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Article

Transcriptome-Based Identification of Novel Transcription Factors Regulating Seed Storage Proteins in Rice

by
Jinpyo So
1,†,
Jong-Yeol Lee
2,†,
Kyoungwon Cho
1,
Suchan Park
1,
Kyuhee Lee
1,
Don-Kyu Kim
1 and
Oksoo Han
1,*
1
Department of Biotechnology and Department of Integrative Food, Bioscience and Biotechnology (BK21 FOUR), Chonnam National University, Gwangju 61186, Republic of Korea
2
Department of Agricultural Biotechnology, National Institute of Agricultural Science, RDA, Jeonju 54874, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(17), 2791; https://doi.org/10.3390/plants14172791
Submission received: 8 August 2025 / Revised: 1 September 2025 / Accepted: 4 September 2025 / Published: 5 September 2025
(This article belongs to the Special Issue Molecular Breeding and Germplasm Improvement of Rice—2nd Edition)
Browse Figures
Versions Notes

Abstract

Seed storage proteins (SSPs) play a pivotal role in determining the development, quality, and nutritional value of rice seeds. In this study, we conducted a transcriptome-based correlation analysis to identify novel transcription factors (TFs) potentially involved in the biosynthesis and accumulation of SSPs. Our analysis revealed nine TFs—OsGATA8, OsMIF1, OsMIF2, OsGZF1, OsbZIP58, OsS1Fa1, OsS1Fa2, OsICE2, and OsMYB24—that exhibit strong co-expression with key SSP genes, including those encoding glutelin and prolamin. Gene expression profiling using quantitative RT-PCR and GUS reporter assays revealed that these TFs are predominantly expressed during seed development, with peak expression observed at 10 days after flowering (DAF). Promoter analysis further demonstrated an enrichment of seed-specific and hormone-responsive cis-regulatory elements, reinforcing the seed-preferential expression patterns of these TFs. Collectively, our findings identify a set of candidate TFs likely involved in SSP regulation and seed maturation, providing a foundation for the genetic enhancement of rice seed quality and nutritional content through targeted breeding and biotechnological approaches.

1. Introduction

Rice (Oryza sativa L.), along with wheat and maize, is among the most extensively cultivated and consumed cereal crops worldwide, serving as a staple food particularly in Asia. It possesses agronomic and nutritional traits critical for both cultivation and human consumption, including yield, grain quality, stress resilience, nutritional composition, and growth duration. Of these, grain quality and nutritional value are primarily dictated by the composition and relative abundance of nutrients in the rice seed [1,2].
Rice seeds serve as a major source of nutrients for humans, predominantly providing energy in the form of starch, along with proteins, lipids, vitamins, and minerals. On a dry weight basis, rice seeds comprise approximately 80% carbohydrates, 8–10% protein, less than 1% lipid, and about 3.5% dietary fiber [2,3]. Seed storage proteins (SSPs), which account for the largest proportion of total protein in rice seeds, are classified into four types based on their solubility: glutelin, prolamin, globulin, and albumin. Among these, glutelin is the most abundant (60–80%), followed by prolamin (20–30%), globulin (~5%), and albumin (~10%) [4,5,6].
SSPs contribute significantly to seed size and weight, and are integral to seed development, nutrient storage, and maturation [1,7]. RNA interference-mediated suppression of glutelin, prolamin, and globulin genes in rice has revealed corresponding reductions in grain weight and starch content, along with altered aleurone and endosperm structures and delayed germination [8]. The knockout of glutelin genes using CRISPR-Cas9 has been shown to produce similar phenotypes, including a reduction in seed weight, morphological changes, and disrupted endosperm structure with lower levels of starch and SSPs [9,10,11]. Furthermore, editing of 13 kDa prolamin genes using CRISPR-Cas9 induced compensatory changes in the expression of other SSPs, with concomitant effects on starch composition and seed structure [12].
The expression and deposition of SSPs are regulated by environmental conditions (e.g., temperature, humidity, nutrient availability) and intrinsic factors such as transcription factors (TFs) and endogenous phytohormones [2,13,14,15]. Several TFs have been identified that modulate SSP expression. For instance, NF-YC12, a component of the NF-Y TF complex, is crucial for starch and SSP accumulation. Loss of its function reduces grain weight and disturbs nutrient deposition [14]. RISBZ1, a type of the basic leucine zipper (bZIP), binds the GCN4 motif to activate endosperm-specific expression of SSP genes and interacts with the rice prolamin box-binding factor (RPBF) to regulate SSP biosynthesis and seed development [16,17]. Conversely, OsGZF1, a CCCH-type zinc finger protein, suppresses GluB-1 expression by inhibiting RISBZ1-mediated activation [18]. OsNAC20 and OsNAC26, members of the NAC TF family, are expressed in the embryo and endosperm and directly enhance transcription of genes involved in starch and SSP biosynthesis. CRISPR-Cas9-mediated knockout of OsNAC20 and OsNAC26 led to a marked decrease in both starch and SSP content [19].
These findings collectively suggest that SSP biosynthesis is intricately coordinated with starch metabolism and seed developmental processes. Notably, mutations in TFs associated with SSP regulation consistently manifest in altered grain morphology and compositional shifts [14,16]. Identification of novel TFs involved in SSP regulation is therefore pivotal for understanding seed development and improving the nutritional quality of rice.
In this study, transcriptomic datasets [20] were employed to identify transcription factors that may potentially be involved in the regulation of seed storage protein expression. Based on these datasets, we constructed a co-expression network to comprehensively examine the relationships between transcription factors and SSP-related genes. Through this network analysis, novel transcription factors closely associated with SSP expression in rice were systematically identified. To support these findings, we validated the expression patterns of the candidate TFs, thereby providing multiple lines of evidence for their potential involvement in SSP regulation.

