II 1st Strand cDNA Synthesis Kit (Takara, Cat. #6210A). Quantitative PCR (qPCR) was performed using 2x Realtime PCR Super mix (Mei5bio, Cat. # MF013-05) with gene-specific primers. \u003cem\u003eSiCullin\u003c/em\u003e was used as the control. Primers are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFor transcriptome sequencing, 16 RNA-seq libraries (four biological replicates per treatment) were constructed and sequenced on an Illumina HiSeq 2500 platform in 150-bp paired-end mode. Clean reads were mapped and analyzed as previously described [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. \u003cem\u003eSiPHO2\u003c/em\u003e is a homologous gene of \u003cem\u003eOsPHO2\u003c/em\u003e and responds to a variety of abiotic stresses.\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ePHO2\u003c/em\u003e encodes a UBC E2 enzyme critical for phosphate homeostasis, characterized by a conserved C-terminal ubiquitin-binding domain. Phylogenetic analysis of UBC family members by conserved UBC domains in Arabidopsis, rice and foxtail millet showed SiPHO2 (\u003cem\u003eSeita.3G150400\u003c/em\u003e) is an ortholog of OsPHO2 (\u003cem\u003eLOC Os05g48390\u003c/em\u003e), and shares 79.04% amino acid sequence identity with OsPHO2 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The genome sequence of \u003cem\u003eSiPHO2\u003c/em\u003e is 2,625 base pair nucleotides, contains eight exons and seven introns, encoding 875 amino acids. SiPHO2 belongs to Group 3 together with AtPHO2 (AT2G33770, UBC24) and OsPHO2 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA).These findings indicated that \u003cem\u003ePHO2\u003c/em\u003e-mediated regulatory mechanisms might be functionally conserved in Pi signaling across monocot and dicot plants.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eSiPHO2\u003c/em\u003e promoter region contains several cis-elements associated with diverse biological processes, including photoresponse, MeJA response, SA response, stress response and meristem development (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). To further characterize its stress-responsive properties, we analyzed the expression pattern of \u003cem\u003eSiPHO2\u003c/em\u003e under various abiotic stress conditions. Our results revealed distinct expression patterns in response to different treatments. Low temperature (4 ℃), drought stress and gibberellic acid (GA) treatment generally suppressed \u003cem\u003eSiPHO2\u003c/em\u003e expression within 24 hours (h). In contrast, MeJA and PEG 6000 treatment induced \u003cem\u003eSiPHO2\u003c/em\u003e expression after 24 h. In addition, we observed significant changes in \u003cem\u003eSiPHO2\u003c/em\u003e expression levels under short-term treatments, ABA (1 h), SA (1 h and 3 h) and NaCl (1 h to 6 h) treatments all elicited rapid responses compared to the 0 h control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These findings indicated that \u003cem\u003eSiPHO2\u003c/em\u003e was involved in dual-phase stress response mechanisms, mediating both rapid transcriptional regulation for immediate stress signaling and sustained induction for long-term adaptive responses.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2.\u003c/b\u003e \u003cb\u003eSiPHO2\u003c/b\u003e \u003cb\u003eis widely expressed in different tissues and localized in the endoplasmic reticulum (ER) of cells.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the tissue-specific expression pattern of \u003cem\u003eSiPHO2\u003c/em\u003e, we generated transgenic plants carrying the GUS reporter gene driven by \u003cem\u003eSiPHO2\u003c/em\u003e promoter (pSiPHO2::GUS). In foxtail millet, histochemical analysis revealed GUS activity in whole seedlings, and in stems, nodes, leaves and panicle of the palnts at reproductive stage (Figure. 1D-I). Similarly, in transgenic rice lines, GUS signals were also observed in seedlings, and roots, stem, leaves, florets and grains during the reproductive stage (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The expression pattern of \u003cem\u003eSiPHO2\u003c/em\u003e was analyzed in tissues at different developmental stages, such as shoot elongation stage, booting stage, panicle developmental stage, flowering stage and grain filling stage. According to the results, \u003cem\u003eSiPHO2\u003c/em\u003e was widely expressed throughout plant development and was highly expressed in seedlings and panicles (Figure. 1J).\u003c/p\u003e \u003cp\u003eThe Ubi::GFP (control) or Ubi::SiPHO2-eGFP vector was constructed and co-transformed with an ER marker, AtWAK2-mCherry [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], in foxtail millet protoplasts to analyze the subcellular location of SiPHO2. The SiPHO2-eGFP fusion protein predominantly colocalized with ER marker, it indicated that SiPHO2 was primarily localized at the ER (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo elucidate the response of \u003cem\u003eSiPHO2\u003c/em\u003e to Pi availability, we performed time-course expression analysis of both shoots and roots under Pi normal and Pi deficient conditions, with sampling intervals at 0, 2, 6, 12, 24 and 72 hours post-treatment [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The results indicated that the expression of \u003cem\u003eSiPHO2\u003c/em\u003e in the shoot remained relatively stable, and it exhibited a downward trend in roots in six hours under different Pi conditions. The results implied that the expression of \u003cem\u003eSiPHO2\u003c/em\u003e might response to Pi deficiency during the short term in roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. \u003cem\u003eSiPHO2\u003c/em\u003e was involved in the Pi homeostasis and development of \u003cem\u003eSetaria italica\u003c/em\u003e.