Dual Transcriptional Circuits: G1-OsMADS34 and G1-TGW2 Cooperatively Regulate Sterile Lemma Identity and Grain Size in Rice

Dual Transcriptional Circuits: G1-OsMADS34 and G1-TGW2 Cooperatively Regulate Sterile Lemma Identity and Grain Size in Rice | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Plant, Cell & Environment This is a preprint and has not been peer reviewed. Data may be preliminary. 2 September 2025 V1 Latest version Share on Dual Transcriptional Circuits: G1-OsMADS34 and G1-TGW2 Cooperatively Regulate Sterile Lemma Identity and Grain Size in Rice Authors : Xuemei Qin , Ping Gan , Jin-Liang Sun , Di Wu , Ru Li , Tianmin Ouyang , Kaichong Teng , Weijian Cen , Baoxiang Qin , Fang Liu , Rongbai Li , and Jijing Luo 0000-0001-5664-0881 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175679682.22419582/v1 Published Plant, Cell & Environment Version of record Peer review timeline 223 views 171 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Rice ( Oryza sativa L.), like other grasses, features a unique organ known as the spikelet within its inflorescence, comprising floret, lemma/palea, sterile lemmas, and rudimentary glumes. Regarding sterile lemma, the molecular regulation underlying its identity remains elusive. Here, we isolated the G1 locus for sterile lemma specification using an F 2 population developed by crossing between Nipponbare and LG7, a variety with a lemma like-sterile lemma ( lsl ). An SNP (+323G/A) in G1 alleles causes a serine to asparagine (S108N) substitution within its ALOG domain, leading to the lsl phenotype. Mechanistically, we found that G1 transactivates the expressions of both OsMADS34 and TGW2 , two genes known to regulate sterile lemma identity and grain size, through binding to the YACTGTW and CArG-box motifs within their promoters, respectively. Intriguingly, the variation in G1 does not disrupt the binding of G1 LG7 (allele from LG7) to both OsMADS34 and TGW2 promoters but affects its transactivation activity. Subsequently, we reveal that the transactivation activity of G1 NIP (allele from Nipponbare) is further enhanced through interactions with either OsMADS34 or TGW2. Furthermore, we demonstrated that G1 specifies sterile lemma identity via OsMADS34 and controls grain size through TGW2 . Our results reveal two transcriptional circuits ( G1 - OsMADS34 and G1 - TGW2 ) that are crucial for determining sterile lemma identity and grain size of rice, providing insights into genetic improvement for breeding programs. Summary statement The isolated G1 gene, carrying an SNP (+323G/A) in its ALOG domain that causes a serine to asparagine substitution (S108N), underlies the lsl phenotype. Mechanistically, G1 transactivates OsMADS34 and TGW2 regulators of sterile lemma identity and grain size by binding to specific motifs (YACTGTW and CArG-box) in their promoters. Although the variant G1 LG7 retains promoter-binding ability, its transactivation activity is impaired. In contrast, G1 NIP exhibits enhanced transactivation through interactions with OsMADS34 or TGW2. We further demonstrate that G1 regulates sterile lemma identity via OsMADS34 and grain size through TGW2, revealing two critical transcriptional circuits coordinating these traits and providing insights for genetic improvement in rice breeding. Dual Transcriptional Circuits: G1-OsMADS34 and G1-TGW2 Cooperatively Regulate Sterile Lemma Identity and Grain Size in Rice Xuemei Qin 1 , Ping Gan 1 , Jinliang Sun 1 , Di Wu 1 , Ru Li 1 , Tianmin Ouyang 1 , Kaichong Teng, Weijian Cen 1 , Baoxiang Qin 1 , Fang Liu 1 , Rongbai Li 1 , Jijing Luo 1✉ 1 College of Life Science and Technology, State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Agriculture, Guangxi University, Nanning 530004, China ✉ Corresponding author: [email protected] The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/) are: Jijing Luo( [email protected] ). ABSTRACT Rice ( Oryza sativa L.), like other grasses, features a unique organ known as the spikelet within its inflorescence, comprising floret, lemma/palea, sterile lemmas, and rudimentary glumes. Regarding sterile lemma, the molecular regulation underlying its identity remains elusive. Here, we isolated the G1 locus for sterile lemma specification using an F 2 population developed by crossing between Nipponbare and LG7, a variety with a lemma like-sterile lemma ( lsl ). An SNP (+323G/A) in G1 alleles causes a serine to asparagine (S108N) substitution within its ALOG domain, leading to the lsl phenotype. Mechanistically, we found that G1 transactivates the expressions of both OsMADS34 and TGW2 , two genes known to regulate sterile lemma identity and grain size, through binding to the YACTGTW and CArG-box motifs within their promoters, respectively. Intriguingly, the variation in G1 does not disrupt the binding of G1 LG7 (allele from LG7) to both OsMADS34 and TGW2 promoters but affects its transactivation activity. Subsequently, we reveal that the transactivation activity of G1 NIP (allele from Nipponbare) is further enhanced through interactions with either OsMADS34 or TGW2. Furthermore, we demonstrated that G1 specifies sterile lemma identity via OsMADS34 and controls grain size through TGW2 . Our results reveal two transcriptional circuits ( G1 - OsMADS34 and G1 - TGW2 ) that are crucial for determining sterile lemma identity and grain size of rice, providing insights into genetic improvement for breeding programs. Summary statement The isolated G1 gene, carrying an SNP (+323G/A) in its ALOG domain that causes a serine to asparagine substitution (S108N), underlies the lsl phenotype. Mechanistically, G1 transactivates OsMADS34 and TGW2 regulators of sterile lemma identity and grain size by binding to specific motifs (YACTGTW and CArG-box) in their promoters. Although the variant G1 LG7 retains promoter-binding ability, its transactivation activity is impaired. In contrast, G1 NIP exhibits enhanced transactivation through interactions with OsMADS34 or TGW2. We further demonstrate that G1 regulates sterile lemma identity via OsMADS34 and grain size through TGW2 , revealing two critical transcriptional circuits coordinating these traits and providing insights for genetic improvement in rice breeding. Keywords: rice; G1 ; lemma-like sterile lemma/grain size regulation 1 INTRODUCTION Rice spikelet, which is a fundamental unit of rice inflorescence, comprises a floret and two empty glumes, also referred to as sterile lemmas. The floret is composed of a lemma, a palea, two lodicules, six stamens, and a carpel ( Yamaki et al., 2011; Lombardo and Yoshida, 2015). Development of rice spikelets is delineated into eight stages (Ikeda et al., 2004). The primordia of rudimentary glumes and sterile lemma emerge from stage Sp1 to Sp2 arranging in a 1/2 alternate pattern. The outer rudimentary glumes are positioned on the adaxial and the inner one on the abaxial sides of the spikelet meristems. In the following stages, the growth of rudimentary glumes is arrested and vestigial. From stages Sp3 to Sp8, lemmas, paleas, lodicules, stamens, carpels, and pollens are sequential formations. So far, more and more attention has been paid to elucidating regulatory mechanisms underlying sterile lemma formation in rice inflorescence development. Sterile lemmas are a common feature across the plants in Gramineae family, suggesting that they might have originated from multiple independent clades (Akiko et al., 2009). In terms of the origin of sterile lemma, building on the “three florets” hypothesis, it has been hypothesized that it originated from the morphologically modified lemmas of the two degenerated lateral florets during the evolution process (Kellogg, 2009; Kobayashi et al., 2010). A number of genes have been documented to regulate sterile lemma development, including LONG STERILE LEMMA ( G1 ), OsMADS34 (Gao et al., 2010; Kobayashi et al., 2010; Lin et al., 2013) , OsMADS1 (Wang et al., 2016) , EXTRA GLUME 1 ( EG1 ) (Li et al., 2009) , INDETERMI NATE SPIKELET 1 ( IDS1 ) (Lee and An, 2011) , MULTI-FLORET SPIKELET 1 ( MFS1 ) (Ren et al., 2013) , NSG1 (Wang et al., 2013; Xu et al., 2020; Zhuang et al., 2020) , and LACKING RUDIMENTARY GLUME 1 ( LRG1 ) (Xu et al., 2020) . Among them, G1 , encoding a DUF640-containing transcription factor with an ALOG ( Arabidopsis LSH1 and Oryza G1) domain, is a critical regulator in the spikelet development of rice, involving in repressing lemma identity to specify the sterile lemma (Akiko et al.,2009). The conserved ALOG domain is unique to G1, and 53 of its residues are highly conserved in the related proteins in rice, Arabidopsis , and Physcomitrella (Akiko et al.,2009). Interestingly, mutation of G1 in rice results in homeotic transformation of sterile lemma to lemma like-sterile lemma (Akiko et al.,2009; Hong et al., 2010; Liu et al., 2016; Yang et al., 2020) . PAP2/OsMADS34 is a gene encoding a functionally diversified SEPALLATA MADS-box protein. It positively regulates spikelet meristem and inflorescence structure (Gao et al., 2010; Ren et al., 2016; Zhu et al., 2021), and was also reported to specify sterile lemma identity in rice (Lin et al., 2013; Meng et al., 2017). Mutation of OsMADS34 not only results in an altered inflorescence but also leads to elongated sterile lemmas with lemma characteristics (Gao et al., 2010; Lin et al., 2013; Ren et al., 2016; Meng et al., 2017). Additionally, double mutant of OsMADS34 and G1 exhibits an additive effect, that is, its sterile