Early transcriptome profiling reveals coordinated epidermal remodeling and chloroplast biogenesis during single-cell C4 priming in Bienertia sinuspersici

Early transcriptome profiling reveals coordinated epidermal remodeling and chloroplast biogenesis during single-cell C4 priming in Bienertia sinuspersici | 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 Early transcriptome profiling reveals coordinated epidermal remodeling and chloroplast biogenesis during single-cell C4 priming in Bienertia sinuspersici Jung Sun Kim, Prabhakaran Soundararajan, Behnam Derakhshani, Inhwang Hwang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8983924/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 Single-cell C₄ (SCC4) photosynthesis in Bienertia sinuspersici requires the formation of dimorphic chloroplasts within a single photosynthetic cell, yet the early developmental transitions preceding this differentiation remain poorly defined. Here, we conducted staged transcriptome profiling of young, intermediate, and mature leaves to characterize transcriptional dynamics associated with early SCC4 establishment, with selected expression patterns validated by qRT-PCR. From 55,257 expressed transcripts, genome-guided filtering identified 199 genes strongly enriched in young leaves and markedly repressed during maturation. Functional annotation revealed coordinated enrichment in plastid biogenesis and protein import, lipid and cuticle remodeling, phenylpropanoid metabolism, transcriptional regulation, auxin-associated pathways, redox homeostasis, and membrane-associated transport. Refinement of this set defined an 81-gene SCC4-associated module and a 33-gene core cohort exhibiting pronounced early-stage dominance. Importantly, these transcriptional shifts preceded detectable induction of canonical C₄ enzyme genes, indicating that structural reorganization and plastid-associated processes are established before biochemical C₄ specialization. Collectively, our results delineate an early transcriptional program underlying SCC4 priming in a non-Kranz C₄ species. single-cell C4 photosynthesis Bienertia sinuspersici early leaf development transcriptome profiling chloroplast biogenesis epidermal remodeling phenylpropanoid metabolism lipid remodeling auxin regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Photosynthetic carbon assimilation in land plants operates through three major pathways: C₃, C₄, and crassulacean acid metabolism (CAM). Among these, C₃ photosynthesis is ancestral but becomes inefficient under high temperature and water-limited conditions due to enhanced photorespiration (Sage, 2004 ; Akhani et al., 2005 ; Sage et al., 2011 ). C₄ photosynthesis evolved repeatedly as a biochemical CO₂-concentrating mechanism that suppresses photorespiration by spatially separating initial CO₂ fixation from the Calvin cycle, classically through Kranz anatomy (Hatch, 1987 ; Edwards et al., 2004 ; Furbank, 2016 ). Exceptionally, a small number of species perform C₄ photosynthesis without Kranz anatomy, instead achieving full C₄ biochemistry within a single photosynthetic cell. This single-cell C₄ (SCC4) strategy, exemplified by Bienertia sinuspersici and Suaeda aralocaspica , represents a striking evolutionary solution to photorespiratory constraint (Edwards et al., 2004 ; Offermann et al., 2015 ; Han et al., 2023 ). In Bienertia, two functionally distinct chloroplast populations are spatially segregated within one cell: peripheral chloroplasts associated with initial carbon fixation and central chloroplasts enriched in Rubisco, separated by a large central vacuole yet connected through cytoplasmic channels (Mai et al., 2019 ). This intracellular organization functionally parallels the mesophyll–bundle sheath division of labor in Kranz-type C₄ species. SCC4 development in Bienertia proceeds gradually during leaf ontogeny. Young leaves exhibit morphologically uniform, C₃-like chloroplasts; intermediate stages initiate intracellular segregation; and mature leaves establish fully differentiated dimorphic chloroplasts and SCC4 physiology (Offermann et al., 2011 ; Koteyeva et al., 2016 ; Han et al., 2023 ). While anatomical, ultrastructural, and physiological aspects of this transition have been well described, the molecular programs that initiate intracellular compartmentation and plastid specialization remain unresolved. Recent genomic and transcriptomic studies have begun to illuminate components of SCC4 regulation, including genome-wide architecture (Kim et al., 2025 ), chloroplast-type-specific carbonic anhydrases (Nguyen et al., 2025 ), and genes involved in organ patterning, chloroplast positioning, and cytoskeletal dynamics (Soundararajan et al., 2019 ; Won et al., 2023 ; Sharpe et al., 2025 ). However, these studies largely capture transcriptional changes coincident with or following visible chloroplast dimorphism, leaving the earliest regulatory events that prime SCC4 development unexplored. In particular, it remains unclear how transcriptional programs associated with plastid biogenesis, membrane and lipid remodeling, structural reinforcement, hormone-associated regulation, redox balance, and metabolite transport are coordinated during early leaf development prior to visible chloroplast differentiation. Understanding these early transitions may help clarify the developmental context preceding SCC4 establishment. By identifying early-enriched transcriptional modules associated with plastid assembly, intracellular architecture, and metabolic processes, we characterize a coordinated transcriptional program that precedes dimorphic chloroplast formation. Our results suggest that SCC4 establishment is associated with early transcriptional pre-patterning prior to the induction of canonical C₄ biochemical genes, thereby defining a developmental context for subsequent intracellular compartmentation and plastid specialization. MATERIALS AND METHODS Plant Material and growth conditions Bienertia plants (BioSample SAMN03290884) were maintained in controlled growth chambers and greenhouses following established procedures (Han et al., 2023). Seedlings were germinated on Murashige and Skoog (MS) medium under a 16 h light / 8 h dark photoperiod at 22 °C until root establishment, then transferred to soil and cultivated in a greenhouse maintained at approximately 28 °C under moderate light intensity with regular irrigation using NaCl-supplemented water. Leaf developmental stages were defined according to Koteyeva et al. (2016) as young (6 mm, approximately 1 cm leaf tips). For each stage, leaf tissues were harvested and immediately frozen in liquid nitrogen. Representative morphological features are shown in Fig. 1. RNA extraction and library preparation Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Germany). Tissues were homogenized in RLT buffer using a TissueLyser II (Qiagen) without thawing. RNA integrity was assessed by agarose gel electrophoresis and NanoDrop spectrophotometry. Genomic DNA was removed using recombinant DNase I (Takara, Japan), and 2 µg of DNase-treated RNA was used for cDNA synthesis. RNA preparation followed protocols previously established for Bienertia (Soundararajan et al., 2019). RNA-seq libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina) and sequenced on an Illumina NovaSeq 6000 platform to generate paired-end 100 bp reads. Biological triplicates were obtained for young, intermediate, and mature leaf stages. All RNA-seq datasets are available in the NCBI Sequence Read Archive under BioProject accession PRJNA917470 (SRP415653). Transcriptome analysis and identification of early-biased genes Raw reads were processed, aligned to the Bienertia reference genome (Kim et al., 2025), and quantified using a standardized RNA-seq analysis pipeline. Differential expression analysis was performed using thresholds of |log₂ fold change| ≥ 2 and adjusted P < 0.05. Genes exhibiting high expression in young leaves, reduced expression in intermediate leaves, and minimal or undetectable expression in mature tissues were classified as early-biased. After genome-based curation and removal of low-confidence loci, 199 early-biased genes were retained. Functional annotation was conducted using Gene Ontology, KEGG pathway mapping, Arabidopsis orthology inference, and literature-based curation. Gene Ontology enrichment analysis was performed using established statistical approaches. Functional categorization is summarized in Supplementary Table S2. qRT-PCR validation cDNA was synthesized using the AmfiRivert II cDNA Synthesis Master Mix (GenDEPOT). Quantitative RT-PCR was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad) using iTaq SYBR Green Supermix under standard cycling conditions (95 °C for 3 min; 40 cycles of 95 °C for 10 s and 60 °C for 30 s). GAPDH served as the internal reference gene. Relative expression levels were calculated using the 2⁻ΔCt and 2⁻ΔΔCt methods, with young leaves used as the reference stage. Genes exhibiting amplification in young and intermediate leaves but not in mature leaves were classified as mature-off. Primer sequences and normalized expression values are provided in Supplementary Tables S3 and S4. Definition of SCC4-ready and core gene sets The 199 early-biased genes were evaluated based on expression magnitude, developmental specificity, qRT-PCR concordance, functional annotation, predicted subcellular localization, and pathway association. From this integrative assessment, 81 genes representing core preparatory pathways related to plastid biogenesis, lipid and cuticle metabolism, cell wall remodeling, hormone-associated regulation, redox buffering, and metabolite transport were designated as the SCC4-ready gene set (Supplementary Table S6). A subset of 33 genes was further defined as high-confidence early regulators based on: (i) ≥10-fold early-to-mature expression contrast or complete repression in mature leaves; (ii) concordant RNA-seq and qRT-PCR expression patterns; and (iii) functional centrality within reconstructed developmental modules. Detailed gene lists are provided in Tables 1 and 2 and Supplementary Tables S5 and S6. RESULTS Transcriptome profiling across leaf developmental stages in Bienertia sinuspersici Young (6 mm) leaves were staged for transcriptome analysis (Fig. 1A). RNA integrity assessment confirmed comparable RNA quality across developmental stages (Fig. 1B). RNA-seq generated a comprehensive dataset comprising 55,257 expressed genes mapped to the Bienertia reference genome (Kim et al., 2025), with expression values provided in Supplementary Table S1. Principal component analysis (Fig. 1C) and hierarchical clustering revealed clear developmental stratification of transcriptomes. Young and mature leaves formed distinct clusters, while intermediate leaves occupied a transitional position. This separation reflects progressive transcriptional reprogramming during leaf maturation and the developmental progression associated with SCC4 establishment. Identification of 199 early-biased genes absent from mature leaves Differential expression analysis identified genes exhibiting high expression in young leaves, reduced expression in intermediate leaves, and minimal or undetectable expression in mature tissues. Using thresholds of |log₂ fold change| ≥ 2 and adjusted P < 0.05, 199 genes met these criteria and were designated as early-biased genes (Supplementary Table S2). These genes represent a developmentally restricted transcriptional cohort active during early leaf stages and largely repressed upon maturation, consistent with roles in early developmental transitions rather than in established SCC4 metabolism. qRT-PCR primer design and validation of early-biased expression patterns Gene-specific primers were designed for all 199 early-biased genes (Supplementary Table S3). Quantitative RT-PCR using the same RNA samples confirmed strong enrichment in young leaves, partial attenuation in intermediate leaves, and near-complete repression in mature tissues (Supplementary Table S4). Expression trends showed high concordance with RNA-seq