\u003c/h2\u003e \u003cp\u003eThe CRISPR/Cas9 system was used to create SiPHO2 loss-of-function mutants in the background of the Ci846 genotype. From ten independent knockout transgenic lines, we identified three distinct editing types: \u003cem\u003eSiPHO2-CR#2\u003c/em\u003e (T insertion), \u003cem\u003eSiPHO2-CR\u003c/em\u003e#\u003cem\u003e3\u003c/em\u003e (C deletion) and \u003cem\u003eSiPHO2-CR\u003c/em\u003e#\u003cem\u003e4\u003c/em\u003e (C insertion) in the coding sequence compared to the Ci846. These frame-shift mutations resulted in premature termination codons, as confirmed by sequence analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsistent with the Pi toxicity phenotype observed in rice [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], the \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants exhibited leaf tip necrotic phenotype during the heading date (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The percentage of leaf tip necrosis area relative to the whole leaf was significantly higher in \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants than that in the Ci846 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). In addition, the Pi concentration in the leaves of \u003cem\u003eSiPHO2-CR\u003c/em\u003e was two to three times higher than that in Ci846 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). These findings suggested that excessive Pi accumulation may lead to leaf damage in \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants.\u003c/p\u003e \u003cp\u003eComprehensive phenotype analysis demonstrated that \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants exhibited significant growth impairments compared to Ci846, including reductions in plant height, leaf length and width, panicle length and width and SPAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-Q). At the seedling stage, the whole plant Pi concentration was higher in \u003cem\u003eSiPHO2-CR\u003c/em\u003e lines compared to Ci846 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Tissue-specific analysis revealed that the shoot to root ratio of Pi concentration was increased in \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). During maturity, while root and stem Pi concentrations remained comparable between \u003cem\u003eSiPHO2-CR\u003c/em\u003e and Ci846, the mutants accumulated significantly higher Pi levels in leaves, particularly in apical young leaves (Figure. 2I). This altered spatial distribution of Pi was associated with the abnormal phenotype, such as, pronounced Pi toxicity symptoms in apical leaves, enhanced basal-to-apical (stem to leaf) Pi translocation and reduced Pi transfer efficiency from apical leaves to panicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Notably, \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants showed 43.60% reduction in panicle weight and 50.54% reduction in grain weight per panicle (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eR-T). The broken of phosphorus homeostasis may be the cause of panicle development defects and yield reduction, suggesting that \u003cem\u003eSiPHO2\u003c/em\u003e plays a crucial role in coordinating both Pi homeostasis and agronomic traits in \u003cem\u003eSetaria italica\u003c/em\u003e through the regulation of interorgan Pi partitioning.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Some Pi transporters and SPX proteins were responsible for root architecture changes of \u003cem\u003eSiPHO2\u003c/em\u003e plants\u003c/h2\u003e \u003cp\u003eRoot architecture plays a crucial role in ion absorption and plant development. Under sufficient Pi condition, \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants exhibited significant alterations in root morphology compared to Ci846, including reduced PR length and LR number, but increased LR length (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, E-G). These architectural changes were associated with Pi distribution patterns. While root Pi concentration was significantly lower in \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants, the higher shoot Pi concentration resulted in an increase shoot to root Pi ratio relative to Ci846 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). These findings demonstrated that \u003cem\u003eSiPHO2\u003c/em\u003e played an integrative role in coordinating root development with Pi acquisition and distribution in \u003cem\u003eSetaria italica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the molecular mechanisms underling root architecture alterations and Pi signal regulation, we analyzed the expression patterns of key genes involved in Pi homeostasis, including high-affinity Pi transporters and \u003cem\u003eSPX\u003c/em\u003e domain-containing