lemmas are turned to be even longer and wider lemma/palea-like organs, indicating that G1 and OsMADS34 synergistically regulate sterile lemma development (Lin et al., 2013; Meng et al., 2017). Moreover, AGL1 (also named LAX1 ) determines the developmental fate of sterile lemmas through positively regulating G1 expression (Yu et al., 2024). A recent report showed that LF2, a gene encoding SNF2 family chromatin remodeling factor, functions upstream of G1 to regulate sterile lemma development (Li et al., 2025). Grain size, a critical factor for yield and visual quality of rice, is defined by its length, width, and thickness (Ruan et al., 2020). Several genes/QTLs controlling grain size are well characterized. Majority of them regulate cell proliferation and/or expansion in glumes to control grain size, which in turn significantly impact both yield and quality (Fan et al., 2006; Song et al., 2007; Hu et al., 2015; Li et al., 2019; Bai et al., 2023). TGW2 encodes a cell number regulator ( OsCNR1 ). A single nucleotide polymorphism (SNP) variation within TGW2 promoter reduces its expression and enhances cell proliferation and expansion in glumes, resulting in increased cell width and length (Ruan et al., 2020), indicating that TGW2 acts as negative regulator of grain size. Although genes/QTLs that control sterile lemma development in rice have been extensively reported, the molecular regulatory networks underlying sterile lemma specification remain unclear. Here, through map-based cloning, we identified a new allele of G1 for controlling the lsl phenotype using an F 2 population developed by crossing between two varieties, Nipponbare (NIP, short sterile lemmas/small grains) and LG7 (long sterile lemmas/large grains). An SNP (+323G/A) in G1 ( Os07g0139300 ) coding sequences (CDSs) results in a non-synonymous substitution (S108N) in LG7, leading to homeotic transformation of sterile lemma to lemma like-sterile lemma in rice grains. Here, a molecular regulatory network was revealed, in which G1 regulates the expressions of OsMADS34 and TGW2 to specify sterile lemma development and control grain size in rice. 2 Results 2.1 Identification of a new allele of G1 An F 2 population was generated by crossing between Nipponbare (NIP) and LG7 (Figure 1A-D and Supplementary Figure 1A, B) to isolate the gene underlying the lsl phenotype of rice. The F 2 plants displayed a phenotypic segregation ratio of approximately 1:3 ( lsl /92: normal/268), which fits to the law of Mendelian inheritance (χ 2 ≈ 0.059 < χ 2 0.05 = 3.84), suggesting that the long sterile lemma trait is controlled by a single recessive gene. In line with previous findings (Akiko et al., 2009; Liu et al., 2016), we found that lsl acquired lemma identity based on the cross-sections and scanning electron microscopic (SEM) observations (Figure 1E-K). Compared with NIP, the cross-section revealed that the lsl of LG7 resembles the lemma, in which five vascular bundles were observed (Figure 1E-H). The outer surface of lsl in LG7 also highly resembles that of the lemma and palea of NIP (Figure 1I-K). To isolate the causal gene for lsl phenotype, we identified critical recombinants from a 3,600-individual F 2 population. Genetic linkage analysis was performed, and the locus was consequently mapped to a 24.32-kb region delimited by molecular markers QX-3 and QX-4 on the short arm of chromosome 7 (Figure 1L). In this genomic region, four genes ( Os07g0139000 , Os07g0139150 , Os07g0139300 , and Os07g0139400 ) were annotated (Figure 1L). Among them, Os07g0139300 ( G1 ) is a gene that regulates sterile lemma identity in rice (Akiko et al., 2009). Therefore, we considered it to be one of the most likely candidates for controlling the lsl phenotype. Sequencing analysis identified only an SNP (+323G/A) in CDS of the two alleles ( G1 NIP and G1 LG7 ), resulting in a substitution of serine to asparagine (S108N) in G1 LG7 protein variant (Figure 1M). As expected, lsl phenotype was observed in G1 knockout mutants ( g1-11 and g1-14 ) (Figure 1N and Supplementary Figure 1C), demonstrating that G1 is the causal gene of the locus. Moreover, disruption of G1 significantly increased grain length and width compared to their wildtype (NIP) (Supplementary Figure 2A-C). Grain size is determined by the size of spikelet hulls (Li et al., 2016), which is intrinsically controlled by both cell division and/or expansion in spikelets. To identify causal factors for the phenotypes, the spikelet hulls of G1 knockout mutants were subjected to cross-sectioning and SEM analyses (Supplementary Figure 2D, G). The results showed no significant difference in outer parenchymal cell number or cell length of the outer glumes between NIP and g1 mutants (Supplementary Figure 2E, H). However, the outer parenchymal cell area and cell width of the outer glumes of g1 mutants were significantly greater than in NIP (Supplementary Figure 2F, I). In summary, it is a newly identified variation in G1 for sterile lemma identity in rice, as well, G1 is probably involved in grain size regulation through enhancing cell expansion of the hulls of spikelets. 2.2 G1 encodes a transcription factor with transactivation activity To explore the expression pattern of G1 , we performed qPCR and in situ hybridization assays. Consistent with recent reports (Fang et al., 2024), G1 expression was significantly higher in young panicles less than 1 cm (Figure 2A). In detail, in situ hybridization assay showed that G1 was specifically detected within the sterile lemma primordia in early developmental stages of spikelets (Sp2-Sp8) (Figure 2B). Considering the presence of lsl in both LG7 and G1 knockout mutants, we assumed that the sterile lemma primordia of these two genotypes may have a high meristematic activity during these developmental stages. To validate this possibility, we performed an in situ hybridization assay with Oryza Sativa Homeobox1 ( OSH1 ) gene as a probe, which is often used to indicate meristematic activity of the shoot apical, inflorescence, and axillary meristems (Sato et al., 1996; Yamaguchi et al., 2004; Li et al., 2025). Interestingly, we found that high intensity of OSH1 signal could still be detected within the sterile lemma primordia at the late spikelet developmental stage (Sp8) of LG7 and g1-11 , however, it could not be detected in the corresponding stage of NIP (Supplementary Figure 3A). qPCR analysis confirmed that OSH1 expression was elevated in the young panicles of LG7 and g1-11 compared to NIP (Supplementary Figure 3B). The results demonstrated high meristematic activity of sterile lemma primordia in these two genotypes throughout the early stages of spikelet development, in agreement with their observed elongation of sterile lemmas. It has been reported that G1 is localized to nucleus and has transactivation activity (Akiko et al., 2009; Hong et al., 2010). Consistently, subcellular localization analysis confirmed its localization: both G1 NIP and G1 LG7 proteins were observed to localize in the nucleus (Figure 2C). To test its transactivation activity, a luciferase reporter assay was performed. G1 CDS was fused with the GAL4 DNA binding domain (BD) in the effector vector (Figure 2D). In the reporter vector, luciferase (LUC) gene is driven by GAL4 mini-promoter. LUC activity assessment was performed after co-transformation of the effector and reporter vectors into rice protoplasts prepared from the sheaths of rice seedlings. The results showed that both G1 NIP and G1 LG7 could be detected to significantly enhance the expression of LUC gene to a certain extent compared with that of the BD vector alone (Figure 2E), indicating G1 functions as a transcription factor with transactivation capability. Furthermore, we confirmed the activity in yeast (Figure 2F). Collectively, the results show that G1 is a transcription factor with transactivation activity. 2.3 G1 directly targets OsMADS34 To identify potential binding sites and downstream targets of G1, we performed ChIP (chromatin immunoprecipitation)-seq analysis and successfully identified 3,699 putative G1-binding sites (Figure 3A). These sites showed uniform chromosomal distribution (Supplementary Figure 4), with about 82% localized to promoter regions (-3000 to 0 bp) (Supplementary Figure 5 and Supplementary Table 2), further indicating potential role of G1 in transcriptional regulation. Importantly, motif enrichment analysis revealed significant overrepresentation of the CArG-box core motif (CC(A/T) 6 GG or C(A/T) 8 G) (Figure 3B), while showing no significant enrichment on the YACTGTW motif (Y = T/C, W = A/T) that is previously reported to be recognized by ALOG-domain proteins (Rieu et al., 2024). Given that OsMADS34 mutations result in the lsl phenotype (Lin et al., 2013), we hypothesized that this gene may be a potential target of G1. This hypothesis further supported by identifying OsMADS34 among the common target genes (Figure 3C and Supplementary Table 2). It is worthy of noting that, both the CArG-box and YACTGTW motifs were identified adjacent to each other in OsMADS34 promoter (Figure 3C, E). Subsequent ChIP-qPCR analysis confirmed G1 significant enriched on the regions containing these two motifs (CTTTAAAATG, 34P2 and YACTGTW/CAAATTTG, 34P3 ) (Figure 3D). Consistent with these findings, Y1H and EMSA analyses confirmed the binding of G1. Moreover, the variation in its ALOG domain was found no significant impact on the binding affinity between the two G1 