data. Functional annotation of early-biased genes Integrated annotation combining Gene Ontology, KEGG pathways, Arabidopsis orthologues, and literature curation revealed enrichment in processes related to epidermal and cuticle development, phenylpropanoid metabolism, lipid modification, plastid biogenesis and protein import, redox balance, hormone-associated regulation, intracellular transport, and cell wall remodeling (Supplementary Table S5). These functional categories indicate coordinated activation of structural, plastidial, and regulatory processes during early leaf development. Definition of 81- gene SCC4-associated module Through integrative evaluation of expression magnitude, developmental specificity, qRT-PCR concordance, and functional annotation, 81 genes were selected as strongly early-enriched candidates (Supplementary Table S6). These genes exhibited robust activation in young leaves, partial reduction in intermediate stages, and near-complete repression in mature tissues. Heatmap visualization revealed highly coordinated expression dynamics across biological replicates, indicating synchronized developmental regulation (Fig. 2). Functional categorization showed that this 81-gene set spans multiple interconnected biological processes, including plastid biogenesis and protein import, lipid and cuticle modification, phenylpropanoid metabolism, transcriptional regulation, hormone-associated pathways, redox balance, and membrane-associated transport (Table 1). Together, these genes define a transcriptional module associated with early SCC4 establishment prior to visible chloroplast dimorphism. Identification of a 33-gene core early-stage cohort To further refine the SCC4-associated module, more stringent criteria emphasizing expression magnitude, early-stage specificity, and reproducibility across platforms were applied. This analysis yielded a subset of 33 genes displaying pronounced enrichment in young leaves and sharp repression during maturation (Fig. 3A; Table 2). Notably, the majority of these genes exhibited minimal residual expression in mature tissues, indicating tightly stage-restricted regulation. Their coordinated activation pattern suggests participation in early structural and plastid-associated transitions that precede chloroplast dimorphism rather than direct involvement in mature SCC4 biochemical processes. Conceptual representation of early SCC4-associated transcriptional organization To facilitate interpretation of the core gene set, representative loci spanning major functional axes were selected for schematic modeling. These loci encompass transcriptional regulators, plastid-associated factors, lipid and cell wall remodeling components, hormone-related elements, redox-associated genes, and membrane-associated transporters. The resulting framework summarizes inferred functional relationships derived from integrated expression and annotation analyses (Fig. 4) and provides a structured representation of transcriptional organization during early SCC4 establishment. Discussion Early transcriptional pre-patterning precedes activation of the C₄ biochemical cycle in Bienertia sinuspersici A central finding of this study is that early leaf development in Bienertia is not characterized by premature activation of the canonical C₄ biochemical cycle, but instead by a coordinated transcriptional program associated with the establishment of cellular and plastidial competence prior to single-cell C₄ (SCC4) differentiation. Consistent with previous anatomical and physiological studies of SCC4 species (Edwards et al., 2004; Offermann et al., 2015; Han et al., 2023; Sharpe et al., 2025), transcripts encoding key C₄ enzymes such as phosphoenolpyruvate carboxylase (PEPC), pyruvate phosphate dikinase (PPDK), and malic enzymes were not enriched in young leaves. Instead, early developmental stages were dominated by genes associated with plastid biogenesis and protein import, membrane lipid remodeling, cell wall modification, transcriptional regulation, hormone-associated processes, and redox-related pathways. Together, these patterns suggest that SCC4 development in Bienertia is preceded by preparatory cellular reorganization rather than early biochemical engagement of C₄ carbon assimilation. Because SCC4 systems must achieve spatial separation of metabolic functions within a single photosynthetic cell, extensive intracellular restructuring is likely required before activation of the C₄ cycle (Koteyeva et al., 2016; Han et al., 2023). Early plastid biogenesis and developmental competence Among the most prominent early-biased modules were genes associated with plastid biogenesis, protein import, and plastid metabolic priming. Early enrichment of plastid ribosomal components such as rpsG , electron transport–related genes such as cob , and MEP/isoprenoid pathway enzymes including DXS-like genes and PSY2 indicates that proplastids in young Bienertia leaves undergo biosynthetic expansion prior to visible chloroplast dimorphism. These transcriptional signatures are consistent with a developmental window in which plastids acquire translational capacity, pigment biosynthetic potential, and protein import competence before divergence into peripheral and central chloroplast types (Jarvis and López-Juez, 2013; Pogson et al., 2015; Cackett et al., 2021). Evidence from other plant systems underscores the importance of this early competence phase. For example, disruption of plastid-localized PPR proteins in barley impairs chloroplast development specifically in young leaves, with only partial recovery at later stages (Huang et al., 2025). Such findings highlight the developmental sensitivity of early plastid maturation. In Bienertia, early transcriptional enrichment of plastid-associated genes may similarly reflect a preparatory phase that precedes biochemical specialization. Redox- and ROS-associated genes were also detected, although their enrichment was more moderate than that of structural and plastid-related genes. This distribution aligns with models in which redox signaling contributes to developmental stabilization and coordination rather than acting as a primary driver of differentiation (Apel and Hirt, 2004; Foyer and Noctor, 2012; Pfannschmidt et al., 2020; Arce et al., 2022). Collectively, these data are consistent with a plastid state primed for later functional divergence without yet expressing the full C₄ enzymatic machinery. Lipid and structural remodeling during early SCC4-associated transitions A notable feature of the early-biased gene set was enrichment of lipid-, cuticle-, phenylpropanoid-, and cell wall–associated pathways. Homologs of WSD-type wax ester synthases, fatty acyl-CoA reductases, lipid-transfer proteins such as EARLI1 and AIR1 , and GDSL lipases exhibited strong early-stage expression. These gene families are traditionally associated with epidermal differentiation and cuticle formation (Samuels et al., 2008; Yeats and Rose, 2013). Recent studies suggest that wax ester biosynthesis also contributes to structural integrity during organ development (Nobuswa et al., 2025; Alotaibi et al., 2020; Bao et al., 2021). In the context of SCC4 development, coordinated activation of these lipid-related pathways in Bienertia may contribute to stabilization of intracellular domains prior to chloroplast repositioning. Although direct biochemical measurements were not conducted here, the transcriptional enrichment observed supports the possibility that structural reinforcement accompanies early cellular reorganization. Transcriptional regulation and hormone-associated processes Transcription factors, including NAC- and MYB-family members, exhibited strong early expression followed by coordinated repression during leaf maturation. This pattern is consistent with involvement in early developmental coordination rather than sustained photosynthetic function. Auxin-associated genes such as GH3.1 and GH3.2 were similarly enriched at early stages, suggesting potential contributions of hormone-regulated growth modulation during intracellular reorganization. Because hormone levels were not directly quantified in this study, the functional contribution of auxin signaling to SCC4 establishment remains to be clarified through targeted experimental approaches. Metabolite transport as a potential amplification layer The membrane-associated gene yjiY displayed the strongest early-to-mature expression ratio among the early-biased cohort. yjiY encodes a small membrane protein lacking catalytic domains, consistent with a role in transport or membrane-associated modulation rather than enzymatic activity. Plastid envelope transport is increasingly recognized as a key regulatory interface in metabolic coordination (Linka and Weber, 2010; Facchinelli and Weber, 2011). The strong early enrichment of yjiY suggests that membrane-associated processes may contribute to metabolic adjustment during early SCC4-associated transitions, although functional validation will be required to determine its precise role. A structured framework for early SCC4-associated development Integrating transcriptomic profiling, qRT-PCR validation, and functional annotation, we propose a structured framework for early SCC4-associated developmental transitions in Bienertia . In this view, early transcriptional regulators and plastid-associated programs coincide with structural remodeling and membrane-associated processes prior to activation of canonical C₄ metabolism. Rather than representing a definitive mechanistic pathway, this framework summarizes coordinated developmental expression dynamics that precede chloroplast dimorphism. This perspective highlights SCC4 as a developmentally organized system in which intracellular competence is established before biochemical specialization, providing a foundation for future experimental studies of single-cell C₄ evolution. Declarations Author contribution J.S.K. conceived and designed the study, performed plant cultivation and sampling, carried out RNA extraction, curated gene sets, conducted data analyses, and wrote the manuscript. P.S. performed qRT-PCR validation. B.D. assisted with data visualization. I.H. coordinated the overall research project and contributed to RNA-seq data generation. All authors reviewed and approved the final manuscript Co mpeting interests The authors declare that they have no competing financial or non-financial interests. Data availability All RNA-seq data supporting the findings of this study are publicly available in the NCBI Sequence Read Archive under BioProject accession PRJNA917470 . Processed expression matrices, qRT-PCR datasets, and functional annotation tables are provided as Supplementary Tables S1–S6. FASTA sequences of the 199 early-biased transcripts are provided in Supplementary data 1. Additional data supporting the findings of this study are available from the corresponding author upon reasonable request. Ethics statement This study did not involve human participants or animals requiring ethical approval. All plant materials used in this study were handled in accordance with institutional and national guidelines. 