2. Results

2.1. Identification of Novel Transcription Factors Correlated with Seed Storage Protein Genes

To identify transcription factors (TFs) potentially involved in the regulation of rice seed storage proteins (SSPs), we constructed a co-expression network based on the rice seed transcriptome derived from the Agilent 4 × 44 K custom oligo-DNA chip (Agilent Technologies, Santa Clara, CA, USA). Annotation of genes using the MapCave tool identified 38 SSP-encoding genes on the array, including 25 prolamins, 11 glutelins, one globulin, and one albumin (Table S1). From a previous RNA-seq dataset of immature seeds from SSP-deficient lines [20], 128 TFs among the top 2000 most abundant transcripts were selected based on functional annotation.
Pearson’s correlation analysis of gene expression profiles across 48 transcriptome datasets revealed strong associations (|r| ≥ 0.7) between 27 SSP and 33 TF genes (Table S1). These interactions were visualized as a co-expression network using Cytoscape (Figure 1). Notably, three TFs—OsMYB24, OsGZF1, and OsICE2—emerged as hubs, correlating with 17, 14, and 12 SSP genes, respectively. These hub TFs shared co-expression with six SSP genes: GluA-2, GluB-2, GluB-5, Pro13b.2, Pro13b.3, and Pro13b.17. Most TFs showed correlation with only a single SSP gene (e.g., nine TFs with GluB-7, five with GluC-1, seven with GluA-1, and one with GluA-3).
Among these, we focused on nine TFs that either functioned as network hubs or were linked to GluA-1, whose functional disruption via CRISPR-Cas9 affects grain traits [10]. These included OsGATA8 (GATA transcription factor 8), OsMIF1 (mini zinc finger 1), OsMIF2 (mini zinc finger 2), OsGZF1 (GluB-1-binding Zinc Finger 1), OsbZIP58 (bZIP transcription factor 58), OsS1Fa1 (site 1 binding factor a1), OsS1Fa2 (site 1 binding factor a2), OsICE2 (Inducer of CBF expression 2) and OsMYB24 (R2R3-MYB transcription factor 24) (Figure 2).
To further validate the regulatory potential of the candidate TFs identified through the co-expression network, we performed a transient overexpression assay in rice protoplasts. The TFs analyzed were OsGATA8, OsMIF1, OsbZIP58, and OsS1Fa1, which are co-expressed with GluA-1. Compared with the control, the expression levels of SSP genes were generally increased in protoplasts overexpressing the TFs. In particular, OsbZIP58 markedly enhanced the expression of genes of GluA group and also led to a pronounced up-regulation of most prolamin genes (except Pro13a-1 and the Pro16 group) as well as the globulin gene (Figure S1). These results confirm that the selected TFs can functionally contribute to the regulation of SSP gene expression.

2.2. Protein–Protein Interaction (PPI) Network Analysis of Candidate TFs

The nine candidate TFs were classified into four families based on Rice Genome Annotation Project, RAP-DB, and InterPro data (Table 1): zinc finger (C3H1: OsGZF1; GATA: OsGATA8; ZF-HD: OsMIF1, OsMIF2), basic domain (bZIP: OsbZIP58; bHLH: OsICE2), S1Fa (OsS1Fa1, OsS1Fa2), and MYB (R2R3: OsMYB24).
PPI predictions using STRING (confidence score ≥ 0.4) revealed diverse molecular interactions (Figure 3). OsbZIP58 interacted with proteins involved in RNA transcription, initiation of protein synthesis, protein kinase, misc, and unknown functions. OsICE2 was associated with components of RNA transcription, ubiquitin, cell organization, and unknown functions. OsGATA8 interacted with proteins linked to RNA transcription, miscellaneous functions, histone, ubiquitin, development, and unknown functions. OsS1Fa1 and OsS1Fa2 were associated with ribosome, RNA transcription, and unknown functions. OsGZF1 interacted with proteins involved in ubiquitin and ribosome. OsMIF1 showed interactions with MYBAS1 and AMY2A, while OsMYB24 interacted with OsALYL1 and MYB-related proteins. No interacting partners were identified for OsMIF2.
Notably, no direct interactions were observed among the nine TFs, consistent with results from the yeast two-hybrid (Y2H) assay (Figure S2). However, OsICE2 and OsbZIP58 commonly interacted with SE14, MPK1, and MPK5, while OsbZIP58 and OsGATA8 shared interactions with CARM1 and Q10SM5_ORYSJ.