alleles (Figure 3E-G). Mutation analysis showed that deletion of either M1 (∆ M1 (-10 bp)) or M2 ( ∆ M2 (-13 bp)) completely abolished G1 binding activity. However, individual deletion of the motif within M2 (∆ M2-1 (-5 bp) or ∆ M2-2 (-8 bp)) resulted in only partial reduction of the binding affinity. These results demonstrate that both the CArG-box and YACTGTW motifs are essential for G1 binding to its downstream targets (Figure 3E-G). Moreover, OsMADS34 could not bind to its own promoter although it is a MADS-box transcription factor (Gao et al., 2010) (Supplementary Figure 6A-C). Several ALOG proteins were shown to directly bind to the promoter regions of their downstream targets and act as transcriptional repressors (Peng et al., 2017; Huang et al., 2021; Vo Phan et al., 2023). Conversely, dual-luciferase reporter (dual-LUC) assays revealed that G1 acts as a transcriptional activator: G1 NIP significantly transactivated LUC reporter gene expression (Figure 3H, I). Deletion of the motifs (CArG-box: ∆ M1 (-10 bp) and YACTGTW motif: ∆ M2-1 (-5 bp)) within OsMADS34 promoter showed no significant reduction in G1 NIP transactivation activity, while deletion of another CArG-box motif (∆ M2-2 (-8 bp)) exhibited significant attenuation of the activity (Figure 3H, I). Collectively, our results demonstrated that the allelic variation within the ALOG domain specifically compromises the transactivation activity of G1, without affecting its DNA-binding affinity to the downstream target OsMADS34 . More importantly, our findings reveal that G1 NIP exerts its transactivation function primarily through recognition and binding to the CArG-box motif (caaatttg). To further confirm the role of G1 in regulating OsMADS34 expression , we performed qPCR and in situ hybridization assays. Expression analysis demonstrated that OsMADS34 expression levels in the young panicles were remarkably reduced in g1 mutant ( g1-11 ) compared to those of NIP (Supplementary Figure 6E). In contrast, in the in situ hybridization assay, G1 expression signal detected in the sterile lemma primordia displayed a similar pattern between osmads34-3 mutant (Supplementary Figure 6D) and NIP throughout Sp2-Sp8 developmental stages (Figures 2B and 3J). These further indicate that G1 acts as an upstream regulator of OsMADS34 . Accordingly, attenuated OsMADS34 signal was observed in the sterile lemma primordia of both LG7 and g1-11 spikelets (Figure 3K-M). Moreover, OsMADS34 and G1 exhibited distinct spatiotemporal expression patterns, consistent with previous reports (Lin et al., 2013), OsMADS34 showed strong expression in Sp2-5 stage spikelet primordia (including rudimentary glumes, sterile lemmas, and other floral organ precursors), while its expression became restricted to stamens and carpel by Sp7-Sp8 stages (Figure 3K). These findings collectively demonstrate that G1 and OsMADS34 coordinately regulate sterile lemma development. 2.4 Interaction between G1 and OsMADS34 enhances G1 transactivation activity To gain further insight into the regulatory network of G1 , we identified G1-interacting proteins through yeast two-hybrid screening. Among the identified genes, once again, OsMADS34 attracted our attention and was confirmed to physically interact with G1 in yeast (Figure 4A). Then, the interaction in vivo was further confirmed through luciferase complementation imaging (LCI), bimolecular fluorescence complementation (BiFC), and Co-immunoprecipitation (Co-IP) assays (Figure 4B-E). Based on the results described above, we speculated that the interaction of G1 with OsMADS34 might have specific effect on the transactivation activity of G1 . To validate this, we performed a dual-LUC assay. The results showed that, in contrast to co-expression of G1 with the reporter alone (b+e), G1 NIP (b+d+e), but not G1 LG7 (c+d+e), could significantly activate the expression of LUC reporter gene in rice protoplasts (Figure 4F-G) and in tobacco leaves (Figure 4H) when co-expressed with OsMADS34 . Collectively, these findings suggest that a positive feedback loop is involved in sterile lemma identity. In this loop, G1 transactivates OsMADS34 expression , and the activity is further enhanced by interacting with OsMADS34. However, the allelic variation of G1 ( G1 LG7 ) impairs the transactivation activity and further leads to a decreased OsMADS34 expression, thereby resulting in lsl phenotype. 2.5 G1 and OsMADS34 synergistically regulate sterile lemma development To elucidate the genetic regulatory relationship between G1 and OsMADS34 , we constructed double mutants of G1 and OsMADS34 . Phenotypic analysis showed that the sterile lemma ( lsl ) length of g1-11 was significantly greater than that of osmads34-3 (Figure 5A-C, G). In the g1/osmads34 double mutant (Supplementary Figure 8A) spikelets, the sterile lemma length was comparable to that of osmads34-3 (Figure 5B-E, G), indicating that G1 function depends on the presence of OsMADS34 . Paraffin sections revealed that the sterile lemmas of g1/osmads34 possessed five vascular bundles (Figure 5E), further confirming that the double mutant phenotype is primarily determined by OsMADS34 . When OsMADS34 was overexpressed in g1-11 mutant (designated as g1/OE-OsMADS34 ) (Supplementary Figure 8A, B), its sterile lemma was significantly shorter than those of the g1-11 and g1/osmads34 double mutant, demonstrating that OsMADS34 overexpression can partially rescue the lsl phenotype resulted from mutation of G1 (Figure 5F-G). These results indicate that OsMADS34 serves as one of a key downstream target of G1 to regulate sterile lemma development in rice. 2.6 G1 - TGW2 forms another transcriptional circuit to regulate grain size In the above-mentioned ChIP-seq analysis, TGW2 was also identified as a potential G1 target (Figure 6A). Further ChIP-PCR analysis showed that G1 also significant enriched on the promoter regions of TGW2 , which contain both CArG-box ( WP2 ) and YACTGTW ( WP3 ) motifs (Figure 6B), and the interaction was further confirmed through Y1H assays (Figure 6C). Remarkably, in agreement with the pattern observed for OsMADS34 , EMSA assays also demonstrated that G1 specifically binds to both the CArG-box and YACTGTW motifs, which was evidence by motif deletion analysis (∆ T1-1 (-9bp) , ∆ T1-2 (-8bp) , and ∆ T2 (-5bp)) (Figure 6D, E), conclusively establishing two motifs are essential for G1’s regulatory function. Previous studies have demonstrated that TGW2 interacts with KRP1, a regulator of cell cycle in plants, to negatively regulate grain width and weight (Ruan et al., 2020). To investigate the role of the G1 protein in regulating TGW2 expression, we conducted dual-LUC assays using rice protoplasts. The results demonstrated that G1 NIP significantly activated TGW2 expression, while the activation capability of G1 LG7 completely abolished (Figure 6F, G). Motif mutation analysis revealed that deletion of the CArG-box motifs (either ∆ T1-1 (-9bp) or ∆ T1-2 (-8bp)) markedly reduced TGW2 transcriptional levels, whereas deletion of the YACTGTW motif (∆ T2 (-5bp)) showed no significant reduction of its expression level (Figure 6G). These findings demonstrate that G1 protein regulates TGW2 expression through binding to the CArG-box motif within TGW2 promoter. Consistently, in situ hybridization and qPCR confirmed reduced TGW2 expression was observed in LG7 and g1-11 lines, of which contain low-transactivation activity of G1 protein variants (Figure 6H and Supplementary Figure 7). To elucidate the genetic relationship between G1 and TGW2 , we overexpressed TGW2 in g1-11 mutant (Supplementary Figure 8A, C). Strikingly, compared with the g1-11 control, the g1/OE-TGW2 plants exhibited significantly smaller grains (Figure 7A-F). These results collectively demonstrate that G1 regulates rice grain development by positively regulating TGW2 expression. 2.7 G1 increases grain size and yield potential Given that G1 not only regulates rice sterile lemma identity but also controls grain size, we investigated the agronomic traits of NIP, g1-11 , g1-14, OE-G1-1 (Supplementary Figure 1C-F) , and g1/OE-TGW2 (Supplementary Figure 8A, C) under natural field conditions. The results showed that the grain width, brown grain length, and brown grain width of OE-G1-1 and g1/OE-TGW2 rice plants significantly reduced compared with g1 mutant, exhibiting a comparable grain size to that of NIP (Figure 7A, B, D and F). Moreover, the size of panicles, 1000-grain area (Figure 7G), brown 1000-grain weight, grain yield per panicle, panicle length and grain yield per plot of the two knockout mutants were considerably higher than those of the wild-type NIP (Figure 7G-K), while in the traits such as grain number per panicle, no of primary branches, and seed setting rate did not show significant difference in these three genotypes (Supplementary Figure 9A-C). This implies that disruption of G1 could increase rice grain size, thereby increasing grain yield of the mutants. Thus, G1 is a gene with potential value for increasing rice yield in breeding. 3 DISCUSSION In this study, we elucidate two transcriptional circuits orchestrated by G1 that controls sterile lemma identity and grain size development in rice through the transcriptional activation of OsMADS34 and TGW2 . The identification of a novel G1 allele ( G1 LG7 ) with S108N substitution in its ALOG domain not only expands the genetic basis of floral organ specification but also provides mechanistic insights into how transcriptional feedback loops integrate developmental and agronomic traits. 