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Module Total Gene Count Biological Role in Early C3→C4 Transition Representative Genes (subset of full list) Phenylpropanoid_lignin_lipid_cuticle 28 Surface cuticle/wax biosynthesis, lipid remodeling, and phenylpropanoid–lignin network formation supporting early epidermal reinforcement GDSL esterases/lipases: GLIP4 , F14O23.4 , dl4155w , CPRD49 , At5g03820 , GDL17_ARATH , GDL14_ARATH , GDL71_ARATH , GDL28_ARATH , GDL56_ARATH O-acyltransferases & lipid enzymes: WSD1 (×4), FAR3 , FAR_SIMCH Phenylpropanoid: COMT (×3), CCoAOMT (×2), OMT1 , SCPL17 Lipid transfer: EARLI1 (×3), AIR1 Transcriptional Control 16 Transcriptional regulators coordinating early leaf fate, epidermal identity, and metabolic reprogramming NAC: NAC021 (×2), NAC031 , NAC098 (×2) MYB: MYB3 , MYB108 , MYB39 (×2) Homeobox/TCP: HAT3 , STM , WOX9 , KNAT2 , TCP12 B3/other TFs: REM9 , VRN1 Chloroplast_biogenesis_import 11 Organelle-associated gene expression, ribosomal components, protein import, and electron transport processes linked to early chloroplast establishment rpsG , rps4 , rplB , ccsA , cob (mitochondrial-encoded), GAPCP1 , PAP26 , HSP17.9-D , PCDH11X , At4g27810 , At5g16860 Cell Wall Expansion & Epidermal Patterning 8 Cell wall loosening, plasmodesmata regulation, and epidermal boundary formation accommodating early structural reorganization PME68 , XTH33 , EPFL2 , EPFL4 , EPFL1 , PDCB3 , CEL3 , 14KD_DAUCA Redox / ROS Energy 7 Redox homeostasis and electron transport–related processes supporting early organelle maturation NUDT20 (×2), CYP77A3 (×2), GRXC3 , PER57 , ND5 (mitochondrial-encoded) Metabolite Transport 5 Membrane-associated solute transport and metabolic balance during early sink–source transition yjiY , SWEET5 , ABCG6 , SLC6A9 , PSOMT1 Isoprenoid / MEP – Carotenoid Pathway 3 MEP-pathway and carotenoid biosynthesis enzymes contributing to plastid differentiation PSY2 , dxs , DXS2-like (Os07g0190000) Auxin Homeostasis 3 Auxin conjugation and homeostasis associated with early leaf patterning and growth modulation GH3.1 , GH3.2 , WTR8_ARATH The 81 SCC4-ready genes were classified into eight functional modules based on integrated annotation using Gene Ontology, KEGG pathway analysis, Arabidopsis orthologues, and literature curation. For each module, the total number of genes, proposed biological roles during the early C3→C4 transition, and representative genes are presented. Together, these modules reflect coordinated transcriptional programs underlying early epidermal remodeling, organelle-associated gene expression, metabolic priming, and regulatory control preceding visible chloroplast dimorphism in Bienertia. The complete gene list is provided in Supplementary Table S6. Table 2. Core early-dominant genes defining the SCC4-ready regulatory framework in Bienertia sinuspersici . Genes Name Description Avr. FPKM q-PCR Ratio (Early/mature) C4_ready_modules Bsv0100-00003862 F14O23.4 GDSL esterase/lipase (lipid remodeling, likely secretory/apoplastic) 2.726 17.507 Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00003864 GDL17_ARATH GDSL esterase/lipase (lipid remodeling) 4.078 FAIL Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00012052 dl4155w GDSL esterase/lipase (lipid remodeling) 19.62 undetected in mature Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00013033 WSD1 Wax ester synthase/acyltransferase (cuticle-associated lipid metabolism) 13.855 31968.006 Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00020061 At5g03820 GDSL esterase/lipase 4.727 undetected in mature Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00020019 FAR_SIMCH Fatty acyl-CoA reductase (cuticular lipid biosynthesis) 6.872 undetected in mature Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00020020 FAR3 Fatty acyl-CoA reductase 2.457 986.415 Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00028292 AIR1 Lipid-transfer protein (likely extracellular/apoplastic) 71.976 2221.656 Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00034628 EARLI1 Lipid transfer protein (stress-responsive, secretory pathway) 2.251 undetected in mature Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00034629 EARLI1 Lipid transfer protein (likely secretory/apoplastic) 1.656 1124.644 Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00036018 WSD1 Wax ester synthase/acyltransferase 3.435 undetected in mature Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00038185 CCoAOMT Caffeoyl-CoA O-methyltransferase (phenylpropanoid pathway) 4.113 9.959 Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00047155 WSD1 Wax ester synthase/acyltransferase 4.074 undetected in mature Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00006302 GDL56_ARATH GDSL esterase/lipase 12.59 9.135 Phenylpropanoid_lignin_lipid_cuticle Bsv0100-00001955 At4g27810 Hypothetical protein (predicted plastid-associated) 3.876 249.375 Chloroplast_biogenesis_import Bsv0100-00022164 rpsG 30S ribosomal protein S7 (chloroplast ribosome-associated) 16.882 undetected in mature Chloroplast_biogenesis_import Bsv0100-00028903 cob Cytochrome b (mitochondrial-encoded, Complex III subunit) 4.172 undetected in mature Redox_ros_energy Bsv0100-00045885 ND5 NADH dehydrogenase subunit 5 (mitochondrial-encoded Complex I) 6.993 2.761 Redox_ros_energy Bsv0100-00018748 PSY2 Phytoene synthase 2 (chloroplastic carotenoid biosynthesis) 2.028 21.730 Chloroplast_biogenesis_import;Isoprenoid_mep_carotenoid Bsv0100-00050947 DXS2-like 1-deoxy-D-xylulose-5-phosphate synthase (plastidial MEP pathway) 1.427 1656.262 Chloroplast_biogenesis_import;Isoprenoid_mep_carotenoid Bsv0100-00030682 NUDT20 Nudix hydrolase 20 (chloroplast-associated redox regulation) 2.881 26.589 Chloroplast_biogenesis_import;Redox_ros_energy Bsv0100-00030683 NUDT20 Nudix hydrolase 20 (chloroplast-associated redox regulation) 5.444 33.973 Chloroplast_biogenesis_import;Redox_ros_energy Bsv0100-00024425 GH3.1 Indole-3-acetic acid-amido synthetase (auxin conjugation) 9.934 695.424 Auxin_homeostasis Bsv0100-00024426 GH3.2 Indole-3-acetic acid-amido synthetase 9.666 83.510 Auxin_homeostasis Bsv0100-00010905 NAC021 NAC-domain transcription factor 2.716 374.971 Transcriptional_control Bsv0100-00010906 NAC021 NAC-domain transcription factor 4.998 602.066 Transcriptional_control Bsv0100-00021511 TCP12 TCP transcription factor 3.252 15.295 Transcriptional_control Bsv0100-00025089 STM Homeobox transcription factor (meristem-related) 2.194 undetected in mature Transcriptional_control Bsv0100-00053610 MYB39 MYB transcription factor 2.209 387.012 Transcriptional_control Bsv0100-00006562 PME68 Pectin methylesterase (cell wall remodeling) 10.164 313.391 Cell_wall_expansion Bsv0100-00030285 XTH33 Xyloglucan endotransglucosylase/hydrolase (cell wall modification) 3.000 21.355 Cell_wall_expansion Bsv0100-00020760 EPF1 Epidermal patterning factor 1 (secreted peptide) 2.935 11.830 Cell_wall_expansion Bsv0100-00009857 yjiY Predicted inner-membrane protein (membrane-associated, non-enzymatic) 718.592 25825.945 Metabolite_transport This table summarizes 33 core early-dominant genes refined from the 81 SCC4-ready gene set using stringent expression and functional criteria: (i) strong enrichment in young leaves with marked repression in mature tissues; (ii) concordant RNA-seq and qRT-PCR expression patterns; and (iii) functional relevance within reconstructed SCC4 preparatory modules. These genes represent central regulatory and structural components underlying early SCC4 priming, including transcriptional control, organelle-associated gene expression, lipid remodeling, cell wall expansion, redox regulation, auxin homeostasis, and membrane-associated transport. Collectively, this 33-gene cohort defines the core regulatory framework preceding dimorphic chloroplast differentiation in Bienertia. Additional Declarations No competing interests reported. Supplementary Files TableS2.xlsx TableS1.xlsx TableS3.xlsx TableS5.xlsx TableS6.xlsx TableS4.xlsx Supplementarydata1.fasta SUPPLEGENDS.docx 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-8983924","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":613991002,"identity":"6424c476-f6fa-4db1-b1e5-febaf6900c7c","order_by":0,"name":"Jung Sun Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYBACxgYGZoYEBhsDCPcA8VrSSNACBMxAfJgELcz9Zw8bPMw5b8wvffjgB4Yz94hw2Iy85ITEbbfNJPvSkiUYbhQTo4XH+ABQi43BGR4DCYYPCURo6T8D0nIOqIX/8w/itDTkGAMddsAMaAsb0GHEaJmRY2yQuC3ZWLKHzcwi4QwRWgyBDpP8uc3OsJ+H+fGND8eI0dKAzCNCAwODPDGKRsEoGAWjYIQDAPcPOgXLh2ZiAAAAAElFTkSuQmCC","orcid":"","institution":"National Institute of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jung","middleName":"Sun","lastName":"Kim","suffix":""},{"id":613991003,"identity":"3d1e1c9c-431d-466e-b598-e20b44786d85","order_by":1,"name":"Prabhakaran Soundararajan","email":"","orcid":"","institution":"National Institute of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Prabhakaran","middleName":"","lastName":"Soundararajan","suffix":""},{"id":613991004,"identity":"ad616c9f-5d7c-4b3f-8fd9-de6b97323cf9","order_by":2,"name":"Behnam Derakhshani","email":"","orcid":"","institution":"National Institute of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Behnam","middleName":"","lastName":"Derakhshani","suffix":""},{"id":613991005,"identity":"d5f1aadc-13bb-4cb8-b89b-ca161a5a2381","order_by":3,"name":"Inhwang Hwang","email":"","orcid":"","institution":"Pohang University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Inhwang","middleName":"","lastName":"Hwang","suffix":""}],"badges":[],"createdAt":"2026-02-27 05:54:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8983924/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8983924/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105846153,"identity":"ea6d1966-39df-4ddc-aa8c-a995ea32344c","added_by":"auto","created_at":"2026-03-31 17:55:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":186753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDefinition of leaf developmental stages and global transcriptome separation during SCC4 initiation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBienertia sinuspersici\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(A) Representative images of leaves at three developmental stages used for transcriptome analysis: young (\u0026lt;3 mm), intermediate (3–6 mm), and mature (\u0026gt;6 mm). Leaves were staged based on length and morphology following Koteyeva et al. (2016).\u003c/p\u003e\n\u003cp\u003e(B) RNA integrity assessment of total RNA isolated from young, intermediate, and mature leaves, showing comparable RNA quality across developmental stages.\u003c/p\u003e\n\u003cp\u003e(C) Principal component analysis (PCA) of RNA-seq expression profiles (FPKM values) from young, intermediate, and mature leaves. Each point represents an independent biological replicate. Samples form distinct clusters along PC1, with intermediate leaves positioned between young and mature stages, reflecting progressive transcriptional reprogramming during leaf development in Bienertia.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/248f9e81295078f216968dc9.png"},{"id":105846157,"identity":"f51a4ede-eb8e-4879-b019-9f1ea4163379","added_by":"auto","created_at":"2026-03-31 17:55:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":290209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional landscape and expression patterns of 199 early-biased genes during early leaf development in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBienertia sinuspersici\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n(A) Functional categorization of 199 early-biased genes identified through differential expression analysis and qRT-PCR validation. Genes were grouped based on integrated annotation using Gene Ontology, KEGG pathways, Arabidopsis orthology, and literature curation. Major functional categories include lipid, cuticle, and wax metabolism; transcriptional control; chloroplast biogenesis and protein import; cell wall expansion and epidermal patterning; phenylpropanoid and lignin metabolism; redox/ROS homeostasis; and auxin homeostasis.\u003c/p\u003e\n\u003cp\u003e(B) Heatmap showing expression profiles of the 81 genes constituting the SCC4-ready module across young (Y), intermediate (I), and mature (M) leaf stages. Most genes exhibit strong enrichment in young leaves followed by marked downregulation during maturation, consistent with roles in early structural reorganization and plastid-associated priming preceding SCC4 differentiation.