2.3. Expression Profiles of TFs During Seed Development

qRT-PCR analysis of TF gene expression across reproductive tissues revealed two major expression patterns (Figure 4A). One group, consisting of OsbZIP58, OsGZF1, OsMYB24, and OsMIF1/2, displayed seed-specific expression peaking at 10 days after flowering (DAF), followed by a decline. The other group, including OsGATA8, OsS1Fa1, and OsICE2, exhibited both seed-specific and leaf-enriched expression profiles.
To further investigate temporal and spatial expression patterns during seed development, four proTF::GUS transgenic lines were generated using 1.5 kb upstream promoter sequences from OsGATA8, OsS1Fa1, OsbZIP58, and OsMIF1 (Figure 4B and Figure S3). GUS staining revealed consistent expression across lines, initially localized to the seed tips until 5 DAF, expanding throughout the seed during development, peaking at 10 DAF, and diminishing as seeds matured and hardened.

2.4. Genome-Wide Enrichment Analysis of Cis-Regulatory Elements (CREs)

To explore regulatory motifs associated with TF expression, genome-wide CRE enrichment analysis was conducted on the 2000 bp upstream promoter regions of the nine candidate TFs using the PLACE database and MEME Suite (Figure S4, Table S5). Ten CREs were significantly enriched (p ≤ 0.1) in five TF promoters: OsMYB24, OsbZIP58, OsGATA8, OsICE2, and OsGZF1.
Specifically, OsMYB24 contained four enriched motifs (ABREZMRAB28, O2F2BE2S1, RYREPEATGMGY2, RYREPEATVFLEB4), OsbZIP58 had three (AMYBOX1, GARE1OSREP1, AUXRETGA2GMGH3), OsGATA8 one (-300ELEMENT), OsGZF1 one (TATABOX1), and OsICE2 one (SEF4MOTIFGM7S) (Figure 5, Table 2). No significant CRE enrichment was observed in OsMIF1, OsMIF2, OsS1Fa1, or OsS1Fa2 promoters.