proteins using qRT-PCR. The \u003cem\u003eOsPHO1\u003c/em\u003e homolog \u003cem\u003eSiPHO1;H1\u003c/em\u003e was up-regulated in shoots, while several Pi transporters (\u003cem\u003eSiPHO1\u003c/em\u003e, \u003cem\u003eSiPHT1\u003c/em\u003e and \u003cem\u003eSiPHF1\u003c/em\u003e) and SPX domain-containing proteins (\u003cem\u003eSiSPX1\u003c/em\u003e and \u003cem\u003eSiMSF\u003c/em\u003e) showed significant up-regulation in roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). The different tissue-specific expression patterns are consistent with the established functional specialization of these genes, where \u003cem\u003ePHO1;H1\u003c/em\u003e primarily regulates shoot Pi homeostasis and \u003cem\u003ePHT1\u003c/em\u003e family members maintain root Pi homeostasis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. These findings suggested that \u003cem\u003eSiPHO2\u003c/em\u003e modulated Pi uptake and translocation by regulating the expression of key Pi homeostasis-related genes, thereby influencing both root architecture and systemic Pi distribution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5. \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants exhibited developmental inhibition during the heading date.\u003c/h2\u003e \u003cp\u003eThe flowering stage is an important period which needs more water and nutrient resources to maintain the crops development, where Pi homeostasis plays pivotal regulatory roles. The phenotype comparison between Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants revealed the mutant lines exhibited a mean 5.81 day delay in heading date under normal Pi condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). It demonstrated the functional involvement of \u003cem\u003eSiPHO2\u003c/em\u003e in reproductive phase regulation. Furthermore, \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants displayed significant alterations in key agronomic traits, including 38.84% reduction in mean plant height (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), 17.33% decrease in leaf length (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and 25.50% reduction in leaf width (Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eTo elucidate the \u003cem\u003eSiPHO2\u003c/em\u003e-mediated heading date regulatory mechanisms, we performed transcriptomic sequencing between Ci846 and \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants. Differential expression analysis (|log\u003csub\u003e2\u003c/sub\u003eFC| \u0026ge; 1; FDR \u0026le; 0.001) identified 1,437 differential expressed genes (DEGs) (813 up-regulated and 624 down-regulated) in \u003cem\u003eSiPHO-CR\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Gene Ontology (GO) enrichment analysis revealed predominant enrichment of these DEGs were mainly enriched in carbohydrate / polysaccharide metabolism, cell wall organization and cellular amide metabolic process (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). In addition, through functional annotation screening of DEGs, 17 heading date related genes exhibiting significant expression alterations in \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants compared to Ci846 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eH) and six key flowering genes were verified by qRT-PCR. The results showed a complete suppression of \u003cem\u003eSiFTc\u003c/em\u003e (paralogous of \u003cem\u003eSiFTa\u003c/em\u003e, 0.15 fold of Ci846), significant down regulation of \u003cem\u003eSiMADS15\u003c/em\u003e and \u003cem\u003eSiMADS3\u003c/em\u003e (MADS-box transcription factor, 0.15-fold and 0.37-fold of Ci846 respectively) and \u003cem\u003eSiNFYA10\u003c/em\u003e (CCAAT-box binding factor, 0.29-fold of Ci846), while up regulation of \u003cem\u003eSiHY5\u003c/em\u003e (photomorphogenesis regulator, 1.65-fold of Ci846) under Pi normal conditions in \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants. However, expression trends of these genes were reversed under Pi deficient conditions, the up-regulation of \u003cem\u003eSiFTc\u003c/em\u003e, \u003cem\u003eSiCOL9\u003c/em\u003e, \u003cem\u003eSiMADS15\u003c/em\u003e, \u003cem\u003eSiMADS3\u003c/em\u003e and \u003cem\u003eSiNFYA10\u003c/em\u003e, while the significant down-regulation of \u003cem\u003eSiHY5\u003c/em\u003e in \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants compared to Ci846 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). These results indicate that \u003cem\u003eSiPHO2\u003c/em\u003e modulates heading date of foxtail millet through transcriptional regulation of conserved flowering pathway factors, particularly via its significant impact on \u003cem\u003eSiFTc\u003c/em\u003e and \u003cem\u003eSiMADS3/15\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis of the \u003cem\u003eFT\u003c/em\u003e gene family showed \u003cem\u003eSiFTc\u003c/em\u003e was a paralogous gene of \u003cem\u003eSiFTa\u003c/em\u003e (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), and SiFTa protein showed significantly higher conservation with OsHd3a (87.71% identity), SiFTc exhibited relatively lower sequence conservation with OsHd3a (75.42% identity). To investigate the functional role of \u003cem\u003eSiFTc\u003c/em\u003e, eight \u003cem\u003eSiFTc\u003c/em\u003e overexpression transgenic lines obtained in ZH11 background, seven of them were transgenic positive lines and showed overexpression (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA-B). The three lines with the highest \u003cem\u003eSiFTc\u003c/em\u003e expression levels for further analysis. The \u003cem\u003eSiFTc-OE\u003c/em\u003e lines exhibited an accelerated floral transition, with heading dates advanced by 28 days compared to ZH11 (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 days vs 50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 days). Concurrently, these transgenic plants displayed pleiotropic morphological changes, including obvious dwarfism and reduced leaf width and length (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, K). It was confirmed that \u003cem\u003eSiFTc\u003c/em\u003e was function as a flowering inducer and integrated into the \u003cem\u003ePHO2\u003c/em\u003e-mediated heading date regulatory network in foxtail millet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Pi deficient conditions influenced the whole plant development of \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants.\u003c/h2\u003e \u003cp\u003eUnder Pi deficient conditions, the plant height of \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants was significantly reduced compared to that of Ci846, both at the seedling stage (47.9% reduction) and at the flowering stage (41.4% reduction) (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B and Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). Moreover, the Pi deficiency response was obvious in the leaf architecture, with \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants exhibiting decreases in both leaf length and width relative to Ci846 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These findings underscored the necessity of functional \u003cem\u003eSiPHO2\u003c/em\u003e for the maintaining of plant development under Pi limited conditions.\u003c/p\u003e \u003cp\u003eTo dissect the molecular mechanisms underlying \u003cem\u003eSiPHO2\u003c/em\u003e-mediated growth regulation under Pi limitation, we conducted comparative transcriptome analysis of \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants and Ci846 at flowering stage under Pi deficient conditions. Differential expression analysis identified 1,146 significantly DEGs (549 up-regulated and 597 down-regulated) in the mutants. GO analysis results showed that the most significant enrichment functional terms was the canonical Pi response pathways, including cell response to phosphate starvation, phospholipid catabolic process and cell response to nutrient level. These genes included some nutrition-related genes, such as phosphorus-related genes (\u003cem\u003ePHT1s\u003c/em\u003e, purple acid phosphatases (\u003cem\u003ePAPs\u003c/em\u003e), sulfoquinovosyl diacylglycerol (\u003cem\u003eSQD\u003c/em\u003e), monogalactosyldiacylglycerol (\u003cem\u003eMGDG\u003c/em\u003e) and \u003cem\u003eSPXs\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results suggested that the \u003cem\u003eSiPHO2\u003c/em\u003e mutation might mimic the natural Pi starvation signal and activate a deficiency Pi response in the \u003cem\u003eSiPHO2\u003c/em\u003e-\u003cem\u003eCR\u003c/em\u003e mutants. In addition, the pathways involved in auxin biosynhetic process regulation, cell communication, cell periphery and ion transmembrane transport activities were enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-H).\u003c/p\u003e \u003cp\u003eAmong them, auxin bioanabolic process and cell periphery are closely related to plant growth and development. In the samples of Ci846 and \u003cem\u003eSiPHO2\u003c/em\u003e-\u003cem\u003eCR\u003c/em\u003e plants cultivated under the Pi deficient condition, the expression of three development-associated DEGs, containing \u003cem\u003eALTERED SEED GERMINATION 3\u003c/em\u003e (\u003cem\u003eSiASG3\u003c/em\u003e), \u003cem\u003eABNORMAL LEAF SHAPE 2\u003c/em\u003e (\u003cem\u003eSiALE2\u003c/em\u003e) and \u003cem\u003eQUANTITATIVE RESISTANCE TO PLECTOSPHAERELLA 1\u003c/em\u003e (\u003cem\u003eSiQRP1\u003c/em\u003e), were selected for qRT-PCR. Consistent with RNA-seq data, \u003cem\u003eSiASG3\u003c/em\u003e and \u003cem\u003eSiALE2\u003c/em\u003e showed suppressed expression in \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants, while \u003cem\u003eSiQRP1\u003c/em\u003e was induced (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). In Arabidopsis, \u003cem\u003eAtASG3\u003c/em\u003e is associate with cell cycle regulation during early leaf and root growth [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], \u003cem\u003eAtALE2\u003c/em\u003e positively regulates protoderm-specific gene expression and for the formation of leaf [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and \u003cem\u003eAtQRP1\u003c/em\u003e negatively regulates epidermal and mesophyll development [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. These results collectively indicated that \u003cem\u003eSiPHO2\u003c/em\u003e played a role in plant growth and development by modulating the activity of numerous genes.