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/ca3baf3b8d286cc7eb37f47b.png"},{"id":105846156,"identity":"8da5ff4f-462f-419f-b1fd-c550033fbfc9","added_by":"auto","created_at":"2026-03-31 17:55:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":176105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCore SCC4-ready gene expression and functional organization during early leaf development in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBienertia sinuspersici\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Heatmap showing expression profiles of the 33 core SCC4-ready genes across young (Y1–Y3), intermediate (I1–I3), and mature (M1–M3) stages. Expression values represent normalized RNA-seq FPKM data. Genes were selected based on strong early-stage enrichment, near-complete repression in mature leaves, and concordance between RNA-seq and qRT-PCR validation. Most genes display sharp early dominance followed by rapid attenuation during maturation, preceding visible chloroplast dimorphism.\u003c/p\u003e\n\u003cp\u003e(B) Functional interaction network summarizing the modular organization of the 33 core SCC4-ready genes. Node size reflects gene number within each functional category. Transcriptional control occupies a central position linking modules associated with phenylpropanoid/lignin/lipid remodeling, organelle-associated gene expression, cell-wall expansion, auxin homeostasis, redox-related processes, and membrane-associated transport. Solid arrows indicate primary inferred relationships; dashed lines indicate supportive associations.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/eeddac79aa4db8ce9186336f.png"},{"id":105846251,"identity":"8304b850-9372-444d-8c10-23de4706aecf","added_by":"auto","created_at":"2026-03-31 17:55:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":478122,"visible":true,"origin":"","legend":"\u003cp\u003eA hierarchical model for early SCC4 priming in \u003cem\u003eBienertia sinuspersici.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSchematic model illustrating the hierarchical regulatory architecture underlying early single-cell C₄ (SCC4) priming in Bienertia. Early transcriptional regulators initiate a preparatory program coordinating structural remodeling and organelle-associated processes prior to visible chloroplast dimorphism. Transcriptional modules activate phenylpropanoid-, lipid-, and cuticle-associated pathways, reinforcing epidermal and peripheral cellular structures while promoting cell-wall reorganization. In parallel, plastid-associated programs establish chloroplast competence before functional specialization. Auxin homeostasis contributes to growth regulation and spatial patterning during early reorganization. Membrane-associated transport processes, exemplified by the inner-membrane protein \u003cem\u003eyjiY\u003c/em\u003e, function downstream as amplification layers integrating structural and metabolic inputs prior to activation of the canonical C₄ biochemical cycle.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/3a9bff4af673a9b55747a3fe.png"},{"id":109335858,"identity":"0d87c425-746d-41f8-9730-3fd671643b27","added_by":"auto","created_at":"2026-05-15 17:09:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1384874,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/778a0626-cb8f-4a3d-9b46-e2c28ddd8f2f.pdf"},{"id":105846213,"identity":"d97cd9f1-7305-41ca-923e-0996bd998a6e","added_by":"auto","created_at":"2026-03-31 17:55:33","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33350,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/eaf8a8c13a61289b5af55a88.xlsx"},{"id":105846234,"identity":"1f08218d-b2b6-46a8-9a21-67344acf357b","added_by":"auto","created_at":"2026-03-31 17:55:34","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5663655,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/1c0e6feeeb00dd2f1efdad9f.xlsx"},{"id":105846211,"identity":"1684f22d-6303-4dbf-9ff5-b3bd1a9dbb0b","added_by":"auto","created_at":"2026-03-31 17:55:33","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":29231,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/d14a1f5e1a65cb8d7b7cf5e2.xlsx"},{"id":105846245,"identity":"5861a712-10d0-4338-840f-21063207d90e","added_by":"auto","created_at":"2026-03-31 17:55:36","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":34182,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/e5c8afb4d538f1a165b69cdc.xlsx"},{"id":105846166,"identity":"23b8c448-3348-4d51-85ed-8bcdcd0be070","added_by":"auto","created_at":"2026-03-31 17:55:29","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":23836,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/32cfa188fc53ff1fc9d6dfe3.xlsx"},{"id":105846250,"identity":"a585c0e3-ff5f-4f15-a948-ca2db6657e90","added_by":"auto","created_at":"2026-03-31 17:55:37","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":63226,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/a343c408f261e20bdd5037c4.xlsx"},{"id":105846154,"identity":"480c879b-d743-48d0-945e-10fa6763c4c3","added_by":"auto","created_at":"2026-03-31 17:55:23","extension":"fasta","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":177501,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata1.fasta","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/9ebd299422aa426417075b4e.fasta"},{"id":105846204,"identity":"acf374fb-de56-4c66-a217-b61fa7fbde46","added_by":"auto","created_at":"2026-03-31 17:55:33","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":14184,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEGENDS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8983924/v1/1ac00656b16b0f2e5750c222.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Early transcriptome profiling reveals coordinated epidermal remodeling and chloroplast biogenesis during single-cell C4 priming in Bienertia sinuspersici","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePhotosynthetic carbon assimilation in land plants operates through three major pathways: C₃, C₄, and crassulacean acid metabolism (CAM). Among these, C₃ photosynthesis is ancestral but becomes inefficient under high temperature and water-limited conditions due to enhanced photorespiration (Sage, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Akhani et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sage et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). C₄ photosynthesis evolved repeatedly as a biochemical CO₂-concentrating mechanism that suppresses photorespiration by spatially separating initial CO₂ fixation from the Calvin cycle, classically through Kranz anatomy (Hatch, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Edwards et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Furbank, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExceptionally, a small number of species perform C₄ photosynthesis without Kranz anatomy, instead achieving full C₄ biochemistry within a single photosynthetic cell. This single-cell C₄ (SCC4) strategy, exemplified by \u003cem\u003eBienertia sinuspersici\u003c/em\u003e and \u003cem\u003eSuaeda aralocaspica\u003c/em\u003e, represents a striking evolutionary solution to photorespiratory constraint (Edwards et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Offermann et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Han et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In Bienertia, two functionally distinct chloroplast populations are spatially segregated within one cell: peripheral chloroplasts associated with initial carbon fixation and central chloroplasts enriched in Rubisco, separated by a large central vacuole yet connected through cytoplasmic channels (Mai et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This intracellular organization functionally parallels the mesophyll\u0026ndash;bundle sheath division of labor in Kranz-type C₄ species.\u003c/p\u003e \u003cp\u003eSCC4 development in Bienertia proceeds gradually during leaf ontogeny. Young leaves exhibit morphologically uniform, C₃-like chloroplasts; intermediate stages initiate intracellular segregation; and mature leaves establish fully differentiated dimorphic chloroplasts and SCC4 physiology (Offermann et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Koteyeva et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Han et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While anatomical, ultrastructural, and physiological aspects of this transition have been well described, the molecular programs that initiate intracellular compartmentation and plastid specialization remain unresolved.\u003c/p\u003e \u003cp\u003eRecent genomic and transcriptomic studies have begun to illuminate components of SCC4 regulation, including genome-wide architecture (Kim et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), chloroplast-type-specific carbonic anhydrases (Nguyen et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and genes involved in organ patterning, chloroplast positioning, and cytoskeletal dynamics (Soundararajan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Won et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sharpe et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, these studies largely capture transcriptional changes coincident with or following visible chloroplast dimorphism, leaving the earliest regulatory events that prime SCC4 development unexplored.\u003c/p\u003e \u003cp\u003eIn particular, it remains unclear how transcriptional programs associated with plastid biogenesis, membrane and lipid remodeling, structural reinforcement, hormone-associated regulation, redox balance, and metabolite transport are coordinated during early leaf development prior to visible chloroplast differentiation. Understanding these early transitions may help clarify the developmental context preceding SCC4 establishment.\u003c/p\u003e \u003cp\u003eBy identifying early-enriched transcriptional modules associated with plastid assembly, intracellular architecture, and metabolic processes, we characterize a coordinated transcriptional program that precedes dimorphic chloroplast formation. Our results suggest that SCC4 establishment is associated with early transcriptional pre-patterning prior to the induction of canonical C₄ biochemical genes, thereby defining a developmental context for subsequent intracellular compartmentation and plastid specialization.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003ePlant Material and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBienertia plants (BioSample SAMN03290884) were maintained in controlled growth chambers and greenhouses following established procedures (Han et al., 2023). Seedlings were germinated on Murashige and Skoog (MS) medium under a 16 h light / 8 h dark photoperiod at 22 \u0026deg;C until root establishment, then transferred to soil and cultivated in a greenhouse maintained at approximately 28 \u0026deg;C under moderate light intensity with regular irrigation using NaCl-supplemented water.\u003c/p\u003e\n\u003cp\u003eLeaf developmental stages were defined according to Koteyeva et al. (2016) as young (\u0026lt;3 mm), intermediate (3\u0026ndash;6 mm), and mature (\u0026gt;6 mm, approximately 1 cm leaf tips). For each stage, leaf tissues were harvested and immediately frozen in liquid nitrogen. Representative morphological features are shown in Fig. 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and library preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Germany). Tissues were homogenized in RLT buffer using a TissueLyser II (Qiagen) without thawing. RNA integrity was assessed by agarose gel electrophoresis and NanoDrop spectrophotometry. Genomic DNA was removed using recombinant DNase I (Takara, Japan), and 2 \u0026micro;g of DNase-treated RNA was used for cDNA synthesis. RNA preparation followed protocols previously established for Bienertia (Soundararajan et al., 2019).\u003c/p\u003e\n\u003cp\u003eRNA-seq libraries were prepared using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina) and sequenced on an Illumina NovaSeq 6000 platform to generate paired-end 100 bp reads. Biological triplicates were obtained for young, intermediate, and mature leaf stages.\u003c/p\u003e\n\u003cp\u003eAll RNA-seq datasets are available in the NCBI Sequence Read Archive under BioProject accession PRJNA917470 (SRP415653).