3.1 G1 acts as Y ACTGTW and CArG-box motif-binding transcription factor G1 is a major gene for specifying sterile lemma identity in rice, which is a member of the plant-specific ALOG gene family (Akiko et al., 2009). It has been reported that both G1 and OsMADS34 play key roles in controlling rice sterile lemma development (Jeon et al., 2000; Akiko et al., 2009; Ren et al., 2016). Here, we further unveiled the regulatory relationships between G1 and OsMADS34 , of which G1 primarily binds to the CArG-box motif within OsMADS34 promoter to transactivate the expression of OsMADS34 although G1 could directly bind to both the YACTGTW and CArG-box motifs in the promoter (Figures 3 and 4). Similar regulatory pattern was also observed in the regulatory circuit of G1 - TGW2 (Figure 6). The identification of these regulatory modules represents a paradigm shift in understanding floral organ specification. While MADS-box transcription factors are classically associated with floral organ identity (Käppel et al., 2018), our findings demonstrate that G1, a non-MADS transcription factor, directly binds CArG-box motif, a hallmark of MADS-domain protein binding site, to activate OsMADS34 and TGW2 expressions. This finding challenges the paradigm that CArG-box recognition is exclusive to MADS-box proteins and suggests convergent evolution in DNA-binding mechanisms. Therefore, G1 is a non-MADS gene that is identified as a CArG-box binding transcription factor in controlling floral organ specification in rice. 3.2 Dual regulatory axes of G1 for trait coordination In the study, we reveal that G1 functions upstream of both OsMADS34 and TGW2 (Figures 3 and 6). Meanwhile, OsMADS34 could physically interact with G1 to form a complex (Figure 4A-E). The interaction further significantly inhibits G1 -mediated transactivation of OsMADS34 (Figure 4F-H). Furthermore, G1 specifies sterile lemma identity via OsMADS34 and controls grain size through TGW2 . These indicate dual regulatory axes mediated by G1 for rice trait coordination. The bifurcation of G1 function addresses a question about how floral development and yield traits are co-regulated. The identified G1 - OsMADS34 / TGW2 positive feedback loops (Figures 3 and 6) highlight a novel mechanism which a single transcription factor coordinates distinct developmental programs through target-specific interactions. Furthermore, the observation of partial rescue of the lsl phenotype in g1 /OE- OsMADS34 double mutants (Figure 5D-G) suggests functional redundancy of G1 downstream targets, likely involving more additional G1 targets, such as OsMADS1 (Fang et al., 2024), to specify sterile lemma identify during the early inflorescence development stages. Therefore, it is worthy of our further analysis. Crucially, the G1 LG7 allele decouples these processes: it retains target promoter binding activity (Figures 3E-G and 6C-E), but fails to transactivate its downstream targets (Figures 3H, I and 6F, G), leading to enlarged grains without compromising panicle architecture of rice (Figure 7 and Supplementary Figure 9). This functional plasticity positions G1 as a prime candidate for breeding programs aiming to improve yield without sacrificing floral integrity. 3.3 Mechanistic insight into G1 -mediated sterile lemma specification and grain size modulation in rice Previous studies have proposed parallel roles for G1 and OsMADS34 in sterile lemma development (Gao et al., 2010). However, our results indicate that, in early spikelet developmental stages, the upregulation of G1 within the sterile lemma primordia transactivates OsMADS34 expression, and the translated protein OsMADS34 interacts with G1 to form complexes to reinforce the transactivation to specify the sterile lemma identity. To our knowledge, the elucidation of the genetic hierarchy and protein interactions (Figures 3 and 4A-E) in our study unequivocally positions G1 as an upstream activator, thereby resolving apparent contradictions. Notably, G1’s ability to form a positive feedback loop with both OsMADS34 and TGW2 (Figure 3H, I) demonstrates its role as a transcriptional amplifier, a mechanism reminiscent of bacterial signaling circuits (Miyashiro and Goulian, 2008) but rarely documented in plants. The regulatory hierarchy clarified here not only solidifies the role of G1 as a key regulator in controlling sterile lemma identity through OsMADS34 and grain size via TGW2 but also provides a coherent framework that integrates these regulatory relationships. 4 Conclusion Here, based on our results, we pictured a regulatory landscape for the regulation of sterile lemma development and grain size in rice. In this regulatory network, G1 functions upstream of OsMADS34 and TGW2 . G1 - OsMADS34 forms a positive feedback regulatory module to enhance OsMADS34 expression in specifying sterile lemma identity, while G1 forms another positive feedback regulatory module with TGW2 to regulate TGW2 expression to control rice grain size. Our findings clearly establish G1 functioning as an upstream activator that influences its downstream targets, thus offering new insights into genetic regulation within this important food crop species. 5 Materials and Methods 5.1 Plant materials The mapping population was generated by crossing Nipponbare with LG7, a variety exhibiting the lsl phenotype, and was used to clone the G1 gene. Nipponbare, LG7, transgenic plants, and the critical recombinants were used for phenotypic investigation, in situ analysis, and genetic analysis. 5.2 Map-based cloning To identify the genetic locus underlying long sterile lemma, an F 2 population of 3,600 plants was generated. Molecular markers were obtained from the public databases Rice Genomic Research Program and Gramene (https://www.gramene.org/). Primer sequences for the molecular markers are listed in Table S1. Informative molecular markers were used for genotyping, and critical recombinants were identified. Genetic linkage analysis was performed to fine map the locus underlying lsl phenotype. Candidates for the locus were determined by referencing the genomic annotation of Nipponbare, and sequence variations in the locus were identified by sequencing the corresponding alleles from Nipponbare and LG7. All the constructed plasmids are listed in Supplementary Table 1. 5.3 Plasmid construction and genetic transformation To generate overexpression constructs, the coding sequence (CDS) of G1 was cloned into the pCAMBIA1300 vector to construct pCAMBIA1300- 35S::G1-GFP , while the OsMADS34 and TGW2 CDS were inserted into the pRHVnGFP vector (He et al., 2018) to construct Ubi::OsMADS34-GFP and Ubi::TGW2-GFP, respectively. G1 and OsMADS34 disruption was achieved using the CRISPR/Cas9 technology as reported previously (Lei et al., 2014; Ma et al., 2015; Liu et al., 2017; Sun et al., 2019; He et al., 2021). The gRNA sequences were cloned into the BsaI restriction sites of the CRISPR/Cas9 plasmid pYLCRISPR/Cas9 - MH. These plasmids were introduced into corresponding recipients via Agrobacterium tumefaciens mediated transformation (Hiei et al., 2003). 5.4 Histological and cytological analysis For paraffin section, the spikelets of NIP, LG7, g1-11 knockout mutants, osmads34-3 knockout mutants, and g1/osmads34 double mutants were collected before flowering and were fixed in Formalin-Acetic-Alcohol (FAA) solution (a mixture of 50 % ethanol, 5 % formaldehyde, and 5 % glacial acetic acid) for more than three days. The fixed samples were then subjected to dehydration using a series of graded ethanol solutions (30%, 40%, 50%, 60%, 75%, 80%, 95%, 100%), cleared in xylene series (50%, 60%, 75%, 80%, 95%, 100%), and embedded in paraffin (Sigma P3683, Germany). Using a microtome, the samples were sectioned to a thickness of 12 μm (Leica RM2235, Germany). The sections were stained with toluidine blue, and observed under a microscope (IMAGER ZI, Germany). The surface of NIP, LG7, and g1-11 sterile lemmas was imaged using a scanning electron microscope (FEI QRATRO, USA). The number and area of parenchyma cells, as well as cell length and width of the outer glumes, were then determined using the ImageJ application (Caroline et al., 2012). 5.5 RNA extraction and qRT–PCR Total RNA was isolated from the leaves, shoots, roots, and young panicles of NIP, LG7 and g1-11 . First-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R312, China). qRT-PCR reactions were performed on the LIGHTCYCLE 480 II using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711, China). Rice ACTIN ( Os03g0718100 ) was used as an internal reference to normalize expression levels. Relative expression levels were determined using the 2 -∆∆Ct method, as described previously (Livak and Schmittgen, 2001), with each analysis conducted in triplicate for both technical and biological replicates. 5.6 Subcellular localization We cloned the full-length CDSs (without the stop codon) of G1 into the pRTVnGFP vector (He et al., 2018). The resulting constructs were introduced into rice protoplasts (young rice leaf sheaths) using a polyethylene glycol-mediated transient expression system, as previously described (Shenng et al., 2014). GFP signal was observed using a laser confocal microscope (LEICA TCS-SP8MP, Germany) with excitation at 488 nm and 552 nm wavelengths, respectively. 