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.1. \u003cem\u003eSiPHO2\u003c/em\u003e modulates root architecture and leaf development through Pi homeostasis.\u003c/h2\u003e \u003cp\u003ePi acquisition and distribution in plants are governed by complex spatial regulation involving root cytoplasm and vascular tissues, a process mediated by the coordinated xylem loading and phloem redistribution [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In poaceae species, adaptive responses to Pi deficiency often include enhanced PR elongation and increased LR formation, as observed in rice and maize [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In addition, the \u003cem\u003esbpho2\u003c/em\u003e mutant showed reduced PR growth under Pi sufficient conditions [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Pi deficiency tolerant genotype showed a lower expression of \u003cem\u003ePvPHO2\u003c/em\u003e, while the Pi content and root biomass increased compared to the sensitive genotype under Pi starvation in bean [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. However, the genetic mechanisms underlying these adaptive responses in \u003cem\u003eSetaria italica\u003c/em\u003e, a model C4 model, remain poorly understood. We investigated the modifications in root system architecture under Pi sufficient conditions and found that \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants exhibited significant alterations in root morphology compared to Ci846, including shortened PR, reduced LR density and elongated individual LR (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, E-G). These architectural variations suggested a crucial role for \u003cem\u003eSiPHO2\u003c/em\u003e in modulating root plasticity and Pi sensitivity, consistent with conserved functions observed in \u003cem\u003eOspho2\u003c/em\u003e and \u003cem\u003eAtpho2\u003c/em\u003e mutants, where \u003cem\u003ePHO2\u003c/em\u003e regulates root elongation, and this regulation is enhanced in the \u003cem\u003eltn1\u003c/em\u003e mutant under Pi starvation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Building on these observations, we systematically characterized tissue-specific Pi redistribution patterns across different developmental stages. The \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants showed root Pi reduced concomitant with shoot hyper-accumulation, particularly in leaves during both vegetative and reproductive phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, H-J). Subsequent analysis revealed an enhanced vascular translocation capacity from stems to leaves, along with preferential Pi transporting to apical leaf regions. Notably, this altered redistribution pattern correlated with progressive chlorosis and necrotic lesion development at leaf tip during maturation, which can be attributed to Pi toxicity resulting from compartmentalization defects. Collectively, these findings underscore the pivotal role of \u003cem\u003eSiPHO2\u003c/em\u003e in maintaining Pi spatial homeostasis through the coordinated regulation of source-to-sink translocation dynamics.\u003c/p\u003e \u003cp\u003ePrevious research has shown that the knockout of \u003cem\u003eTaPHO2\u003c/em\u003e-A1 in wheat led to a significantly enhancement in Pi uptake and grain yield, with no discernible adverse effects under high Pi conditions [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In contrast, our study observed that \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants displayed impaired panicle development, marked by substantial reductions in panicle length, width and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eP-T). Notably, our analysis of Pi absorption and transport dynamics revealed a significant decrease in Pi translocation efficiency from leaves to panicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ), which may be responsible for the observed abnormalities in panicle development. This finding highlights the importance of \u003cem\u003eSiPHO2\u003c/em\u003e in maintaining the balance of Pi distribution and the crucial role in panicle development of \u003cem\u003eSetaita italica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.2.\u003c/b\u003e \u003cb\u003eSiPHO2\u003c/b\u003e \u003cb\u003eplays a crucial role in plant growth and development through both Pi signaling pathways and other gene regulatory mechanisms.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePHR1\u003c/em\u003e-miR399-\u003cem\u003ePHO2\u003c/em\u003e signaling pathway, a well-established regulatory network for Pi homestasis, has been extensively characterized in rice and Arabidopsis [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, this pathway remains to be elucidated in foxtail millet. Our study has uncovered distinct molecular responses in foxtail millet under Pi sufficient conditions. While the expression profiles of key Pi-starvation-responsive genes were found to be relatively stable in the shoots, a significant up-regulation of numerous genes associated with Pi starvation was observed in the roots. Notably, we identified two pivotal transporters showed a increased expression in roots of \u003cem\u003eSiPHO2-CR\u003c/em\u003e lines: \u003cem\u003eSiPHT1\u003c/em\u003e (\u003cem\u003eSeita.9G238300\u003c/em\u003e), a homolog of the rice Pi transporter \u003cem\u003eOsPT2\u003c/em\u003e, which is implicated in root-to-shoot translocation of Pi [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], \u003cem\u003eSiPHO1\u003c/em\u003e (\u003cem\u003eSeita.1G360400\u003c/em\u003e), the homolog of \u003cem\u003eOsPHO1\u003c/em\u003e, which is known to facilitate Pi transport from root to shoot in rice [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Our research indicated that \u003cem\u003eSiPHT1\u003c/em\u003e and \u003cem\u003eSiPHO1\u003c/em\u003e might act in concert to regulate the translocation of Pi from the root to the shoot in foxtail millet, potentially accounting for the higher Pi levels observed in the leaves of \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants compared to Ci846 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, I). This finding aligns with the role of these transporters in other species, such as rice, where they are involved in Pi translocation. Furthermore, we have identified \u003cem\u003eSiPHF1\u003c/em\u003e (\u003cem\u003eSeita.2G065200\u003c/em\u003e), a homolog of \u003cem\u003eOsPHF1\u003c/em\u003e in rice, which is known to regulate the plasma membrane localization of both high and low affinity Pi transporters, thereby controlling Pi uptake and transport [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], was higher expressed in \u003cem\u003eSiPHO2-CR\u003c/em\u003e lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). This suggests that \u003cem\u003eSiPHF1\u003c/em\u003e may play a similar role in foxtail millet with that of rice, facilitating Pi trafficking from the endoplasmic reticulum to the plasma membrane. Under Pi deficient conditions, we observed significant differential expression of Pi starvation responsive genes. GO analysis revealed a prominent clustering of DEGs within the Pi starvation response module (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), which is consistent with previous observations of \u003cem\u003ePHO2\u003c/em\u003e-mediated gene expression changes in rice and Arabidopsis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These findings collectively demonstrated that \u003cem\u003eSiPHO2\u003c/em\u003e mediated conserved regulatory functions in plant growth and development through Pi signaling pathways in foxtail millet.\u003c/p\u003e \u003cp\u003eGO enrichment analysis of DEGs in \u003cem\u003eSiPHO2-CR\u003c/em\u003e plants under Pi deficient conditions also revealed significant enrichment of terms associated with auxin biosynthesis and metabolism, cell periphery organization and nutrient homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-H). Considering the key role of hormonal regulation in root development [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], we conducted a detail investigation into auxin signaling dynamics in the \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutant. Notably, key auxin biosynthesis genes, such as those in the YUC family, which are responsive to Pi starvation, exhibited differently regulation. In rice, auxin derived from YUC activates the transcription of \u003cem\u003eOsWOX11\u003c/em\u003e, thereby promoting crown root development [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Similarly, the up regulated of \u003cem\u003eSiPGP\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), encoding an auxin efflux carrier involved in auxin polar transport [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], suggests an enhanced redistribution of auxin in \u003cem\u003eSiPHO2-CR\u003c/em\u003e. Collectively, these findings indicate that \u003cem\u003eSiPHO2\u003c/em\u003e modulates auxin pathway activity to influence growth architecture. Mechanistic investigations uncovered multiple developmental regulators responsive to Pi starvation. For instance, \u003cem\u003eASG3\u003c/em\u003e, which regulates leaf and root growth early in the cell cycle [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], \u003cem\u003eQRP1\u003c/em\u003e, which affects epidermal and mesophyll development [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], \u003cem\u003eALE2\u003c/em\u003e, involved in the regulation of the formation of leafy organs [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], \u003cem\u003eBRL2\u003c/em\u003e, which controls cell division and elongation to regulate organ development [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], the homolog of these genes showed increased expression in \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants under Pi starvation, suggesting that they might be regulated by \u003cem\u003eSiPHO2.