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome analysis and identification of early-biased genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw reads were processed, aligned to the \u003cem\u003eBienertia\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ereference genome (Kim et al., 2025), and quantified using a standardized RNA-seq analysis pipeline. Differential expression analysis was performed using thresholds of |log₂ fold change| \u0026ge; 2 and adjusted P \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003eGenes exhibiting high expression in young leaves, reduced expression in intermediate leaves, and minimal or undetectable expression in mature tissues were classified as early-biased. After genome-based curation and removal of low-confidence loci, 199 early-biased genes were retained.\u003c/p\u003e\n\u003cp\u003eFunctional annotation was conducted using Gene Ontology, KEGG pathway mapping, Arabidopsis orthology inference, and literature-based curation. Gene Ontology enrichment analysis was performed using established statistical approaches. Functional categorization is summarized in Supplementary Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ecDNA was synthesized using the AmfiRivert II cDNA Synthesis Master Mix (GenDEPOT). Quantitative RT-PCR was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad) using iTaq SYBR Green Supermix under standard cycling conditions (95 \u0026deg;C for 3 min; 40 cycles of 95 \u0026deg;C for 10 s and 60 \u0026deg;C for 30 s). GAPDH served as the internal reference gene.\u003c/p\u003e\n\u003cp\u003eRelative expression levels were calculated using the 2⁻\u0026Delta;Ct and 2⁻\u0026Delta;\u0026Delta;Ct methods, with young leaves used as the reference stage. Genes exhibiting amplification in young and intermediate leaves but not in mature leaves were classified as mature-off. Primer sequences and normalized expression values are provided in Supplementary Tables S3 and S4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDefinition of SCC4-ready and core gene sets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 199 early-biased genes were evaluated based on expression magnitude, developmental specificity, qRT-PCR concordance, functional annotation, predicted subcellular localization, and pathway association.\u003c/p\u003e\n\u003cp\u003eFrom this integrative assessment, 81 genes representing core preparatory pathways related to plastid biogenesis, lipid and cuticle metabolism, cell wall remodeling, hormone-associated regulation, redox buffering, and metabolite transport were designated as the SCC4-ready gene set (Supplementary Table S6).\u003c/p\u003e\n\u003cp\u003eA subset of 33 genes was further defined as high-confidence early regulators based on:\u003cbr\u003e\u0026nbsp;(i) \u0026ge;10-fold early-to-mature expression contrast or complete repression in mature leaves;\u003cbr\u003e\u0026nbsp;(ii) concordant RNA-seq and qRT-PCR expression patterns; and (iii) functional centrality within reconstructed developmental modules.\u003c/p\u003e\n\u003cp\u003eDetailed gene lists are provided in Tables 1 and 2 and Supplementary Tables S5 and S6.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eTranscriptome profiling across leaf developmental stages in \u003cem\u003eBienertia sinuspersici\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYoung (\u0026lt;3 mm), intermediate (3\u0026ndash;6 mm), and mature (\u0026gt;6 mm) leaves were staged for transcriptome analysis (Fig. 1A). RNA integrity assessment confirmed comparable RNA quality across developmental stages (Fig. 1B). RNA-seq generated a comprehensive dataset comprising 55,257 expressed genes mapped to the Bienertia reference genome (Kim et al., 2025), with expression values provided in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003ePrincipal component analysis (Fig. 1C) and hierarchical clustering revealed clear developmental stratification of transcriptomes. Young and mature leaves formed distinct clusters, while intermediate leaves occupied a transitional position. This separation reflects progressive transcriptional reprogramming during leaf maturation and the developmental progression associated with SCC4 establishment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof 199 early-biased genes absent from mature leaves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferential expression analysis identified genes exhibiting high expression in young leaves, reduced expression in intermediate leaves, and minimal or undetectable expression in mature tissues. Using thresholds of |log₂ fold change| \u0026ge; 2 and adjusted P \u0026lt; 0.05, 199 genes met these criteria and were designated as early-biased genes (Supplementary Table S2).\u003c/p\u003e\n\u003cp\u003eThese genes represent a developmentally restricted transcriptional cohort active during early leaf stages and largely repressed upon maturation, consistent with roles in early developmental transitions rather than in established SCC4 metabolism.\u003c/p\u003e\n\u003ch3\u003eqRT-PCR primer design and validation of early-biased expression patterns\u003c/h3\u003e\n\u003cp\u003eGene-specific primers were designed for all 199 early-biased genes (Supplementary Table S3). Quantitative RT-PCR using the same RNA samples confirmed strong enrichment in young leaves, partial attenuation in intermediate leaves, and near-complete repression in mature tissues (Supplementary Table S4). Expression trends showed high concordance with RNA-seq data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional annotation of early-biased genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntegrated annotation combining Gene Ontology, KEGG pathways, \u003cem\u003eArabidopsis\u003c/em\u003e orthologues, and literature curation revealed enrichment in processes related to epidermal and cuticle development, phenylpropanoid metabolism, lipid modification, plastid biogenesis and protein import, redox balance, hormone-associated regulation, intracellular transport, and cell wall remodeling (Supplementary Table S5).\u003c/p\u003e\n\u003cp\u003eThese functional categories indicate coordinated activation of structural, plastidial, and regulatory processes during early leaf development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDefinition of 81-\u003c/strong\u003e\u003cstrong\u003egene SCC4-associated module\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough integrative evaluation of expression magnitude, developmental specificity, qRT-PCR concordance, and functional annotation, 81 genes were selected as strongly early-enriched candidates (Supplementary Table S6). These genes exhibited robust activation in young leaves, partial reduction in intermediate stages, and near-complete repression in mature tissues. Heatmap visualization revealed highly coordinated expression dynamics across biological replicates, indicating synchronized developmental regulation (Fig. 2).\u003c/p\u003e\n\u003cp\u003eFunctional categorization showed that this 81-gene set spans multiple interconnected biological processes, including plastid biogenesis and protein import, lipid and cuticle modification, phenylpropanoid metabolism, transcriptional regulation, hormone-associated pathways, redox balance, and membrane-associated transport (Table 1). Together, these genes define a transcriptional module associated with early SCC4 establishment prior to visible chloroplast dimorphism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of a 33-gene core early-stage cohort\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further refine the SCC4-associated module, more stringent criteria emphasizing expression magnitude, early-stage specificity, and reproducibility across platforms were applied. This analysis yielded a subset of 33 genes displaying pronounced enrichment in young leaves and sharp repression during maturation (Fig. 3A; Table 2).\u003c/p\u003e\n\u003cp\u003eNotably, the majority of these genes exhibited minimal residual expression in mature tissues, indicating tightly stage-restricted regulation. Their coordinated activation pattern suggests participation in early structural and plastid-associated transitions that precede chloroplast dimorphism rather than direct involvement in mature SCC4 biochemical processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptual representation of early SCC4-associated transcriptional organization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo facilitate interpretation of the core gene set, representative loci spanning major functional axes were selected for schematic modeling. These loci encompass transcriptional regulators, plastid-associated factors, lipid and cell wall remodeling components, hormone-related elements, redox-associated genes, and membrane-associated transporters. The resulting framework summarizes inferred functional relationships derived from integrated expression and annotation analyses (Fig. 4) and provides a structured representation of transcriptional organization during early SCC4 establishment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003ch3\u003eEarly transcriptional pre-patterning precedes activation of the C₄ biochemical cycle in \u003cem\u003eBienertia sinuspersici\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eA central finding of this study is that early leaf development in Bienertia is not characterized by premature activation of the canonical C₄ biochemical cycle, but instead by a coordinated transcriptional program associated with the establishment of cellular and plastidial competence prior to single-cell C₄ (SCC4) differentiation. Consistent with previous anatomical and physiological studies of SCC4 species (Edwards et al., 2004; Offermann et al., 2015; Han et al., 2023; Sharpe et al., 2025), transcripts encoding key C₄ enzymes such as phosphoenolpyruvate carboxylase (PEPC), pyruvate phosphate dikinase (PPDK), and malic enzymes were not enriched in young leaves. Instead, early developmental stages were dominated by genes associated with plastid biogenesis and protein import, membrane lipid remodeling, cell wall modification, transcriptional regulation, hormone-associated processes, and redox-related pathways.\u003c/p\u003e\n\u003cp\u003eTogether, these patterns suggest that SCC4 development in Bienertia is preceded by preparatory cellular reorganization rather than early biochemical engagement of C₄ carbon assimilation. Because SCC4 systems must achieve\u0026nbsp;spatial separation of metabolic functions within a single photosynthetic cell, extensive intracellular restructuring is likely required before activation of the C₄ cycle (Koteyeva et al., 2016; Han et al., 2023).\u003c/p\u003e\n\u003ch3\u003eEarly plastid biogenesis and developmental competence\u003c/h3\u003e\n\u003cp\u003eAmong the most prominent early-biased modules were genes associated with plastid biogenesis, protein import, and plastid metabolic priming. Early enrichment of plastid ribosomal components such as \u003cem\u003erpsG\u003c/em\u003e, electron transport\u0026ndash;related genes such as \u003cem\u003ecob\u003c/em\u003e, and MEP/isoprenoid pathway enzymes including DXS-like genes and \u003cem\u003ePSY2\u003c/em\u003e indicates that proplastids in young Bienertia leaves undergo biosynthetic expansion prior to visible chloroplast dimorphism. These transcriptional signatures are consistent with a developmental window in which plastids acquire translational capacity, pigment biosynthetic potential, and protein import competence before divergence into peripheral and central chloroplast types (Jarvis and L\u0026oacute;pez-Juez, 2013; Pogson et al., 2015; Cackett et al., 2021).\u003c/p\u003e\n\u003cp\u003eEvidence from other plant systems underscores the importance of this early competence phase. For example, disruption of plastid-localized PPR proteins in barley impairs chloroplast development specifically in young leaves, with only partial recovery at later stages (Huang et al., 2025). Such findings highlight the developmental sensitivity of early plastid maturation. In Bienertia, early transcriptional enrichment of plastid-associated genes may similarly reflect a preparatory phase that precedes biochemical specialization.