5.7 In situ hybridization Samples were freshly collected and fixed in freshly prepared glutaraldehyde-based fixative (2.5% glutaraldehyde in PBS containing 0.1% Tween-20 and 0.1% Triton X-100, v/v) solution stored at 4 °C, followed by dehydration through a graded ethanol series, xylene infiltration, and embedding in Paraplast Plus. The samples were then sectioned into 12-mm thick slices using a rotary microtome (Leica, RM2245, Germany). Probes for G1 , OsMADS34 , TGW2 , and OSH1 were synthesized for hybridization according to previously described methods (Akiko et al., 2009; Gao et al., 2010; Hu et al., 2015). The Digoxigenin (DIG)-labeled kit was used to transcribe the probes in vitro using either the T7 or T3 promoter with RNA polymerase (Roche, 11175025910, Switzerland). Hybridization of digoxygenin-labeled RNA and detection of the hybridized probe were performed according to the previous protocol (Zhang et al., 2017). A hybridization signal was detected under a microscope. 5.8 Transcriptional and transactivation activity assays Transcriptional activity assays were conducted in rice protoplasts (Guo et al., 2013). In this system, the GAL4 DNA-binding domain (BD) was utilized. Effector plasmids with in-frame fusions of BD and G1 and OsMADS34 were constructed, respectively. The pUbiquitin::REN reporter plasmid was used as an internal control. HOS15 and ARF5M served as transcription suppression and activation controls, respectively. The Firefly (LUC) and Renilla (REN) Luciferase reporter plasmids were co-transformed respectively with the BD-G1 and BD-OsMADS34 effector plasmids into rice protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured using a GloMax 20/20 luminometer (Promega, Madison WI) according to the manufacturer’s protocol (LABLEAD, DR075), with three biological replicates. LUC activity was used to assess the transcriptional effects of the effector, while REN activity served as an internal control for normalization to minimize experimental variability. For transactivation assay in yeast cells, the bait plasmid pGBKT7 containing G1 CDS was introduced into the yeast AH109 strain. Transformed yeast cells were first cultured on synthetic dropout (SD) medium plates lacking tryptophan (SD/-Trp) at 30 °C for 3 days, and then transferred to SD agar medium lacking tryptophan, histidine, and adenine (SD/-Trp/-His/-Ade). Transactivation activity was assessed based on yeast growth status and β -galactosidase activity. 5.9 Yeast one-hybrid and two-hybrid assays For yeast two-hybrid assay (Y2H), the full-length or truncated CDSs of G1 gene were ligated into the EcoRI / BamHI restriction sites of the pGBKT7 vector (In-Fusion ® HD Cloning, Takara, Japan) to generate the pGBKT7- G1 and pGADT7- OsMADS34 constructs. The resulting plasmids, or a control plasmid were co-transformed into yeast Y2H Golden strain. The transformed yeast cells were grown on the SD/-Trp and SD/-Trp/-His/-Ade medium with 100 µg/mL AbA (Aureobasidin A) and 200 mg/L X - gal (5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside). The plates were incubated at 30 °C for 3 days. For yeast one-hybrid assay (Y1H), we amplified the promoter regions of OsMADS34 ( pMADS34 -1: -2232 to -1417 bp and pMADS34 -2: -864 to -238 bp) and TGW2 ( pTGW2 -1: -2054 to -1463 bp, pTGW2 -2: -1399 to -809 bp, and pTGW2 -3: -808 to 0 bp) by PCR using genomic DNA of Nipponbare as a template. The amplified PCR products were inserted into the pHIS2 vector, which is upstream of HIS3 reporter gene, to generate the pHIS- pOsMADS34 and pHIS- pTGW2 constructs. The G1 CDS was cloned into pGADT7 and fused in-frame with its activation domain (AD) to construct the pGADT7- G1 . The plasmid pairs (pHIS2- pOsMADS34 /pGADT7- G1 , pHIS2- pTGW2 /pGADT7- G1 , and the control pHIS2/pGADT7) were co-transformed into yeast AH109 strain. The transformed yeast cells were cultured on the SD/-Leu/-Trp and SD/-Leu/-Trp/-His+50 mM 3-AT (3-amino-1, 2, 4-triazole) plates at 30 °C for 3-4 days. 5.10 Bimolecular fluorescence complementation (BiFC) assay For the BiFC assay in tobacco (N. benthamiana) leaves , the coding sequences (CDSs) of G1 and OsMADS34 were cloned into the pRHVn-mVenus-N and pRHVn-mVenus-C vectors (He et al., 2018), respectively. The resulting constructs were introduced into Agrobacterium strain GV3101, which was used for co-infiltrating into tobacco leaves. GFP signal was detected using a laser confocal microscope (LEICA-TCS-SP8MP, Germany). 5.11 Luciferase complementation imaging (LCI) assay The LCI assay was performed as described previously (Chen et al., 2008), the CDS of G1 was inserted into the plasmid pRHVc-cLUC, and the CDSs of OsMADS34 were fused in-frame with nLUC in the plasmid pRHVc-nLUC, respectively, to examine the interaction of G1 with either OsMADS34 . The resulting constructs were co-infiltrated into tobacco leaves and cultured for 54 h. Dual-Luciferase (Abcam, Ab143655, UK) was sprayed onto the surface of tobacco leaves. LUC signal was detected using a Plant In Vivo Imaging System (IVISL ® umina LT). 5.12 Co-immunoprecipitation (Co-IP) assay To validate the interaction between G1 and OsMADS34, we co-expressed G1-HA with OsMADS34-GFP under the control of the ubiquitin ( Ubi ) promoter in a transient expression assay. After co-transformation of the plasmids, rice protoplasts were harvested by centrifugating at 200 g for 5 minutes after 16-hour incubation. The collected protoplasts were lysed with CelLytic IP buffer (Biosharp; BL509A; China) and incubated on ice for 30 minutes, followed by centrifugation at 13,000 g for 10 minutes at 4 °C to eliminate aggregates. The supernatant was incubated with 1 µL of GFP-tag antibody (Thermo Fisher Scientific, LOT2339829, USA) for 8 hours at 4 °C with gentle shaking (40-50 rpm). Protein A/G Agarose beads (LABLEAD; P0233; China) were added and incubated for 3 hours at 4 °C with shaking. The beads were collected by centrifugation at 200 g for 1 minute at 4 °C and washed five times with ice-cold CelLytic IP buffer. Proteins were eluted from the beads by boiling in the SDS-PAGE sample buffer for 10 minutes and analyzed via Western blotting. 5.13 Dual-Luciferase (Dual-LUC) transient expression assay Fragments (2000 bp) of the OsMADS34 , OsMADS34 ∆ M1 (∆ M1 : ctttaaaatg deletion), OsMADS34 ∆ M2-1 (∆ M2-1 : actgt deletion), OsMADS34 ∆ M2-2 (∆ M2-2 : caaatttg deletion), TGW2, TGW2 ∆ T1-1 (∆ T1-1 : cattattag deletion), TGW2 ∆ T1-2 (∆ T1-2 : cattaatg deletion) and TGW2 ∆ T2 (∆ T2 : actgt deletion) promoters were amplified and inserted into the pGreen II0800-LUC vector. The CDSs of G1 , OsMADS34 , and TGW2 (excluding stop codons) were amplified and cloned into the pRTVnGFP vector (He et al., 2018) as effectors. These resulting constructs were then introduced into rice protoplasts. Equal amounts of plasmid were introduced into rice protoplasts. Relative LUC activity (LUC/REN) was calculated to assess Dual-LUC transient expression assay. 5.14 Electrophoretic mobility shift assay (EMSA) The unlabeled and biotin-labeled DNA probes were synthesized by Nanning GenSys biotechnology (www.gensysbio.com). The GST-G1 NIP and GST-G1 LG7 proteins were isolated and purified as described above. EMSA/Gel-shift binding buffer (Beyotime, GS005, China) was used for carrying out protein-DNA binding reactions at room temperature (RT) for 30 minutes. A 6% native polyacrylamide gel was then used to separate the reaction samples. The separated samples were transferred to a nylon membrane. The signal was detected using a chemiluminescent EMSA Kit (Beyotime, GS009, China). The binding specificity of the protein-DNA interaction was determined by adding varying amounts of the unlabeled probes. GST protein served as a negative control in the experiments. 5.15 Chromatin immunoprecipitation (ChIP) assay Following the method described previously (Zhao et al., 2020), approximately 2 g of young panicles (1-2 cm) of G1 overexpression plants were collected and subjected to cross-linking in a 1% formaldehyde solution under vacuum for 30 minutes. To avoid the presence of air bubbles, non-contact ultrasonic interruptions were made at high power settings (30s on, 60s off, 6 cycles), and the chromatin was fragmented at 4 ℃. Chromatin immunoprecipitations (IPs) were carried out with 2 ug GFP-tag antibody (Thermo Fisher Scientific, LOT2339829, USA) at 4 ℃ overnight. The IPs without antibody applied were served as the negative control. The bound DNA was isolated and then used for ChIP-qPCR. Meanwhile, the eluted DNA was used to construct the sequencing library with the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB, E7335S, E7645S, USA). The ChIP-seq DNA libraries were sequenced using the Illumina NovaSeq platform. For ChIP-seq data analysis, MACS2 software (Awdeh et al., 2021) was used for peak calling across the genome, with a P < 0.01 for screening significant peaks. The images were visualized using IGV 2.18.4 software and R package ChIPseeker (Thorvaldsdottir et al., 2012; Yu et al., 2015). Motif enrichment analysis among G1 binding peaks was performed using Homer (Fan et al., 2022). 