3. Discussion

Efforts to suppress the synthesis of seed storage proteins (SSPs) in rice—through CRISPR-Cas9-mediated gene editing or transcriptional silencing via RNA interference—have often resulted in compensatory upregulation of non-targeted SSPs, thereby maintaining total seed protein content [12,35,36]. These findings underscore the necessity for a deeper understanding of SSP regulatory networks.
It has been revealed that SSP biosynthesis influences starch metabolism by altering carbon–nitrogen partitioning, modulating the sucrose–hexose balance, and providing metabolic signals that regulate starch biosynthetic pathways [37]. This suggests that a deeper understanding of SSP regulatory networks may not only improve knowledge of protein accumulation but also provide critical insights into the coordinated synthesis and deposition of multiple storage reserves in seeds.
In this study, we conducted a comprehensive co-expression analysis and identified nine transcription factors (TFs)—OsICE2, OsGZF1, OsMYB24, OsMIF1, OsMIF2, OsGATA8, OsbZIP58, OsS1Fa1, and OsS1Fa2—that are strongly associated with SSP gene expression (Figure 1). Expression profiling using qRT-PCR and promoter-driven GUS reporter assay in transgenic rice confirmed that these TFs are highly expressed during seed development (Figure 4). Furthermore, transient overexpression assays in rice protoplasts demonstrated that selected TFs, particularly OsbZIP58, can directly activate multiple SSP genes, providing functional support for our network-based predictions (Figure S1). However, as these assays were limited to a protoplast system, further in planta validation such as stable transformation or mutant analysis will be required to confirm their regulatory roles during seed development.
Among them, OsbZIP58 and OsGZF1 have previously been identified as regulators of seed-specific gene expression [16,17,18]. OsbZIP58, a member of the bZIP family, contains a basic DNA-binding domain and a leucine zipper domain mediating DNA binding and protein–protein interactions, respectively [38]. As a transcriptional activator, it regulates genes involved in starch biosynthesis and SSP accumulation during seed development [39,40]. SSP gene promoters commonly harbor cis-regulatory elements (CREs) such as AACA, ACGT, GCN4, and PROL. OsbZIP58, predominantly expressed in the endosperm and aleurone layer, binds to GCN4, ACGT, and ATGA motifs to activate seed-specific gene expression [17,41,42]. It also interacts synergistically with RPBF (Rice Prolamin Box Binding Factor), a member of the Dof family, to promote storage protein gene expression [16,43].
In this study, the OsbZIP58 promoter was found to contain the seed-specific motif AMYBOX1 (TAACARA) and the auxin-responsive element AUXRETGA2GMGH3 (TGACGTGGC) (Figure 5, Table 2). Co-expression and yeast two-hybrid (Y2H) analyses showed that OsbZIP58 is closely correlated with GluA-1, but not with other TFs (Figure 1, Figure S2). However, PPI analysis indicated that SE14, MPK1, and MPK5 bind with both OsbZIP58 and OsICE2, and Q10SM5_ORYSJ and CARM1 interact with both OsbZIP58 and OsGATA8, suggesting that the interaction between the TFs is mediated by other proteins (Figure 3). To provide more definitive evidence of these interactions, further experimental validation such as Y2H or BiFC will be necessary.
OsICE2, a TF of the bHLH family that includes a total of 167 genes in rice [44,45], contains a single helix–loop–helix (HLH) motif and an ACT_UUR-ACR-like domain in its C-terminal region (Figure 2). OsICE2 plays an important role in plant growth and development, particularly in response to cold stress and in stomatal development [22,23,46,47]. As shown in Figure 4A, OsICE2 expression gradually increases until 10 DAF and then declines, exhibiting a pattern similar to that of 12 SSP genes (Figure 1). Promoter analysis further revealed significant enrichment of the SEF4MOTIFGM7S element (RTTTTTR), a known cis-regulatory element associated with seed storage protein gene expression [33].
OsGATA8 is a TF belonging to the GATA-type zinc finger protein family, which recognizes GATA motifs in DNA and plays a crucial role in signaling pathways related to abiotic stresses, including salt, drought, and exogenous abscisic acid (ABA) [48,49]. Previous studies have linked OsGATA8 to water stress responses and tiller formation [21,50]. Co-expression network analysis revealed that OsGATA8 exhibits a transcriptional pattern similar to that of GluA-1, a major SSP gene (Figure 1).
Promoter analysis showed that the OsGATA8 promoter is significantly enriched in the -300ELEMENT motif (TGHAAARK), an enhancer element identified in glutenin promoters that drives endosperm-specific expression [32]. Furthermore, both the expression profile of OsGATA8 during seed maturation and GUS reporter activity driven by the OsGATA8 promoter::GUS transgenic rice supports its involvement in seed development.