\u003c/p\u003e\n\u003cp\u003eRedox- and ROS-associated genes were also detected, although their enrichment was more moderate than that of structural and plastid-related genes. This distribution aligns with models in which redox signaling contributes to developmental stabilization and coordination rather than acting as a primary driver of differentiation (Apel and Hirt, 2004; Foyer and Noctor, 2012; Pfannschmidt et al., 2020; Arce et al., 2022). Collectively, these data are consistent with a plastid state primed for later functional divergence without yet expressing the full C₄ enzymatic machinery.\u003c/p\u003e\n\u003ch3\u003eLipid and structural remodeling during early SCC4-associated transitions\u003c/h3\u003e\n\u003cp\u003eA notable feature of the early-biased gene set was enrichment of lipid-, cuticle-, phenylpropanoid-, and cell wall\u0026ndash;associated pathways. Homologs of WSD-type wax ester synthases, fatty acyl-CoA reductases, lipid-transfer proteins such as \u003cem\u003eEARLI1\u003c/em\u003e and \u003cem\u003eAIR1\u003c/em\u003e, and GDSL lipases exhibited strong early-stage expression. These gene families are traditionally associated with epidermal differentiation and cuticle formation (Samuels et al., 2008; Yeats and Rose, 2013).\u003c/p\u003e\n\u003cp\u003eRecent studies suggest that wax ester biosynthesis also contributes to structural integrity during organ development (Nobuswa et al., 2025; Alotaibi et al., 2020; Bao et al., 2021). In the context of SCC4 development, coordinated activation of these lipid-related pathways in Bienertia may contribute to stabilization of intracellular domains prior to chloroplast repositioning. Although direct biochemical measurements were not conducted here, the transcriptional enrichment observed supports the possibility that structural reinforcement accompanies early cellular reorganization.\u003c/p\u003e\n\u003ch3\u003eTranscriptional regulation and hormone-associated processes\u003c/h3\u003e\n\u003cp\u003eTranscription factors, including NAC- and MYB-family members, exhibited strong early expression followed by coordinated repression during leaf maturation. This pattern is consistent with involvement in early developmental coordination rather than sustained photosynthetic function. Auxin-associated genes such as \u003cem\u003eGH3.1\u003c/em\u003e and \u003cem\u003eGH3.2\u003c/em\u003e were similarly enriched at early stages, suggesting potential contributions of hormone-regulated growth modulation during intracellular reorganization.\u003c/p\u003e\n\u003cp\u003eBecause hormone levels were not directly quantified in this study, the functional contribution of auxin signaling to SCC4 establishment remains to be clarified through targeted experimental approaches.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolite transport as a potential amplification layer\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe membrane-associated gene \u003cem\u003eyjiY\u003c/em\u003e displayed the strongest early-to-mature expression ratio among the early-biased cohort. \u003cem\u003eyjiY\u003c/em\u003e encodes a small membrane protein lacking catalytic domains, consistent with a role in transport or membrane-associated modulation rather than enzymatic activity.\u003c/p\u003e\n\u003cp\u003ePlastid envelope transport is increasingly recognized as a key regulatory interface in metabolic coordination (Linka and Weber, 2010; Facchinelli and Weber, 2011). The strong early enrichment of \u003cem\u003eyjiY\u003c/em\u003e suggests that membrane-associated processes may contribute to metabolic adjustment during early SCC4-associated transitions, although functional validation will be required to determine its precise role.\u003c/p\u003e\n\u003ch3\u003e\u0026nbsp;A structured framework for early SCC4-associated development\u003c/h3\u003e\n\u003cp\u003eIntegrating transcriptomic profiling, qRT-PCR validation, and functional annotation, we propose a structured framework for early SCC4-associated developmental transitions in \u003cem\u003eBienertia\u003c/em\u003e. In this view, early transcriptional regulators and plastid-associated programs coincide with structural remodeling and membrane-associated processes prior to activation of canonical C₄ metabolism. Rather than representing a definitive mechanistic pathway, this framework summarizes coordinated developmental expression dynamics that precede chloroplast dimorphism.\u003c/p\u003e\n\u003cp\u003eThis perspective highlights SCC4 as a developmentally organized system in which intracellular competence is established before biochemical specialization, providing a foundation for future experimental studies of single-cell C₄ evolution.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.S.K. conceived and designed the study, performed plant cultivation and sampling, carried out RNA extraction, curated gene sets, conducted data analyses, and wrote the manuscript. P.S. performed qRT-PCR validation. B.D. assisted with data visualization. I.H. coordinated the overall research project and contributed to RNA-seq data generation. All authors reviewed and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo\u003c/strong\u003e\u003cstrong\u003empeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial or non-financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll RNA-seq data supporting the findings of this study are publicly available in the NCBI Sequence Read Archive under BioProject accession \u003cstrong\u003ePRJNA917470\u003c/strong\u003e. Processed expression matrices, qRT-PCR datasets, and functional annotation tables are provided as Supplementary Tables S1\u0026ndash;S6. FASTA sequences of the 199 early-biased transcripts are provided in Supplementary data 1. Additional data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve human participants or animals requiring ethical approval. All plant materials used in this study were handled in accordance with institutional and national guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Institute of Agricultural Sciences (PJ017445012026) of the Rural Development Administration, Republic of Korea\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAkhani H, Barroca J, Koteeva N, Voznesenskaya E, Franceschi V, Edwards G, Ghaffari SM, Ziegler H. 2005. \u003cem\u003eBienertia sinuspersici\u003c/em\u003e (Chenopodiaceae): a new species from Southwest Asia and discovery of a third terrestrial C₄ plant without Kranz anatomy. Systematic Botany 30, 290\u0026ndash;301.\u003c/li\u003e\n \u003cli\u003eAlotaibi S, Elseehy MM, Aljuaid BS, El-Shehawi AM. 2020. 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Journal of Experimental Botany 67, 2587\u0026ndash;2601.\u003c/li\u003e\n \u003cli\u003eLinka N, Weber APM. 2010. Intracellular metabolite transporters in plants. Molecular Plant 3, 21\u0026ndash;53.\u003c/li\u003e\n \u003cli\u003eMai KKK, Yeung W-T, Han S-Y, Cai X, Hwang I, Kang B-H. 2019. Electron tomography analysis of thylakoid assembly and fission in chloroplasts of a single-cell C₄ plant, \u003cem\u003eBienertia sinuspersici\u003c/em\u003e. Scientific Reports 9, 19640.\u003c/li\u003e\n \u003cli\u003eMoreno-S\u0026aacute;nchez R, Saavedra E, Rodr\u0026iacute;guez-Enr\u0026iacute;quez S, Ol\u0026iacute;n-Sandoval V. 2008. Metabolic control analysis: a tool for designing strategies to manipulate metabolic pathways. BioMed Research International 2008, 597913.\u003c/li\u003e\n \u003cli\u003eNguyen T, Lee N, Fr\u0026ouml;mling FJ, Meister TL, Kim JS, Offermann S, Hwang I. 2025. Expression and localization of two beta-carbonic anhydrases in \u003cem\u003eBienertia\u003c/em\u003e, a single-cell C₄ plant. Frontiers in Plant Science 15, 1506375.\u003c/li\u003e\n \u003cli\u003eNobuswa T, Sasaki-Sekimoto Y, Ohta H, Kusaba M. 2025. The WSD-type wax ester synthase is widely conserved in streptophytes and crucial for floral organ formation under high humidity in land plants. Journal of Plant Research 138, 497\u0026ndash;509.\u003c/li\u003e\n \u003cli\u003eOffermann S, Okita TW, Edwards GE. 2011. Resolving the compartmentation of C₄ photosynthesis in single cells of \u003cem\u003eBienertia sinuspersici\u003c/em\u003e. Plant Physiology 155, 1612\u0026ndash;1628.\u003c/li\u003e\n \u003cli\u003eOffermann S, Friso G, Doroshenk KA, Sun Q, Sharpe RM, Okita TW, Wimmer D, Edwards GE, van Wijk KJ. 2015. Developmental and subcellular organization of single-cell C₄ photosynthesis in \u003cem\u003eBienertia sinuspersici\u003c/em\u003e determined by large-scale proteomics and cDNA assembly. Journal of Proteome Research 14, 2090\u0026ndash;2108.\u003c/li\u003e\n \u003cli\u003eOlsen AN, Ernst HA, Leggio LL, Skriver K. 2005. NAC transcription factors: structurally distinct, functionally diverse. Trends in Plant Science 10, 79\u0026ndash;87.\u003c/li\u003e\n \u003cli\u003ePfannschmidt T, Terry MJ, Akem OV, Quiros PM. 2020. Retrograde signals from endosymbiotic organelles: a common control principle in eukaryotic cells. Philosophical Transactions of the Royal Society B 375, 20190396.\u003c/li\u003e\n \u003cli\u003ePogson BJ, Ganguly D, Albrecht-Borth V. 2015. Insights into chloroplast biogenesis and development. Biochimica et Biophysica Acta 1847, 1017\u0026ndash;1024.\u003c/li\u003e\n \u003cli\u003eSage RF. 2004. The evolution of C₄ photosynthesis. New Phytologist 161, 341\u0026ndash;370.\u003c/li\u003e\n \u003cli\u003eSage RF, Christin P-A, Edwards EJ. 2011. The C₄ plant lineages of planet Earth. Journal of Experimental Botany 62, 3155\u0026ndash;3169.\u003c/li\u003e\n \u003cli\u003eSamuels L, Kunst L, Jetter R. 2008. Sealing plant surfaces: cuticular wax formation by epidermal cells. Annual Review of Plant Biology 59, 683\u0026ndash;707.\u003c/li\u003e\n \u003cli\u003eSharpe RM, Hewitt S, Edwards G, Dhingra A. 2025. Comparative transcriptome analysis of emerging young and mature leaves of \u003cem\u003eBienertia sinuspersici\u003c/em\u003e, a single-cell C₄ plant. PeerJ 13, e19282.\u003c/li\u003e\n \u003cli\u003eSoundararajan P, Won SY, Park DS, Lee Y-H, Kim JS. 2019. Comparative analysis of the YABBY gene family of \u003cem\u003eBienertia sinuspersici\u003c/em\u003e, a single-cell C₄ plant. Plants 8, 536.\u003c/li\u003e\n \u003cli\u003eWon SY, Soundararajan P, Irulappan V, Kim JS. 2023. In silico, evolutionary, and functional analysis of CHUP1 and its related proteins in \u003cem\u003eBienertia sinuspersici\u003c/em\u003e: a comparative study across C₃, C₄, CAM, and SCC4 model plants. Frontiers in Plant Science 14, 1225951.\u003c/li\u003e\n \u003cli\u003eYeats TH, Rose JKC. 2013. The formation and function of plant cuticles. Plant Physiology 163, 5\u0026ndash;20.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"1149\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" colspan=\"7\" valign=\"bottom\" style=\"width: 100%;\"\u003e\n \u003cp\u003eTable 1. Functional classification of SCC4-ready genes enriched in young \u003cem\u003eBienertia sinuspersici\u0026nbsp;\u003c/em\u003eleaves.