5.16 Analysis of the performance of agronomic traits A series of agronomic traits were assessed based on the field trials on the campus of Guangxi University (Nanning, Guangxi, China). These included the number of grains per panicle, 1000-grain weight, seed-setting rate, panicle length, number of primary branches per panicle, and grain yield per plot. The seed-setting rate was determined by manually separating the filled and unfilled grains from the main panicle. The rate was calculated using the formula: (filled grain number / (filled grain number + unfilled grain number)) × 100 %. The grain yield per plant was measured after all filled grains from each plant dried at 42 °C in an oven. A random sample of filled grains was used to determine the 1,000-grain weight. The area of each plot was 100 cm × 100 cm, and all grains from the plot were collected to evaluate the yield of plot trials. Acknowledgments We thank Dr. Zhongquan Cai (Guangxi University) for kindly providing the variety LG7 for G1 cloning. We thank Prof. Yuese Ning (Institute of Plant Protection, CAAS, China) for kindly providing the plasmids for the study. We highly appreciate the editors and reviewers for reviewing the manuscript and for their insightful comments. Funding This work was supported by the Natural Science Foundation of Guangxi Province (2024GXNSFGA010003, Guike-AD25069107, 2018GXNSFDA138004), the National Natural Science Foundation of China (CN) (32260446, 31671646), Guangxi Central Guidance for Local Science and Technology Development Funds (GuiKe-ZY23055027), and the State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources (SKLCUSA-a08). Author contributions J. L conceived and designed the studies. X. Q, P. G, J. S, D. W and K. T performed all the lab experiments. X. Q carried out the data processing. J. L and X. Q drafted the manuscript. J. L, X. Q, J. S, R.B. L, T. OY, B. Q, F. L, R. L revised the draft. All the authors approved the final version of the manuscript. Conflict of interest statement. The authors declare that they have no competing interests. REFERENCES T. SuzakiW. Tanaka H. Y. Hirano Akiko, Y.,. 2009. “The homeotic gene long sterile lemma ( G1 ) specifies sterile lemma identity in the rice spikelet.” Proceedings of the National Academy of Sciences of the United States of America 106:20103-20108.Awdeh, A., M. Turcotte, T. J. Perkins. 2021. “WACS: improving ChIP-seq peak calling by optimally weighting controls.” BMC Bioinformatics 22:69.Bai, F., H. Ma, Y. Cai, M. Q. Shahid, Y. Zheng, C. Lang, Z. Chen, J. Wu, X. Liu, L. Wang. 2023. “Natural allelic variation in GRAIN SIZE AND WEIGHT 3 of wild rice regulates the grain size and weight.” Plant Physiology 193:502-518.Caroline, A., W. S. Rasband, K. W. Eliceiri. 2012. “NIH Image to ImageJ: 25 years of image analysis.” Nature Methods 9:671-675.Chen, H., Y. Zou, Y. Shang, H. Lin, Y. Wang, R. Cai, X. Tang, J. M. Zhou. 2008. “Firefly luciferase complementation imaging assay for protein-protein interactions in plants.” Plant Physiology 146:368-376.Fan, C., Y. Xing, H. Mao, T. Lu, B. Han, C. Xu, X. Li, Q. Zhang. 2006. “ GS3 , a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein.” Theoretical and Applied Genetics 112:1164-1171.Fan, G., Z. Tao, S. Chen, H. Zhang, R. Yu. 2022. “Positive allosteric regulation of PAC1-R up-regulates PAC1-R and its specific ligand PACAP .” Acta Biochimica et Biophysica Sinica 54:657-672.Fang, H., H. Chen, J. Wang, N. Li, L. Zhang, C. Wei. 2024. “ G1 interacts with OsMADS1 to regulate the development of the sterile lemma in rice.” Plants 13:505.Gao X, W Liang, C. Yin, S. Ji, H. Wang, X. Su, C. Guo, H. Kong, H. Xue, D. Zhang. 2010. “The SEPALLATA -Like gene OsMADS34 is required for rice inflorescence and spikelet development.” Plant Physiology 153:728-740.Guo, S., Y. Xu, H. Liu, Z. Mao, C. Zhang, Y. Ma, Q. Zhang, Z. Meng, K. Chong. 2013. “The interaction between OsMADS57 and OsTB1 modulates rice tillering via DWARF14 .” Nature Communications 4:1566.He, C., H. Liu, D. Chen, W. Z. Xie, M. Wang, Y. Li, X. Gong, W. Yan, L. L. Chen. 2021. “CRISPR‐Cereal: a guide RNA design tool integrating regulome and genomic variation for wheat, maize and rice.” Plant Biotechnol Journal 19:2141-2143.He, F., F. Zhang, W. Sun, Y. Ning, G. L. Wang. 2018. “A versatile vector toolkit for functional analysis of rice genes.” Rice 11:27.Hiei, Y., S. Ohta, T. Komari, T. Kumashiro. 2003. “Efficient transformation of rice ( Oryza sativa L .) mediated by Agrobacterium and sequence analysis of the boundaries of the T‐DNA.” The Plant Journal 6:271-282.Hong, L., Q. Qian, K. Zhu, D. Tang, Z. Huang, L. Gao, M. Li, M. Gu, Z. Cheng. 2010. “ ELE restrains empty glumes from developing into lemmas.” Journal of Genetics and Genomics 37:101-115.Hu, J., Y. Wang, Y. Fang, L. Zeng, J. Xu, H. Yu, Z. Shi, J. Pan, D. Zhang, S. Kang, L. Zhu, G. Dong, L. Guo, D. Zeng, G. Zhang, L. Xie, G. Xiong, J. Li, Q. Qian. 2015. “A rare allele of GS2 enhances grain size and grain yield in rice.” Molecular Plant 8:1455-1465.Hu, Y., W. Liang, C. Yin, X. Yang, B. Ping, A. Li, R. Jia, M. Chen, Z. Luo, Q. Cai, X. Zhao, D. Zhang, Z. Yuan. 2015. “Interactions of OsMADS1 with floral homeotic genes in rice flower development.” Molecular Plant 8:1366-1384.Huang, X., S. Chen, W. Li, L. Tang, Y. Zhang, N. Yang, Y. Zou, X. Zhai, N. Xiao, W. Liu, P. Li, C. Xu. 2021. “ROS regulated reversible protein phase separation synchronizes plant flowering.” Nature Chemical Biology 17 : 549-557.Jeon, J.S., S. Jang, J. Nam, C. Kim, Gynheung. An. 2000. “ Leafy hull sterile 1 is a homeotic mutation in a rice MADS Box gene affecting rice flower.” The Plant Cell 12:871-884.Shen, J., J. Fu, M. Jin, X. Wang, C. Gao, C. X. Zhuang, J. Wan, L. Jiang. 2014. “Isolation, culture, and transient transformation of plant protoplasts.” Current Protocols in Cell Biology 63 : 2.8.1-17.Käppel, S., R. Melzer, F. Rümpler, C. Gafert, G. Theißen. 2018. “The floral homeotic protein SEPALLATA3 recognizes target DNA sequences by shape readout involving a conserved arginine residue in the MADS‐domain.” The Plant Journal 95:341-357.Kellogg, E. A. 2009. “The evolutionary history of ehrhartoideae, Oryzeae , and Oryza .” Rice 2:1-14.Kobayashi, K., M. Maekawa, A. Miyao, H. Hirochika, J. Kyozuka. 2010. “ PANICLE PHYTOMER2 ( PAP2 ), encoding a SEPALLATA subfamily MADS-box protein, positively controls spikelet meristem identity in rice.” Plant Cell Physiology 51:47-57.Ikeda K, H., H. Sunohara, Y. Nagato. 2004. “Developmental course of inflorescence and spikelet in rice.” Breeding Science 54:147-156.Lee, D. Y., G. An. 2011. “Two AP2 family genes, SUPERNUMERARY BRACT ( SNB ) and OsINDETERMINATE SPIKELET 1 ( OsIDS1 ), synergistically control inflorescence architecture and floral meristem establishment in rice.” The Plant Journal 69:445-461.Lei, Y., L. Lu, H. Y. Liu, S. Li, F. Xing, L. L. Chen. 2014. “CRISPR-P: A web tool for synthetic single-guide RNA design of CRISPR-System in plants.” Molecular Plant 7:1494-1496.Li, H., D. Xue, Z. Gao, M. Yan, W. Xu, Z. Xing, D. Huang, Q. Qian, Y. Xue. 2009. “A putative lipase gene EXTRA GLUME1 regulates both empty‐glume fate and spikelet development in rice.” The Plant Journal 57:593-605.Li, N., Y. Li. 2016. “Signaling pathways of seed size control in plants.” Current Opinion in Plant Biology 33:23-32.Li, N., R. Xu, Y. Li. 2019. “Molecular networks of seed size control in plants.” Annual Review of Plant Biology 70:435-463.Li, Z., Z. Wang, C. Wang, X. Zeng, J. Tang, H. Zhu, H. Lin, S. Zhu, Y. Li, P. Yao, Y. Gao, G. He, H. Zhuang, Y. Li. 2025. “LATERAL FLORET 2 encoding a sucrose non-fermenting 2 chromatin remodeling factor regulates axillary meristem of spikelet development in rice ( Oryza sativa ).” New Phytologist 246 : 598-615.Lin, X., F. Wu, X. Du, X. Shi, Y. Liu, S. Liu, Y. Hu, G. Theißen, Z. Meng. 2013. “The pleiotropic SEPALLATA ‐like gene OsMADS34 reveals that the ‘empty glumes’ of rice ( Oryza sativa ) spikelets are in fact rudimentary lemmas.” New Phytologist 202:689-702.Liu, H., Y. Ding, Y. Zhou, W. Jin, K. Xie, L.L. Chen. 2017. “CRISPR-P 2.0: An improved CRISPR-Cas9 tool for genome editing in plants.” Molecular Plant 10:530-532.Liu, M., H. Li, Y. Su, W. Li, C. Shi. 2016. “ G1/ELE functions in the development of rice lemmas in addition to determining identities of empty glumes.” Frontiers in Plant Science 7:1006.Livak, K. J., T. D. Schmittgen. 2001. “Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔCT method.” Methods 25:402-408.Lombardo, F., H. Yoshida. 2015. “Interpreting lemma and palea homologies: a point of view from rice floral mutants.” Frontiers in Plant Science 6:61.Ma, X., Q. Zhang, Q. Zhu, W. Liu, Y. Chen, R. Qiu, B. Wang, Z. Yang, H. Li, Y. Lin, Y. Xie, R. Shen, S. Chen, Z. Wang, Y. Chen, J. Guo, L. Chen, X. Zhao, Z. Dong, Y. G. Liu. 2015. “A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.” Molecular Plant 8:1274-1284.Meng, Q., X. Li, W. Zhu, L. Yang, W. Liang, L. Dreni, D. Zhang. 2017. “Regulatory network and genetic interactions established by OsMADS34 in rice inflorescence and spikelet morphogenesis.” Journal of Integrative Plant Biology 59:693-707.Miyashiro, T., M. Goulian. 2008. “High stimulus unmasks positive feedback in an autoregulated bacterial signaling circuit.” Proceedings of the National Academy of Sciences of the United States of America 105:17457-17462.Peng, P., L. Liu, J. Fang, J. Zhao, S. Yuan, X. Li. 2017. “The rice TRIANGULAR HULL1 protein acts as a transcriptional repressor in regulating lateral development of spikelet.” Scientific Reports 7 : 13712.Ren, D., Y. Li, F. Zhao, X. Sang, J. Shi, N. Wang, S. Guo, Y. Ling, C. Zhang, Z. Yang, G. He. 2013. “ MULTI-FLORET SPIKELET1 , which encodes an AP2/ERF protein, determines spikelet meristem fate and sterile lemma identity in rice.” Plant Physiology 162:872-884.Ren, D., Y. Rao, Y. Leng, Z. Li, Q. Xu, L. Wu, Z. Qiu, D. Xue, D. Zeng, J. Hu, G. Zhang, L. Zhu, Z. Gao, G. Chen, G. Dong, L. Guo, Q. Qian. 2016. “Regulatory role of OsMADS34 in the determination of glumes fate, grain yield, and quality in rice.” Frontiers in Plant Science 7:1853.Rieu, P., V. M. Beretta, F. Caselli, E. Thévénon, J. Lucas, M. Rizk, E. Franchini, E. Caporali, C. Paleni, M. H. Nanao, M. M. Kater, R. Dumas, C. Zubieta, F. Parcy, V. Gregis. 