\u003c/em\u003e This \u003cem\u003eSiPHO2\u003c/em\u003e-mediated transcriptional network likely drives the observed phenotypic reductions in plant height and leaf width in \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants. These findings demonstrate that \u003cem\u003eSiPHO2\u003c/em\u003e orchestrates plant growth through dual mechanisms: the first being canonical Pi signaling and the second involving a multi-layered regulatory encompassing auxin signaling, cell cycle control and tissue morphogenesis. This highlights the integrative role of \u003cem\u003eSiPHO2\u003c/em\u003e in coordinating developmental plasticity under Pi stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.3. \u003cem\u003eSiPHO2\u003c/em\u003e regulates heading date through modulation of \u003cem\u003eSiFTc\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eFlowering time in plants is regulated by a variety of abiotic stresses, including cold and nutrient deficiencies, such as nitrogen, phosphorus and potassium [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Flowering integration genes expressed predominantly in the companion cells of the leaf phloem, particularly in the distal secondary vein of leaves [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. The FT protein is transported from the leaf to the shoot apical meristem through the phloem to execute its biological functions [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. It is well established that the \u003cem\u003eGI-CO-FT\u003c/em\u003e module constitutes a classic flowering pathway across different species [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Several regulatory factors can modulate flowering by influencing the expression of \u003cem\u003eFT\u003c/em\u003e. For instance, \u003cem\u003eCO\u003c/em\u003e, a B-box zinc finger transcription factor, promotes \u003cem\u003eFT\u003c/em\u003e expression in sieve tube companion cell under long-day conditions in Arabidopsis leaves, leading to the formation of an FT protein complex that activates downstream genes such as \u003cem\u003eSUPPRESSOR OF OVEREXPRESSION OF CONSTANS1\u003c/em\u003e (\u003cem\u003eSOC1\u003c/em\u003e) and \u003cem\u003eAPETALA1\u003c/em\u003e (\u003cem\u003eAP1\u003c/em\u003e), thereby promoting flowering [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Overexpression of \u003cem\u003eFT\u003c/em\u003e in Arabidopsis results in extremely early flowering, while the \u003cem\u003eft\u003c/em\u003e mutant exhibits a delayed-flowering phenotype [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Arabidopsis, both \u003cem\u003emiR399-OE\u003c/em\u003e plants and \u003cem\u003epho2\u003c/em\u003e mutants exhibited an early flowering phenotype under normal temperature conditions (23\u0026deg;C) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the early flowering phenotype is abolished in \u003cem\u003epho2\u003c/em\u003e mutant plants when grown under low Pi conditions, with delayed flowering even being observed [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. In contrast, our study demonstrated that \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants displayed a delayed heading date under Pi sufficient conditions, whereas under Pi deficiency cultivation, the heading date of \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants was significantly earlier compared to that of Ci846. Notably, under Pi sufficient conditions, the expression of \u003cem\u003eSiFTc\u003c/em\u003e in \u003cem\u003eSiPHO2-CR\u003c/em\u003e mutants was lower than that of Ci846, but the expression pattern was reversed under Pi deficiency cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). These findings suggest that \u003cem\u003eSiPHO2\u003c/em\u003e plays a critical role in the crosstalk between Pi homeostasis and the regulation of flowering time. The loss of \u003cem\u003eOsPHO2\u003c/em\u003e function resulted in a delayed flowering phenotype under Pi normal conditions in rice, it is similar with our results [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These results further supporting the hypothesis that the regulatory mechanisms governing flowering time in response to Pi availability differ between monocots and dicots.\u003c/p\u003e \u003cp\u003eThe regulatory network between Pi signaling and flowering time involves complex molecular crosstalk across plant species. In Arabidopsis, \u003cem\u003ePHO1\u003c/em\u003e modulates the floral transition through jasmonic acid (JA) dependent suppression of \u003cem\u003eFT\u003c/em\u003e and \u003cem\u003eTWIN SISTER OF FT\u003c/em\u003e (\u003cem\u003eTSF\u003c/em\u003e), a paralogue of \u003cem\u003eFT\u003c/em\u003e [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], while \u003cem\u003eOsPHO2\u003c/em\u003e coordinates Pi homeostasis and flowering time via interaction with \u003cem\u003eGI\u003c/em\u003e, a core flowering regulator [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Building on these models, our transcriptome analysis in foxtail millet uncovered a distinct mechanism: \u003cem\u003eSiPHO2\u003c/em\u003e deficiency specifically disrupts the expression of \u003cem\u003eSiFTc\u003c/em\u003e (an ortholog of \u003cem\u003eOsHd3a\u003c/em\u003e), while was not \u003cem\u003eFTa\u003c/em\u003e, which is the most ortholog of \u003cem\u003eOsHd3a\u003c/em\u003e and is a flowering inducer [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This reveals evolutionary divergence in Pi responsive flowering regulation and establishes \u003cem\u003eSiFTc\u003c/em\u003e as a novel Pi-stress-responsive flowering determinant. This finding contrasts with the conserved \u003cem\u003eGI-CO-FT\u003c/em\u003e module[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Our results thus delineated a \u003cem\u003eSiPHO2-SiFTc\u003c/em\u003e regulatory module that reconfigures heading date pathways under Pi