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 231px;\"\u003e\n \u003cp\u003eModule\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003eTotal Gene Count\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274px;\"\u003e\n \u003cp\u003eBiological Role in Early C3\u0026rarr;C4 Transition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003eRepresentative Genes (subset of full list)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"66\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 231px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 81px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 274px;\"\u003e\n \u003cp\u003eSurface cuticle/wax biosynthesis, lipid remodeling, and phenylpropanoid\u0026ndash;lignin network formation supporting early epidermal reinforcement\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003eGDSL esterases/lipases:\u0026nbsp;\u003cem\u003eGLIP4\u003c/em\u003e, \u003cem\u003eF14O23.4\u003c/em\u003e, \u003cem\u003edl4155w\u003c/em\u003e, \u003cem\u003eCPRD49\u003c/em\u003e, \u003cem\u003eAt5g03820\u003c/em\u003e, \u003cem\u003eGDL17_ARATH\u003c/em\u003e, \u003cem\u003eGDL14_ARATH\u003c/em\u003e, \u003cem\u003eGDL71_ARATH\u003c/em\u003e, \u003cem\u003eGDL28_ARATH\u003c/em\u003e, \u003cem\u003eGDL56_ARATH\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"76\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003eO-acyltransferases \u0026amp; lipid enzymes:\u0026nbsp;\u003cem\u003eWSD1\u003c/em\u003e (\u0026times;4), \u003cem\u003eFAR3\u003c/em\u003e, \u003cem\u003eFAR_SIMCH\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"57\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003ePhenylpropanoid:\u0026nbsp;\u003cem\u003eCOMT\u003c/em\u003e (\u0026times;3), \u003cem\u003eCCoAOMT\u003c/em\u003e (\u0026times;2), \u003cem\u003eOMT1\u003c/em\u003e, \u003cem\u003eSCPL17\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"45\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003eLipid transfer:\u0026nbsp;\u003cem\u003eEARLI1\u003c/em\u003e (\u0026times;3), \u003cem\u003eAIR1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"32\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" style=\"width: 231px;\"\u003e\n \u003cp\u003eTranscriptional Control\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 81px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 274px;\"\u003e\n \u003cp\u003eTranscriptional regulators coordinating early leaf fate, epidermal identity, and metabolic reprogramming\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003eNAC:\u0026nbsp;\u003cem\u003eNAC021\u003c/em\u003e (\u0026times;2), \u003cem\u003eNAC031\u003c/em\u003e, \u003cem\u003eNAC098\u003c/em\u003e (\u0026times;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"33\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003eMYB:\u0026nbsp;\u003cem\u003eMYB3\u003c/em\u003e, \u003cem\u003eMYB108\u003c/em\u003e, \u003cem\u003eMYB39\u003c/em\u003e (\u0026times;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"33\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003eHomeobox/TCP: \u003cem\u003eHAT3\u003c/em\u003e, \u003cem\u003eSTM\u003c/em\u003e, \u003cem\u003eWOX9\u003c/em\u003e, \u003cem\u003eKNAT2\u003c/em\u003e, \u003cem\u003eTCP12\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"33\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003eB3/other TFs: \u003cem\u003eREM9\u003c/em\u003e, \u003cem\u003eVRN1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"33\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 231px;\"\u003e\n \u003cp\u003eChloroplast_biogenesis_import\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 81px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eOrganelle-associated gene expression, ribosomal components, protein import, and electron transport processes linked to early chloroplast establishment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003e\u003cem\u003erpsG\u003c/em\u003e, \u003cem\u003erps4\u003c/em\u003e, \u003cem\u003erplB\u003c/em\u003e, \u003cem\u003eccsA\u003c/em\u003e, \u003cem\u003ecob\u003c/em\u003e (mitochondrial-encoded), \u003cem\u003eGAPCP1\u003c/em\u003e, \u003cem\u003ePAP26\u003c/em\u003e, \u003cem\u003eHSP17.9-D\u003c/em\u003e, \u003cem\u003ePCDH11X\u003c/em\u003e, \u003cem\u003eAt4g27810\u003c/em\u003e, \u003cem\u003eAt5g16860\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"53\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"53\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 231px;\"\u003e\n \u003cp\u003eCell Wall Expansion \u0026amp; Epidermal Patterning\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274px;\"\u003e\n \u003cp\u003eCell wall loosening, plasmodesmata regulation, and epidermal boundary formation accommodating early structural reorganization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003e\u003cem\u003ePME68\u003c/em\u003e, \u003cem\u003eXTH33\u003c/em\u003e, \u003cem\u003eEPFL2\u003c/em\u003e, \u003cem\u003eEPFL4\u003c/em\u003e, \u003cem\u003eEPFL1\u003c/em\u003e, \u003cem\u003ePDCB3\u003c/em\u003e, \u003cem\u003eCEL3\u003c/em\u003e, \u003cem\u003e14KD_DAUCA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"17\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 231px;\"\u003e\n \u003cp\u003eRedox / ROS Energy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274px;\"\u003e\n \u003cp\u003eRedox homeostasis and electron transport\u0026ndash;related processes supporting early organelle maturation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003e\u003cem\u003eNUDT20\u003c/em\u003e (\u0026times;2), \u003cem\u003eCYP77A3\u003c/em\u003e (\u0026times;2), \u003cem\u003eGRXC3\u003c/em\u003e, \u003cem\u003ePER57\u003c/em\u003e, \u003cem\u003eND5\u003c/em\u003e (mitochondrial-encoded)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"66\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 231px;\"\u003e\n \u003cp\u003eMetabolite Transport\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274px;\"\u003e\n \u003cp\u003eMembrane-associated solute transport and metabolic balance during early sink\u0026ndash;source transition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003e\u003cem\u003eyjiY\u003c/em\u003e, \u003cem\u003eSWEET5\u003c/em\u003e, \u003cem\u003eABCG6\u003c/em\u003e, \u003cem\u003eSLC6A9\u003c/em\u003e, \u003cem\u003ePSOMT1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"7\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 231px;\"\u003e\n \u003cp\u003eIsoprenoid / MEP \u0026ndash; Carotenoid Pathway\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274px;\"\u003e\n \u003cp\u003eMEP-pathway and carotenoid biosynthesis enzymes contributing to plastid differentiation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003e\u003cem\u003ePSY2\u003c/em\u003e, \u003cem\u003edxs\u003c/em\u003e, \u003cem\u003eDXS2-like\u003c/em\u003e (Os07g0190000)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"7\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 231px;\"\u003e\n \u003cp\u003eAuxin Homeostasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 274px;\"\u003e\n \u003cp\u003eAuxin conjugation and homeostasis associated with early leaf patterning and growth modulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 331px;\"\u003e\n \u003cp\u003e\u003cem\u003eGH3.1\u003c/em\u003e, \u003cem\u003eGH3.2\u003c/em\u003e, \u003cem\u003eWTR8_ARATH\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 233px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"7\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 99.8808%;\" colspan=\"7\"\u003e\u003cbr\u003eThe 81 SCC4-ready genes were classified into eight functional modules based on integrated annotation using Gene Ontology, KEGG pathway analysis, Arabidopsis orthologues, and literature curation. For each module, the total number of genes, proposed biological roles during the early C3\u0026rarr;C4 transition, and representative genes are presented. Together, these modules reflect coordinated transcriptional programs underlying early epidermal remodeling, organelle-associated gene expression, metabolic priming, and regulatory control preceding visible chloroplast dimorphism in Bienertia. The complete gene list is provided in Supplementary Table S6.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2. Core early-dominant genes defining the SCC4-ready regulatory framework in \u003cem\u003eBienertia sinuspersici\u003c/em\u003e.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"913\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eGenes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003eAvr. FPKM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eq-PCR Ratio (Early/mature)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eC4_ready_modules\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00003862\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eF14O23.4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eGDSL esterase/lipase (lipid remodeling, likely secretory/apoplastic)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.726\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e17.507\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00003864\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eGDL17_ARATH\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eGDSL esterase/lipase (lipid remodeling)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e4.078\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eFAIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00012052\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003edl4155w\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eGDSL esterase/lipase (lipid remodeling)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e19.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eundetected in mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00013033\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eWSD1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eWax ester synthase/acyltransferase (cuticle-associated lipid metabolism)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e13.855\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e31968.006\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00020061\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eAt5g03820\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eGDSL esterase/lipase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e4.727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eundetected in mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00020019\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eFAR_SIMCH\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eFatty acyl-CoA reductase (cuticular lipid biosynthesis)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e6.872\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eundetected in mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00020020\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eFAR3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eFatty acyl-CoA reductase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.457\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e986.415\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00028292\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eAIR1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eLipid-transfer protein (likely extracellular/apoplastic)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e71.976\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e2221.656\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00034628\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eEARLI1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eLipid transfer protein (stress-responsive, secretory pathway)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eundetected in mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00034629\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eEARLI1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eLipid transfer protein (likely secretory/apoplastic)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.656\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e1124.644\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00036018\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eWSD1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eWax ester synthase/acyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e3.435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eundetected in mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00038185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eCCoAOMT\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eCaffeoyl-CoA O-methyltransferase (phenylpropanoid pathway)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e4.113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e9.959\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00047155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eWSD1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eWax ester synthase/acyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e4.074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eundetected in mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00006302\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eGDL56_ARATH\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eGDSL esterase/lipase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e12.