OsGZF1 is a TF belonging to the C3H zinc finger protein family, which plays crucial roles in stress responses and developmental processes in roots, leaves, flowers, and seeds [51]. OsGZF1 has been reported to repress the expression of GluB-1 by binding to AACA, ACGT, GCN4, and PROL motifs within its promoter, thereby interfering with the activation of GluB-1 by OsbZIP58 [17,41]. In OsGZF1 RNAi mutants, down-regulation of OsGZF1 led to increased grain nitrogen content, suggesting that OsGZF1 binds to promoters of glutelin genes, including that of GluB-1, to repress their expression [18]. In this study, PPI and Y2H analysis showed no direct interaction between OsGZF1 and OsbZIP58 (Figure 3, Figure S2). Considering that both OsbZIP58 and OsGZF1 bind to the same cis-elements on the promoter of SSP genes and OsbZIP58 activates the expression of SSPs but OsGZF1 represses it, the results suggest that OsbZIP58, as an activator, and OsGZF1, as a repressor, might compete to regulate the expression of SSPs. Promoter analysis of OsGZF1 (Figure 5, Table 2) revealed a significant enrichment of TATABOX1 (CTATAAATAC), a motif highly conserved in the promoters of SSP genes in the Poaceae family [52]. Furthermore, co-expression network analysis showed that OsGZF1 shares expression patterns with 14 SSP genes, including GluA-2, GluB-2, GluB-4, GluB-5, GluD-1, Pro10.2, Pro13a.2, Pro13b.1, Pro13b.2, Pro13b.3, Pro13b.5, Pro13b.14, Pro13b.17, and Pro13b.19, whereas OsbZIP58 is more specifically co-expressed with GluA-1 (Figure 1). The result suggests that the existence of at least two regulatory modes for controlling SSP content: One is that SSP content could be properly controlled by the co-occurrence of a large number of SSP gene expression activities with OsGZF1. The other is that the expression activities of a small number of SSP genes, including GluA-1, might occur with that of OsbZIP58, activating the expression of a large number of SSP genes.
OsMYB24 is a TF belonging to the R2R3-type MYB family, characterized by two tandem MYB domains (R2 and R3). Each domain consists of a three-helix structure comprising 51 to 53 amino acids (Figure 2), and specifically recognizes the YAAC(G/T)G DNA sequence [53,54], potentially playing a crucial role in gene expression for plant development, cell differentiation, and specialized metabolism such as lignin and flavonoid biosynthesis. During seed development, co-expression analysis (Figure 1) revealed that OsMYB24 is co-expressed with three prolamin genes (Pro13b.9, Pro13b.21, and Pro16.2) as well as 14 SSP genes that also show co-expression with OsGZF1. Promoter analysis of OsMYB24 showed significant enrichment of three seed-specific CREs—O2F2BE2S1 (GCCACCTCAT), RYREPEATGMGY2 (CATGCAT), and RYREPEATVFLEB4 (CATGCATG)—as well as an ABA/water–stress-responsive CRE, ABREZMRAB28 (CCACGTGG). Expression profiling during seed maturation (Figure 4A) demonstrated that OsMYB24 exhibits a transcriptional pattern similar to that of OsGZF1. These findings suggest a potential role for OsMYB24 in regulating seed storage protein expression, highlighting the need for further functional studies to elucidate its involvement in seed development.
OsS1Fa1 and OsS1Fa2 are small proteins composed of 76 and 80 amino acids, respectively (Figure 2), and belong to the S1Fa TF family, which is characterized by a nuclear localization signal and a putative DNA-binding helix [55,56]. The S1Fa family is known to specifically recognize the negative promoter element S1F, repressing the expression of RPS1, a gene encoding the chloroplast ribosomal protein S1, particularly in non-photosynthetic tissues such as roots. Additionally, S1Fa TFs are highly expressed under drought stress conditions, where they contribute to the suppression of RPS1 expression as part of the drought response [24,57]. In this study, OsS1Fa1 showed elevated expression in the rice flag leaf at 21 DAF compared to other tissues, including roots, stems, and seeds. In developing seeds, OsS1Fa1 expression peaked at 10 DAF and declined thereafter. These results suggest that OsS1Fa1 may be involved in seed developmental regulation, in addition to its known role in stress responses. Moreover, PPI network analysis of OsS1Fa1 and OsS1Fa2 (Figure 2) predicted potential interactions with ribosomal proteins (Figure 3, Tables S3 and S4), implying a possible role in the post-translational regulation of ribosome biogenesis.
OsMIF1 and OsMIF2 are homeodomain-deficient TFs that belong to the plant-specific ZF-HD TF family, which is known to play critical roles in environmental stress responses, as well as the growth and development of floral and seed structures [58,59]. In rice, the ZF-HD family comprises 11 canonical ZF-HD members, each containing both a C5H3-type zinc finger domain and a homeodomain, and 4 MIFs (mini zinc finger proteins, OsMIF1–4), which lack the homeodomain. Although the roles of OsMIF1 and OsMIF2 in rice are currently unclear, their homologs in other species, AtMIF2 in Arabidopsis, SlIMA in tomato, and GhMIF in Gerbera, have been reported to be involved in floral meristem termination, floral development, and ovule development [60,61]. In this study, GUS reporter assays driven by the OsMIF1 promoter and qRT-PCR analysis of OsMIF1 and OsMIF2 expression (Figure 4) suggest that both TFs may play a role in the reproductive stage of rice development.
Our results provided important clues for identifying candidate TFs involved in the regulation of SSPs. These approaches open the possibility of uncovering novel regulatory mechanisms of SSP expression that have not been previously reported, thereby offering new directions for future research. In particular, for the TFs whose functions have not yet been characterized, our results indirectly suggest their potential roles in seed development and SSP regulation. Nevertheless, these results do not provide direct evidence that the identified TFs regulate SSP expression. Future functional studies may help to establish their precise contributions to SSP biosynthesis and grain development, thereby validating and extending the hypotheses proposed in this study.