limitation, providing mechanistic insights into nutrient-environment coordination of heading date in \u003cem\u003eSetaria italica\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePi deficiency is indeed a major yield-limiting factor in crop production, as it influences crop yield and development [\u003cspan additionalcitationids=\"CR76 CR77\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. The excessive reliance on Pi fertilizers to address Pi deficiency is unsustainable, and while the molecular mechanisms of enhancing yield stability under Pi starvation remain poorly understood. Our results showed that \u003cem\u003eSiFTc\u003c/em\u003e overexpression accelerated the heading date, providing critical insights into Pi-responsive developmental regulation. Therefore, we speculate that \u003cem\u003eSiFTc\u003c/em\u003e suppression or loss-of-function under Pi limitation will delay the heading date while enhancing yield potential. These findings establish \u003cem\u003eSiFTc\u003c/em\u003e as a key regulator of Pi starvation adaptation and provide a targeted genetic strategy to develop more resilient crop varieties in the face of Pi scarcity through optimized heading date regulation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eXing GF and Zhang JW supervised the study; Li YQ, Wang HL and Fei HM performed experiments; Jia DD generated transgenetic plants; Qu RF and Wei R organized the vectors of transgenetic, Zhang YH, Liao HZ, Wei JH and Zhao XW provided technical assistance; Wang HL and Li YQ analysed the whole data and writing the manuscript; Xing GF revised the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was funded by Beijing Natural Science Foundation (6234044), the National Natural Science Foundation of China (32372062, 32241041), the Youth Scientific Research Foundation of Beijing Academy of Agriculture and Forestry Sciences (QNJJ202414), the National Key R\u0026amp;D Program of China (No. 2023YFD1200704, 2023YFD1200700), the Shanxi Province Postgraduate Innovation Project (2023KY328, 2021Y335).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDe Bang TC, Husted S, Laursen KH, Persson DP, Schjoerring JK (2021) The molecular-physiological functions of mineral macronutrients and their consequences for deficiency symptoms in plants. 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Plants (Basel). 14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Li S, An X, Song Z, Zhu Y, Tan Y, Guo X, Wang D (2025) Correction: Effects of nitrogen, phosphorus and potassium formula fertilization on the yield and berry quality of blueberry, PLoS One. 20 e0318032\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Setaria italica, Pi homeostasis, SiPHO2, Heading date","lastPublishedDoi":"10.21203/rs.3.rs-6218826/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6218826/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhosphorus (P) is a crucial macronutrient and its deficiency severely limits plant growth and yield. Although multiple inorganic phosphate (Pi) signaling regulators have been identified, the function of them in plant development and flowering time regulatory remains inadequately characterized in C4 model species like \u003cem\u003eSetaria italica\u003c/em\u003e. Here, CRISPR/Cas9-generated \u003cem\u003eSiPHO2\u003c/em\u003e knockout lines exhibited disrupted Pi homeostasis, and the lines showed shoot Pi accumulation, leaf tip necrosis, modified root architecture and reduced yield compared with wildtype (Ci846) under Pi deficient conditions. Transcriptome analysis suggested these phenotypic abnormalities might due to expression patterns alteration of Pi starvation-responsive genes. Notably, \u003cem\u003eSiPHO2\u003c/em\u003e knockout lines displayed earlier heading date under Pi deficiency but delayed heading date under normal conditions compared to Ci846 plants. Expression profiling and transgenic functional verification revealed that the heading date reversal correlated with the expression pattern of \u003cem\u003eFLOWERING LOCUS T c\u003c/em\u003e (\u003cem\u003eSiFTc\u003c/em\u003e), rather than \u003cem\u003eSiFTa\u003c/em\u003e, which is the closest homolog of \u003cem\u003eHeading date 3a\u003c/em\u003e (\u003cem\u003eOsHd3a\u003c/em\u003e). This study identifies a novel flowering regulator as a potential target for coordinating phosphorus-mediated heading date regulation and yield production. Our findings elucidate genetic mechanisms underlying phosphorus-dependent developmental regulation and propose a strategic approach for improving crop yield under Pi starvation.\u003c/p\u003e","manuscriptTitle":"SiPHO2 orchestrates phosphorus homeostasis and regulates heading date through SiFTc-dependent pathway in foxtail millet (Setaria italica)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 06:36:19","doi":"10.21203/rs.3.rs-6218826/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dc36d4fb-e4e5-4059-a5e0-3fc725ef57c1","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-07T07:54:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-24 06:36:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6218826","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6218826","identity":"rs-6218826","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}