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e9.135\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003ePhenylpropanoid_lignin_lipid_cuticle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00001955\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eAt4g27810\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eHypothetical protein (predicted plastid-associated)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e3.876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e249.375\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eChloroplast_biogenesis_import\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00022164\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003erpsG\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003e30S ribosomal protein S7 (chloroplast ribosome-associated)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e16.882\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eundetected in mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eChloroplast_biogenesis_import\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00028903\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003ecob\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eCytochrome b (mitochondrial-encoded, Complex III subunit)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e4.172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eundetected in mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eRedox_ros_energy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00045885\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eND5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eNADH dehydrogenase subunit 5 (mitochondrial-encoded Complex I)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e6.993\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e2.761\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eRedox_ros_energy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00018748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003ePSY2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003ePhytoene synthase 2 (chloroplastic carotenoid biosynthesis)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e21.730\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eChloroplast_biogenesis_import;Isoprenoid_mep_carotenoid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00050947\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eDXS2-like\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 226px;\"\u003e\n \u003cp\u003e1-deoxy-D-xylulose-5-phosphate synthase (plastidial MEP pathway)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e1.427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e1656.262\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eChloroplast_biogenesis_import;Isoprenoid_mep_carotenoid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00030682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eNUDT20\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eNudix hydrolase 20 (chloroplast-associated redox regulation)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.881\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e26.589\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eChloroplast_biogenesis_import;Redox_ros_energy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00030683\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eNUDT20\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eNudix hydrolase 20 (chloroplast-associated redox regulation)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e5.444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e33.973\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eChloroplast_biogenesis_import;Redox_ros_energy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00024425\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eGH3.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eIndole-3-acetic acid-amido synthetase (auxin conjugation)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e9.934\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e695.424\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eAuxin_homeostasis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00024426\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eGH3.2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 226px;\"\u003e\n \u003cp\u003eIndole-3-acetic acid-amido synthetase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e9.666\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e83.510\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eAuxin_homeostasis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00010905\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eNAC021\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eNAC-domain transcription factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.716\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e374.971\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eTranscriptional_control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00010906\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eNAC021\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eNAC-domain transcription factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e4.998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e602.066\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eTranscriptional_control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00021511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eTCP12\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eTCP transcription factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e3.252\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e15.295\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eTranscriptional_control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00025089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eSTM\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eHomeobox transcription factor (meristem-related)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003eundetected in mature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eTranscriptional_control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00053610\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eMYB39\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eMYB transcription factor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e387.012\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eTranscriptional_control\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00006562\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003ePME68\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003ePectin methylesterase (cell wall remodeling)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e10.164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e313.391\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eCell_wall_expansion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00030285\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eXTH33\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003eXyloglucan endotransglucosylase/hydrolase (cell wall modification)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e3.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e21.355\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eCell_wall_expansion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003eBsv0100-00020760\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eEPF1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 226px;\"\u003e\n \u003cp\u003eEpidermal patterning factor 1 (secreted peptide)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e2.935\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e11.830\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eCell_wall_expansion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBsv0100-00009857\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eyjiY\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 226px;\"\u003e\n \u003cp\u003ePredicted inner-membrane protein (membrane-associated, non-enzymatic)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 51px;\"\u003e\n \u003cp\u003e718.592\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 119px;\"\u003e\n \u003cp\u003e25825.945\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 320px;\"\u003e\n \u003cp\u003eMetabolite_transport\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;This table summarizes 33 core early-dominant genes refined from the 81 SCC4-ready gene set using stringent expression and functional criteria: (i) strong enrichment in young leaves with marked repression in mature tissues; (ii) concordant RNA-seq and qRT-PCR expression patterns; and (iii) functional relevance within reconstructed SCC4 preparatory modules. These genes represent central regulatory and structural components underlying early SCC4 priming, including transcriptional control, organelle-associated gene expression, lipid remodeling, cell wall expansion, redox regulation, auxin homeostasis, and membrane-associated transport. Collectively, this 33-gene cohort defines the core regulatory framework preceding dimorphic chloroplast differentiation in Bienertia.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"single-cell C4 photosynthesis, Bienertia sinuspersici, early leaf development, transcriptome profiling, chloroplast biogenesis, epidermal remodeling, phenylpropanoid metabolism, lipid remodeling, auxin regulation","lastPublishedDoi":"10.21203/rs.3.rs-8983924/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8983924/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSingle-cell C₄ (SCC4) photosynthesis in \u003cem\u003eBienertia sinuspersici\u003c/em\u003e requires the formation of dimorphic chloroplasts within a single photosynthetic cell, yet the early developmental transitions preceding this differentiation remain poorly defined. Here, we conducted staged transcriptome profiling of young, intermediate, and mature leaves to characterize transcriptional dynamics associated with early SCC4 establishment, with selected expression patterns validated by qRT-PCR.\u003c/p\u003e \u003cp\u003eFrom 55,257 expressed transcripts, genome-guided filtering identified 199 genes strongly enriched in young leaves and markedly repressed during maturation. Functional annotation revealed coordinated enrichment in plastid biogenesis and protein import, lipid and cuticle remodeling, phenylpropanoid metabolism, transcriptional regulation, auxin-associated pathways, redox homeostasis, and membrane-associated transport. Refinement of this set defined an 81-gene SCC4-associated module and a 33-gene core cohort exhibiting pronounced early-stage dominance.\u003c/p\u003e \u003cp\u003eImportantly, these transcriptional shifts preceded detectable induction of canonical C₄ enzyme genes, indicating that structural reorganization and plastid-associated processes are established before biochemical C₄ specialization. Collectively, our results delineate an early transcriptional program underlying SCC4 priming in a non-Kranz C₄ species.\u003c/p\u003e","manuscriptTitle":"Early transcriptome profiling reveals coordinated epidermal remodeling and chloroplast biogenesis during single-cell C4 priming in Bienertia sinuspersici","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-31 17:54:34","doi":"10.21203/rs.3.rs-8983924/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":"b89f0548-16eb-4431-b2fc-fbc6befe5a9a","owner":[],"postedDate":"March 31st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-15T17:05:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-12T13:57:49+00:00","index":33,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T17:09:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-31 17:54:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8983924","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8983924","identity":"rs-8983924","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}