4. Materials and Methods

4.1. Correlation Analysis of Transcription Factor and Seed Storage Protein Genes Using Rice Seed Transcriptome

The 2000 most abundant transcripts in immature seeds of rice (Oryza sativa cv. Ilmi) were selected from previously published RNA-Seq data [20] and annotated using MSU locus identifiers from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/, accessed on 7 August 2025). Functional categorization was performed based on reference annotations from the MapManStore database for the O. sativa Japonica group (https://mapman.gabipd.org/mapmanstore, accessed on 7 August 2025).
To evaluate gene co-expression between transcription factors (TFs) and seed storage proteins (SSPs)—including glutelins, prolamins, and globulins—Pearson’s correlation coefficients were calculated (cut-off: |r| ≥ 0.7) using Microsoft Excel (Table S1). The rice seed transcriptome was compiled from 46 publicly available microarray datasets (GEO Series: GSE30462, GSE37478, GSE37650, GSE49963, and GSE79405) and 43 in-house microarray experiments based on the Agilent Rice 4 × 44 K platform (Agilent Technologies, Santa Clara, CA, USA). Co-expression networks were visualized using Cytoscape (v3.10.2).

4.2. Information on Selected Transcription Factors

Genomic loci and sequences of selected TFs were collected from the Rice Genome Annotation Project (http://rice.uga.edu, accessed on 7 August 2025) and RAP-DB (https://rapdb.dna.affrc.go.jp, accessed on 7 August 2025). Functional annotations were based on literature cited in Table 1. Protein domains were predicted using InterPro (https://www.ebi.ac.uk/interpro, accessed on 7 August 2025).

4.3. Protein–Protein Interaction (PPI) Network Analysis

To infer functions of hub genes co-expressed with SSP genes, PPI networks were constructed using the STRING database (https://string-db.org, accessed on 7 August 2025) with a minimum confidence score of 0.4. Network visualizations were generated in Cytoscape (v3.10.2) based on STRING outputs.

4.4. Plant Materials and Growth Conditions

Seeds of O. sativa L. ssp. Japonica cv. Ilmi were sourced from the Korea Seed & Variety Service (Gimcheon, Korea). Plants were cultivated in paddy fields under standard agronomic conditions.

4.5. Rice Protoplast Transient Expression

Rice protoplasts were isolated from 8–12 days old seedlings by enzymatic digestion (cellulase and macerozyme) and purified using W5/MMG buffers. For transient overexpression, 5–10 µg of plasmid DNA carrying OsGATA8, OsbZIP58, OsS1Fa1, or OsMIF1 was introduced into protoplasts via PEG-mediated transformation, and cells were incubated for 16–24 h at room temperature. Total RNA was extracted from transformed protoplasts.

4.6. RNA Extraction and Quantitative RT-PCR

Total RNA was isolated from leaves, roots, and stems at 21 days after flowering (DAF), as well as from immature seeds at various developmental stages, using a modified protocol optimized for starchy tissues [62]. The cDNA library was synthesized by using 1 µg of RNA with QuantiTect Reverse Transcription Kit (Cat. #205311, Qiagen, Hilden, Germany) according to the manufacturer’s protocol. qRT-PCR was conducted on a Rotor-Gene Q (Qiagen) using 10 ng of cDNA, 0.5 µM gene-specific primers, and 10 µL of 2× SYBR Green Master Mix (Qiagen, Cat. #204343, Hilden, Germany) in a 20 µL total volume. Thermal cycling was as follows: 95 °C for 10 min; 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; followed by melting curve analysis from 72 to 95 °C. Gene expression was normalized to Ubiquitin 5 (Os01g0328400) [63]. Primer sequences are provided in Table S2.

4.7. GUS Staining Assay

Genomic DNA was extracted from 21-day-old seedlings as described by [64]. A 1.5 kb upstream promoter region of each TF gene was amplified from 5 ng of genomic DNA and cloned into the pCAMBIA1201 vector. Constructs were introduced into the Agrobacterium tumefaciens strain EHA101 and subsequently transformed into embryogenic rice calli derived from mature seeds [65]. Immature seeds at various stages were harvested from transgenic lines for GUS staining, which was conducted following [66] to minimize false negatives. Samples were pretreated in ice-cold 90% acetone (5–15 min), then incubated in staining buffer [50 mM sodium phosphate buffer (pH 7.2), 2 mM each of K4[Fe(CN)6] and K3[Fe(CN)6], 2% Triton X-100, 2 mM X-Gluc] under vacuum for 5 min, and then incubated overnight at 37 °C in the dark. Samples were rinsed in 70% ethanol and stored at 4 °C.

4.8. Yeast Two-Hybrid Assay

The interactions between TFs were examined using the ProQuest™ Two-Hybrid System with Gateway Technology (Invitrogen, Cat. #PQ1000101, Carlsbad, CA, USA). Full-length cDNAs of each TF were cloned into the bait (pDEST32) and prey (pDEST22) vectors via Gateway cloning. Cloning primers are listed in Table S2. Yeast strain MaV203 was used for transformation and interaction screening.

4.9. Cis-Regulatory Element (CRE) Frequency Analysis

The 2000 bp promoter regions upstream of the start codon for 55,802 annotated rice genes (MSU RGAP; http://rice.uga.edu, accessed on 7 August 2025) were extracted and screened for cis-regulatory elements using the PLACE database (https://www.dna.affrc.go.jp/PLACE, accessed on 7 August 2025) via Find Individual Motif Occurrence (FIMO) in the MEME Suite (https://meme-suite.org, accessed on 7 August 2025). Frequencies of individual CREs were computed genome-wide and specifically for nine selected TF promoters. Enrichment was expressed as log2 fold change (log2FC), and statistical significance was determined using a normal distribution model based on genome-wide CRE occurrence.