2024. “The ALOG domain defines a family of plant-specific transcription factors acting during Arabidopsis flower development.” Proceedings of the National Academy of Sciences of the United States of America 121 : e2310464121.Ruan, B., L. Shang, B. Zhang, J. Hu, Y. Wang, H. Lin, A. Zhang, C. Liu, Y. Peng, L. Zhu, D. Ren, L. Shen, G. Dong, G. Zhang, D. Zeng, L. Guo, Q. Qian, Z. Gao. 2020. “Natural variation in the promoter of TGW2 determines grain width and weight in rice.” New Phytologist 227:629-640.Song, X. J., W. Huang, M. Shi, M. Z. Zhu, H. X. Lin. 2007. “A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase.” Nature Genetics 39:623-630.Sun, J., H. Liu, J. Liu, S. Cheng, Y. Peng, Q. Zhang, J. Yan, H. J. Liu, L. L. Chen, I. Birol 2019. “CRISPR-Local: a local single-guide RNA (sgRNA) design tool for non-reference plant genomes.” Bioinformatics 35:2501-2503.Thorvaldsdottir, H., J. T. Robinson, J. P. Mesirov. 2012. “Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration.” Brief Bioinform 14:178-192.Vo Phan, M. S., I. Keren, P. T. Tran, M. Lapidot, V. Citovsky. 2023. “Arabidopsis LSH10 transcription factor and OTLD1 histone deubiquitinase interact and transcriptionally regulate the same target genes.” Communications Biology 6 : 58.Wang, L., X. Q. Zeng, H. Zhuang, Y. L. Shen, H. Chen, Z. W. Wang, J. C. Long, Y. H. Ling, G. H. He, Y. F. Li. 2016. “Ectopic expression of OsMADS1 caused dwarfism and spikelet alteration in rice.” Plant Growth Regulation 81:433-442.Wang, N., Y. Li, X. Sang, Y. Ling, F. Zhao, Z. Yang, G. He. 2013. “Nonstop glumes ( nsg ), a novel mutant affects spikelet development in rice.” Genes & Genomics 35:149-157.Xu, Q., H. Yu, S. Xia, Y. Cui, X. Yu, H. Liu, D. Zeng, J. Hu, Q. Zhang, Z. Gao, G. Zhang, L. Zhu, L. Shen, L. Guo, Y. Rao, Q. Qian, D. Ren. 2020. “The C2H2 zinc-finger protein LACKING RUDIMENTARY GLUME 1 regulates spikelet development in rice.” Science Bulletin 65:753-764.Yamaguchi, T., N. Nagasawa, S. Kawasaki, M. Matsuoka, Y. Nagato, H. Y. Hirano. 2004. “The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa .” The Plant Cell 16:500-509.Yamaki, S., Y. Nagato, N. Kurata, K. I. Nonomura. 2011. “Ovule is a lateral organ finally differentiated from the terminating floral meristem in rice.” Developmental Biology 351:208-216.Yang, D., N. He, X. Zheng, Y. Zhen, Z. Xie, C. Cheng, F. Huang. 2020. “Cloning of long sterile lemma ( lsl2 ), a single recessive gene that regulates spike germination in rice ( Oryza sativa L .).” BMC Plant Biology 20:561.Yu, G., L. G. Wang, Q. Y. He. 2015. “ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization.” Bioinformatics 31:2382-2383.Yu, H., A. Wang, G. Zhang, G. Dong, L. Guo, Q. Qian, D. Ren. 2024. “Rice AGL1 determines grain size and sterile lemma identity.” The Crop Journal 12:630-634. Sato, Y., SK Hong, A. Tagiri, H. Kitano, N. Yamamoto, Yasuo Nagato and Makoto Matsuoka. 1996. “A rice homeobox gene, OSH1 , is expressed before organ differentiation in a specific region during early embryogenesis.” Proceedings of the National Academy of Sciences of the United States of America 93:8117-8122.Zhang, T., Y. Li, L. Ma, X. Sang, Y. Ling, Y. Wang, P. Yu, H. Zhuang, J. Huang, N. Wang, F. Zhao, C. Zhang, Z. Yang, L. Fang, G. He. 2017. “ LATERAL FLORET 1 induced the three-florets spikelet in rice.” Proceedings of the National Academy of Sciences of the United States of America 114:9984-9989.Zhao, L., L. Xie, Q. Zhang, W. Ouyang, L. Deng, P. Guan, M. Ma, Y. Li, Y. Zhang, Q. Xiao, J. Zhang, H. Li, S. Wang, J. Man, Z. Cao, Q. Zhang, Q. Zhang, G. Li, X. Li. 2020. “Integrative analysis of reference epigenomes in 20 rice varieties.” Nature Communications 11:2658.Zhu, W., L. Yang, D. Wu, Q. Meng, X. Deng, G. Huang, J. Zhang, X. Chen, C. Ferrándiz, W. Liang, L. Dreni, D. Zhang. 2021. “Rice SEPALLATA genes OsMADS5 and OsMADS34 cooperate to limit inflorescence branching by repressing the TERMINAL FLOWER1 ‐like gene RCN4 .” New Phytologist 233:1682-1700.Zhuang, H., H. L. Wang, T. Zhang, X. Q. Zeng, H. Chen, Z. W. Wang, J. Zhang, H. Zheng, J. Tang, Y. H. Ling, Z. L. Yang, G. H. He, Y. F. Li. 2020. “ NONSTOP GLUMES1 encodes a C2H2 zinc finger protein that regulates spikelet development in rice.” The Plant Cell 32:392-413. Dual Transcriptional Circuits: G1-OsMADS34 and G1-TGW2 Cooperatively Regulate Sterile Lemma Identity and Grain Size in Rice Figure Legends Figure 1. Map-based cloning of G1 . (A-D) Spikelets of Nipponbare (A-B) and LG7 (C-D). (E-H) Cross-sections of the spikelets and the sterile lemmas of Nipponbare (E-F) and LG7 (G-H). (I-K) Epidermal cells of the sterile lemmas (I) and lemmas (J) of Nipponbare and sterile lemmas of LG7 (K). (L) Fine mapping of the G1 locus. Numerals on the linkage map indicate the number of recombinants. (M) Gene structure of G1 and the variation in the two alleles of the varieties. (N) Spikelets of the CRISPR/Cas9-edited G1 plants. Red stars indicate vascular bundles (E-H). le, lemma; pa, palea; sl, sterile lemma; lsl, long sterile lemma. Scale bars, 500 µm in A, B, C, D, and N; 200 µm in E and G; 100 µm in F, H, I, J and K. Figure 2. Characterization of G1 . (A) Expression pattern of G1 in different tissues of Nipponbare. YP, young panicle. Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (B) Spatial expression of G1 in the young panicles of Nipponbare and LG7. le, lemma; pa, palea; sl, sterile lemma; st, stamen; fm, floral meristem. Scale bar, 200 µm. (C) Subcellular localization of G1 in rice protoplasts. Green fluorescence shows EGFP and red fluorescence shows mCherry, NLM, nuclear localization marker. Scale bar, 7.5 µm. (D-E) Transcriptional activity assay of G1 in rice protoplasts. The effector plasmids encode BD-G1 NIP and BD-G1 LG7 fusion proteins, which bind to the promoter of the reporter plasmid. pUbiquitin::REN was used as an internal control. ARF5M and HOS15 were used as positive and negative controls, respectively. Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (F) Analysis of G1 transactivation activity in yeast. Figure 3. G1 binds to the OsMADS34 promoter and activates its transcription. (A) A Venn diagram illustrating the common putative G1 target genes identified in the IP-G1-3 and IP-G1-4 samples through ChIP-seq. (B) Motif analysis using HOMER was performed to identify core motifs enriched within the G1-binding regions determined by ChIP-seq. (C) G1 binds to the OsMADS34 promoter by both ChIP-seq and ChIP-qPCR. The diagrams depict the putative promoter region of the OsMADS34 gene. 34P1 , 34P2 , 34P3 and 34P4 indicate the regions used for designing primers for ChIP-qPCR. The 34P2 and 34P3 regions contain the YACTGTW and CArG-box motifs, respectively. (D) ChIP-qPCR analysis showing G1 binding sites. 34 P1 , 34 P2 , 34 P3, and 34 P4 indicate analyzed fragments as shown in (C). Values are means ± SD (n = 3). (E) Y1H assay to test the binding of G1 NIP and G1 LG7 to the promoter of OsMADS34 . (F-G) EMSA assay showing the binding of G1 to the YACTGTW and CArG-box motifs (CTTTAAAATG and CAAATTTG) within OsMADS34 promoter. The cold probe was added at 10- and 20-fold molar excess of labeled probes, respectively. Red arrows indicate DNA-protein complexes and blue arrows indicate the free probes. (H-I) G1 transactivates transcription of OsMADS34 as shown by dual-luciferase transcriptional activity assay in rice protoplasts. The CTTTAAAATG, YACTGTW, and CAAATTTG motifs in the M1 , M2-1, and M2-2 sequences of the OsMADS34 promoter were deleted to generate ∆M1 , ∆M2-1 and ∆M2-2, respectively. Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (J) Spatial expression of G1 in early developmental stages of spikelets in the OsMADS34 mutant (os mads34-3 ). (K-M) Spatial expression of OsMADS34 in early developmental stages of spikelets in Nipponbare, LG7, and g1-11 . ca, carpel; fm, floral meristem; le, lemma; pa, palea; sl, sterile lemma; spm, spikelet meristem; st, stamen. Scale bar, 100 µm in (J-M). Figure 4. Identification of interacting protein of G1 . (A) G1 NIP and G1 LG7 interact with OsMADS34, as determined by the Yeast two-hybrid (Y2H) assay. The combination of pGBKT7-53 and pGADT7-T served as positive controls, while pGBKT7-λ and pGADT7-T were used as negative controls. Bait, protein fused with binding domain; Prey, protein fused with activation domain; EV, empty vector; SD/-L-T-H-A/X-α-gal, synthetic dropout (SD) media lacking Leu, Trp, His, and Ade; SD/-L-T, SD media lacking Leu, Trp; AbA, Aureobasidin A; X - α -gal, 5-Bromo-4-chloro-3-indolyl α -D-galactopyranoside. (B) BIFC assay showing the interaction of G1 NIP with OsMADS34 in tobacco leaves. The empty vector with the N- or C-terminus of mVenus was used as negative control. Scale bar, 7.5 μm. (C-D) LCI assay showing the interaction of G1 NIP and G1 LG7 with OsMADS34 in tobacco leaves. Indicated plasmid pairs were co-transformed into the tobacco leaves. The empty vector containing nLUC or cLUC was used as a negative control. (E) In vivo co-immunoprecipitation (Co-IP) assay: GFP / G1 - HA and G1 - HA / OsMADS34 - GFP were separately co-transformed into rice protoplasts. Protein samples were detected with anti-GFP and anti-HA antibodies. IP, immunoprecipitation. (F-H) G1 NIP interacts with OsMADS34 and activates transcription of OsMADS34 as shown by dual-luciferase transcriptional activity assay in rice protoplasts (G) and tobacco leaves (H). Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). Figure 5. Phenotypic analysis of the spikelets of g1-11 , osmads34-3 , and g1/osmads34 in the flowering stage. (A) Morphologies of g1-11 spikelets. (B-C) Spikelets of osmads34-3 (B) and cross-section of its spikelet hulls (C). (D-E) Spikelets of g1/osmads34 (D) and cross-sections of spikelet hulls of g1/osmads34 (E). (F) Spikelets of g1/OE-OsMADS34. (G) Sterile lemma length/grain length ratio of Nipponbare, g1-11 , osmads34-3 , g1/osmads34 and g1/OE-OsMADS34 . Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). Scale bars, 500 µm in A; 1mm in B, D, and F; 200 µm in C and E. Figure 6. Binding of G1 to the TGW2 promoter activates its expression to control the grain size. (A) Detection of G1 enrichment on TGW2 promoter by ChIP-seq. The diagrams depict the putative promoter region of the TGW2 gene. W P1 , W P2 , W P3, and W P4 indicate the regions used for designing primers for ChIP-qPCR. The WP2 and WP3 regions contain YACTGTW and CArG-box motifs, respectively. (B) ChIP-qPCR analysis showing G1 binding sites within TGW2 promoter. W P1 , W P2 , W P3, and W P4 indicate analyzed fragments within the four target loci as shown in (A). Values are means ± SD (n = 3). (C) Y1H assay to test the binding of G1 NIP and G1 LG7 to the promoter of TGW2 . (D-E) EMSA assay showing the binding of G1 to the YACTGTW and CArG motifs (CATTATTAG and CATTAATG) within TGW2 promoter. The cold probe was added at 10- and 20-fold molar excess of labeled probes, respectively. Red arrows indicate DNA-protein complexes and blue arrows indicate the free probes. (F-G) G1 NIP transactivates transcription of TGW2 as shown by dual-luciferase transcriptional activity assay in rice protoplasts. The YACTGTW, CATTATTAG, and CATTAATG motifs in the T1 , T2-1, and T2-2 sequences of TGW2 promoter were deleted to generate ∆T1 , ∆T2-1 and ∆T2-2, respectively. Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (H) Spatial expression of TGW2 in early developmental stages of spikelets in NIP, LG7, and g1-11 . ca, carpel; fm, floral meristem; le, lemma; pa, palea; sl, sterile lemma; spm, spikelet meristem; st, stamen. Scale bar, 100 µm. Figure 7. Genetics of grain size and knockout of G1 increases grain yield. (A) Grains of NIP, g1-11 , OE-G1-1 and g1/OE-TGW2 . (B) Brown grains of NIP, g1-11 , OE-G1-1 and g1/OE-TGW2 . (C-D) Grain length and width of NIP, g1-11 , OE-G1-1 and g1/OE-TGW2 plants. (E-F) Grain length and width of brown rice of NIP, g1-11 , OE-G1-1 and g1/OE-TGW2 plants. Data are means ± SD (n = 10). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (G) Panicle and area of 1000 grains of NIP, g1-11 , and g1-14 . Scale bar, 1 cm. (H) 1000-grain weight of brown rice. Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (I-K) Grain yield per panicle (I), panicle length (J), and grain yield per plot (K) of NIP, g1-11 , and g1-14 . In (I and J), data are means ± SD (n = 10). In (K), data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (L) A working model was proposed to depict the G1 -mediated sterile lemma identity and grain controlling regulatory network with the involvement of OsMADS34 and TGW2 . Scare bar, 1 cm in A and B, 0.5 cm in G. Supporting Information Supplementary Figure 1. Plant materials used in the study. (A) Spikelets of NIP and LG7. (B) Comparison of the sterile lemma length/grain length ratio of NIP and LG7. Data are means ± SD (n = 10) (* P < 0.05, ** P < 0.01, t -test). (C) Sequencing of the CRISPR/Cas9-edited G1 in g1-11 and g1-14 mutants. (D) Schematic diagram of plasmid construction for generating G1 NIP overexpression lines and grain phenotypes of G1 overexpression lines, OE-G1-1 and OE-G1-2, driven by CaM35S promoter. (E) Relative expression of G1 in NIP, OE-G1-1 and OE-G1-2 . Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (F) Western blot analysis with anti-GFP was performed to detect p35S::G1 NIP -TurboID protein expression levels in OE-G1-1 and OE-G1-2. Scale bar, 1 cm in A and D. Supplementary Figure 2. Phenotypic and agronomic trait analysis, along with the cytological examination of glumes in NIP, g1-11 and g1-14 plants. (A) Grains of Nipponbare and CRISPR/Cas9-edited G1 mutants g1-11 and g1-14 . (B-C) Comparison of the brown grain width and brown grain length of the Nipponbare, g1-11 and g1-14 . Data are means ± SD (n = 10). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (D) Comparison of the cell number in the cross-sections of the outer parenchyma layer of Nipponbare (up), g1-11 , and g1-14 (down) lemma. OPC, outer parenchyma cells; IPC, inner parenchyma cells. Magnified views of the red boxed areas are shown on the right. (E-F) Comparison of outer parenchymal cell number and outer parenchymal cell area. In E), data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). In F), data are means ± SD (n = 10). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (G) Scanning electron microscope image of the outer glumes from the Nipponbare, g1-11, and g1-14 plants. Red boxes are the bulge cells of the outer lemma glumes. (H-I) Comparison of the cell length and width of outer lemma glumes. Data are means ± SD (n = 10). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). Scale bars, 200 µm in D (left); 50 µm in G and D (right); 1 cm in A. Supplementary Figure 3. Expression analysis of OSH1 . (A) Spatial expression of OSH1 in the early stages of the young panicles in NIP, LG7, and g1-11 . fm, floral meristem; le, lemma; pa, palea; sl, sterile lemma; spm, spikelet meristem; st, stamen. Scale bars,100 µm. (B) Expression pattern of OSH1 in YP 1.5-2 cm of NIP, LG7, and g1-11 . YP, young panicles. Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). Supplementary Figure 4. ChIP peaks over chromosomes. Supplementary Figure 5. Genome-wide analyses of G1 downstream regulatory gene network. (A-B) Distribution of read count frequency relative to genic region in IP-G1-3 (A) and IP-G1-4 (B). (C-D) Distribution of candidate G1 -binding regions across the rice genome, as determined by ChIP-seq, shows their association with key genomic landmarks, including the transcription start site (TSS). Supplementary Figure 6. Examination of OsMADS34 expression levels. (A) Y1H assay to test the binding of OsMADS34 to the promoter of OsMADS34 . (B-C) Transactivation activity of OsMADS34 assay using rice protoplasts. The effector plasmids with BD can bind to the GAL4 mini promoter in the reporter plasmid. pUbiquitin::REN was used as an internal control. ARF5M and HOS15 were used as positive and negative controls, respectively. Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). (D) Mutation sites in the osmads34 mutant are identified. (E) Expression levels of OsMADS34 in different tissues of NIP and g1-11 . YP, young panicle. Data are means ± SD (n = 3) (* P < 0.05, ** P < 0.01, t -test). Supplementary Figure 7. Expression pattern of TGW2 in different tissues of NIP and g1-11 . YP, young panicles. Data are means ± SD (n = 3) (* P < 0.05, ** P < 0.01, t -test). Supplementary Figure 8. Generation of double mutants. (A) Sequencing of the mutation sites in the g1/osmads34 double mutant. (B-C) Relative expressions of OsMADS34 and TGW2 genes in g1/OE-OsMADS34 and g1/OE-TGW2 transgenic lines, respectively. Data are means ± SD (n = 3). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). Supplementary Figure 9. Statistical analysis of agronomic traits. (A-C) Grain numbers per panicle (A), No. of primary branches per panicle (B), and seed setting rate (C). Data are means ± SD (n = 10). Different letters indicate significant differences ( P < 0.05, one-way ANOVA, Tukey’s test). Information & Authors Information Version history V1 Version 1 02 September 2025 Peer review timeline Published Plant, Cell & Environment Version of Record 4 Jan 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Plant, Cell & Environment Keywords g1 development genome lemma-like sterile lemma/grain size regulation rice Authors Affiliations Xuemei Qin Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Ping Gan Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Jin-Liang Sun Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Di Wu Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Ru Li Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Tianmin Ouyang Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Kaichong Teng Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Weijian Cen Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Baoxiang Qin Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Fang Liu Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Rongbai Li Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Jijing Luo 0000-0001-5664-0881 [email protected] Guangxi University State Key Laboratory for Conservation and Utilization of Subtropical Agro BioResources View all articles by this author Metrics & Citations Metrics Article Usage 223 views 171 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Xuemei Qin, Ping Gan, Jin-Liang Sun, et al. Dual Transcriptional Circuits: G1-OsMADS34 and G1-TGW2 Cooperatively Regulate Sterile Lemma Identity and Grain Size in Rice. Authorea . 02 September 2025. DOI: https://doi.org/10.22541/au.175679682.22419582/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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