5. Conclusions

In this study, we identified nine TFs that are co-expressed with major SSP genes in rice through transcriptome-based co-expression network analysis. These TFs—OsGATA8, OsMIF1, OsMIF2, OsGZF1, OsbZIP58, OsS1Fa1, OsS1Fa2, OsICE2, and OsMYB24—exhibited seed-preferential expression patterns, with peak expression during early seed development. Promoter analysis revealed significant enrichment of seed-specific and hormone-responsive CREs, supporting their potential roles in SSP transcriptional regulation. Functional domain prediction, protein–protein interaction analysis, and GUS reporter assays further supported the relevance of these TFs to seed development and nutrient storage. Collectively, these findings provide valuable insights into the transcriptional networks governing rice SSP biosynthesis and offer molecular targets for genome editing to enhance seed quality and nutrient content in rice.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14172791/s1. Figure S1. Transient over-expression of candidate TFs in rice protoplasts and their effects on seed storage protein (SSP) gene expression.; Figure S2. Interaction between TFs in yeast two-hybrid assays.; Figure S3. Confirmation of pTF::GUS transgenic rice.; Figure S4. Volcano plot of cis-element enrichment in TF promoters.; Table S1. Pearson’s correlation values between genes assigned to transcription factor and seed storage proteins.; Table S2. Primer list; Table S3. Proteins interacting with the selected transcription factors; Table S4. Annotation of proteins interacting with the selected transcription factors; Table S5. Cis-regulatory elements in the 2000 bp region upstream of the translation start codon of selected TF genes in rice identified through genome-wide frequency comparison; Table S6. Cis-regulatory element localization within promoter regions of selected TF genes.

Author Contributions

Conceptualization, J.S., J.-Y.L., K.C. and O.H.; methodology, J.S., J.-Y.L. and K.C.; software, K.C., J.-Y.L.; validation, J.S., J.-Y.L. and K.L.; formal analysis, J.S., S.P. and K.C.; investigation, J.S. and J.-Y.L.; resources, K.C. and O.H.; writing—original draft preparation, J.S. and J.-Y.L.; writing—review and editing, D.-K.K., K.C. and O.H.; visualization, J.S., J.-Y.L. and K.C.; supervision, O.H.; funding acquisition, J.-Y.L., K.C. and O.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by a grant from the National Institute of Agricultural Science (RDA PJ01720903), Rural Development Administration, Republic of Korea, and the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-RS-2023-00248217 to K Cho, 2023R1A2C1002936, 2024H1A7A2A02000017, and BK21 FOUR (Fostering Outstanding Universities for Research, No. 4120240915070) to O Han).

Data Availability Statement

All data generated and analyzed during this study are included in this published article and its additional files.

Acknowledgments

Authors thank NRF, RDA, and BK21 FOUR for financial support.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ABAAbscisic acid
bHLHBasic helix–loop–helix
bZIPBasic leucine zipper
CRECis-regulatory element
CRISPR-Cas9Clustered regularly interspaced short palindromic repeats–CRISPR associated protein 9
DAFDays after flowering
FIMOFind Individual Motif Occurrence
GATAGATA-type zinc finger protein
GEOGene Expression Omnibus
GUSβ-glucuronidase
MIFMini zinc finger protein
MSUMichigan State University
MYBMyeloblastosis-related transcription factor
PPIProtein–protein interaction
PLACEPlant cis-acting regulatory DNA elements database
RAP-DBRice Annotation Project Database
RGAPRice Genome Annotation Project
RNAiRNA interference
RPBFRice Prolamin Box Binding Factor
S1FSite 1 Factor
SSPSeed storage proteins
STRINGSearch Tool for the Retrieval of Interacting Genes/Proteins
TFTranscription factor
Y2HYeast two-hybrid
ZF-HDZinc finger homeodomain

References

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Plants 14 02791 g001
Figure 1. Co-expression network between transcription factor- and seed storage protein-related genes. The hexagonal boxes represent glutelin proteins, the circular boxes represent prolamin proteins, and the square boxes represent transcription factors. The red lines indicate positive correlation coefficiency, while the blue lines indicate negative correlation coefficiency. The proteins highlighted in red are the selected transcription factors.
Figure 1. Co-expression network between transcription factor- and seed storage protein-related genes. The hexagonal boxes represent glutelin proteins, the circular boxes represent prolamin proteins, and the square boxes represent transcription factors. The red lines indicate positive correlation coefficiency, while the blue lines indicate negative correlation coefficiency. The proteins highlighted in red are the selected transcription factors.
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Figure 2. Schematic representation of the domain predicted to be present in selected TF proteins.
Figure 2. Schematic representation of the domain predicted to be present in selected TF proteins.
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Figure 3. Protein–protein interaction network of each TF. The hexagonal boxes filled with yellow represent the selected TF proteins, and the square-shaped boxes represent proteins predicted to interact with the TF.
Figure 3. Protein–protein interaction network of each TF. The hexagonal boxes filled with yellow represent the selected TF proteins, and the square-shaped boxes represent proteins predicted to interact with the TF.
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Figure 4. Expression patterns of TFs in different tissues and stages. (A) qRT-PCR analysis of TFs. The expression of TFs was normalized to that of OsUBI5. (B) Histochemical assay of GUS activity in developing seeds of transgenic rice plants expressing four TF promoter-GUS fusion constructs. Rice seeds were collected 1–21 days after flowering (DAF).
Figure 4. Expression patterns of TFs in different tissues and stages. (A) qRT-PCR analysis of TFs. The expression of TFs was normalized to that of OsUBI5. (B) Histochemical assay of GUS activity in developing seeds of transgenic rice plants expressing four TF promoter-GUS fusion constructs. Rice seeds were collected 1–21 days after flowering (DAF).
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Figure 5. Distribution of significantly enriched cis-regulatory elements (p ≤ 0.1) in the 2 kb upstream promoter regions of TF genes.
Figure 5. Distribution of significantly enriched cis-regulatory elements (p ≤ 0.1) in the 2 kb upstream promoter regions of TF genes.
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Table 1. Information of TFs selected by correlation analysis.
Table 1. Information of TFs selected by correlation analysis.
SuperclassClassGene NameMSU ID aRAP Locus ID bFunctional DescriptionReference
Zinc fingerGATA typeOsGATA8LOC_Os01g24070Os01g0343300Response salinity, drought stress, ABA[21]
ZF-HD typeOsMIF1LOC_Os11g03420Os11g0128300
OsMIF2LOC_Os12g03110Os12g0124500
C3H typeOsGZF1LOC_Os07g47240Os07g0668600Binding with the GluB-1 promoter and controls glutelin content[18]
Basic domainsBasic leucine zipper factors (bZIP)OsbZIP58, RISBZ1LOC_Os07g08420Os07g0182000Regulate seed storage protein (SSP) and starch synthesis genes during grain filling stage[16,17]
Basic helix–loop–helix factors (bHLH)OsbHLH001, OsICE2LOC_Os01g70310Os01g0928000Response cold stress and regulate stomatal development[22,23]
S1FaS1FaOsS1Fa1LOC_Os04g33420Os04g0408900Response drought stress[24]
OsS1Fa2LOC_Os04g33440Os04g0408700
MYBR2R3 typeOsMYB24LOC_Os01g74590Os01g0977300
a Rice Genome Annotation Project (http://rice.uga.edu/, accessed on 7 August 2025). b The Rice Annotation Project (RAP, https://rapdb.dna.affrc.go.jp/, accessed on 7 August 2025).
Table 2. Enriched cis-regulatory elements in promoters of selected transcription factor genes.
Table 2. Enriched cis-regulatory elements in promoters of selected transcription factor genes.
NO.GeneCis elementConsensus
Sequence (5′-3′)		DescriptionReference
1OsMYB24ABREZMRAB28CCACGTGGABA and water–stress response element[25]
2OsMYB24O2F2BE2S1GCCACCTCATSeed-specific expression element[26]
3OsMYB24RYREPEATGMGY2CATGCATSeed-specific expression element[27]
4OsMYB24RYREPEATVFLEB4CATGCATGABA response and Seed storage protein expression element[28]
5OsbZIP58AMYBOX1TAACARASeed-specific expression element[29]
6OsbZIP58AUXRETGA2GMGH3TGACGTGGCAuxin-responsive element[30]
7OsbZIP58GARE1OSREP1TAACAGASeed-specific expression and GA-responsive element[31]
8OsGATA8-300ELEMENTTGHAAARKSeed storage protein expression element[32]
9OsGZF1SEF4MOTIFGM7SRTTTTTRSeed storage protein expression element[33]
10OsICE2TATABOX1CTATAAATACTATA box-like and Seed-specific expression element[34]
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So, J.; Lee, J.-Y.; Cho, K.; Park, S.; Lee, K.; Kim, D.-K.; Han, O. Transcriptome-Based Identification of Novel Transcription Factors Regulating Seed Storage Proteins in Rice. Plants 2025, 14, 2791. https://doi.org/10.3390/plants14172791

AMA Style

So J, Lee J-Y, Cho K, Park S, Lee K, Kim D-K, Han O. Transcriptome-Based Identification of Novel Transcription Factors Regulating Seed Storage Proteins in Rice. Plants. 2025; 14(17):2791. https://doi.org/10.3390/plants14172791

Chicago/Turabian Style

So, Jinpyo, Jong-Yeol Lee, Kyoungwon Cho, Suchan Park, Kyuhee Lee, Don-Kyu Kim, and Oksoo Han. 2025. "Transcriptome-Based Identification of Novel Transcription Factors Regulating Seed Storage Proteins in Rice" Plants 14, no. 17: 2791. https://doi.org/10.3390/plants14172791

APA Style

So, J., Lee, J.-Y., Cho, K., Park, S., Lee, K., Kim, D.-K., & Han, O. (2025). Transcriptome-Based Identification of Novel Transcription Factors Regulating Seed Storage Proteins in Rice. Plants, 14(17), 2791. https://doi.org/10.3390/plants14172791

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