Protein synthesis is a complex process that can be categorized into four distinct stages: initiation, elongation, termination, and ribosome recycling (Sonenberg and Hinnebusch, 2009). Translation initiation represents the crucial phase of protein synthesis, where the ribosome assembles on the mRNA and initiates the synthesis of the polypeptide chain (Merrick and Pavitt, 2018). In eukaryotes, this process is regulated and carried out by a series of protein complexes known as eukaryotic translation initiation factors (eIFs) (Gebauer and Hentze, 2004; Hinnebusch, 2006). These factors orchestrate the assembly of the translation initiation complex, which includes the small ribosomal subunit, initiator tRNA, and mRNA (Merrick and Pavitt, 2018). The eIFs not only facilitate the correct positioning of the ribosome on the mRNA but also regulate the rate and efficiency of translation initiation (Roy and von Arnim, 2013; Raabe et al., 2019). As such, they serve as important regulators of gene expression and can influence various aspects of plant growth and development.

The types of eIFs are diverse and complex, and at least 29 have been identified, including eIF1, eIF1A, eIF2, eIF2α, eIF2B, eIF3a-m, eIF4A1, eIF4A2, eIF4B, eIF4E, eIF4G, eIF4F, eIF5, eIF5A, eIF5B, eIF6.1and eIF6.2. Each exerting distinct roles during the translation initiation process (Jackson et al., 2010; Guo et al., 2011; Aitken and Lorsch, 2012; Hinnebusch and Lorsch, 2012; Raabe et al., 2019; Castellano and Merchante, 2021; Singha et al., 2021; Ma et al., 2022). Among them, eIF6 is an essential protein that possesses a distinctive anti-association activity. As it binds to immature large ribosomal subunits (pre-60S) in the nucleolus, it prevents their premature association with 40S subunits (Miluzio et al., 2009). Subsequently, eIF6 is detected in the nucleoplasm during pre-60S subunit maturation and is exported to the cytosol where it releases the 60S ribosomal subunit. The 60S subunits then join with 40S subunits to form the 80S ribosome complex (Si and Maitra, 1999; Basu et al., 2001; Ceci et al., 2003; Gandin et al., 2008; Miluzio et al., 2009).

Over the past decades, extensive research has been devoted to exploring the function of eIFs in animals and yeast (Kapp and Lorsch, 2004; Sonenberg and Hinnebusch, 2009; Aitken and Lorsch, 2012). Indeed, recent research has suggested that eIFs play a crucial role in regulating cell differentiation, cell cycle progression, and stress responses in plants (Rausell et al., 2003; Thompson et al., 2004; Diédhiou et al., 2008; Singh et al., 2013; Chen et al., 2019; Raabe et al., 2019; Castellano and Merchante, 2021). For example, Fumonisin B1-resistant 12 (FBR12) encodes a putative eIF-5A-2 protein that regulates growth and development of floral organs and sporogenesis by influencing cell division, cell growth, and cell death in Arabidopsis (Feng et al., 2007). In addition, eIF5A played a significant role in the process of cadmium (Cd) accumulation and sensitivity in Arabidopsis. The ateif5a mutant exhibited a higher level of Cd accumulation in both roots and shoots compared to the wild type. Moreover, AteIF5A was found to impact Cd sensitivity by modulating Cd uptake, accumulation, and detoxification (Xu et al., 2015). Furthermore, eIFs are involved in the processes of plant growth and development, such as embryogenesis, flowering, and organogenesis. For example, mutations in the eIF3 subunits eIF3e, eIF3f, and eIF3h in Arabidopsis do not impact pollen formation or maturation. However, they do cause deficiencies in pollen germination and/or pollen tube growth, resulting in reduced efficiency of male gamete transmission (Xia et al., 2010; Roy et al., 2011). Rice eIF3 subunit f has been reported to play an important role in post-meiotic pollen formation, knockdown of OseIF3f showed a large reduction in seed setting and pollen fertility (Li et al., 2016). The expression levels of OseIF3f were significantly higher in unicellular microspores and bicellular pollen than in mature tricellular pollen or germinated pollen, with at least a three-fold increase observed, suggesting that OseIF3f might play a more crucial role in microgametogenesis rather than pollen germination (Wei et al., 2010). Interestingly, OseIF3e interacted with OseIF3f and OseIF6, respectively. OseIF3e-RNAi plants exhibited stunted growth during both the seedling and vegetative stages, and displayed defects in pollen maturation and small grains (Wang et al., 2016).

eIF6 was first discovered and characterized as a wheat protein that associates with the 60S ribosome in crop plants (Russell and Spremulli, 1980). The study on eIF6 in yeast (TIF6) demonstrated its crucial role in ribosome biogenesis. When TIF6 is depleted, it causes abnormal processing of ribosomal RNA (rRNA) precursors and a decrease in the abundance of 60S ribosomal subunits, leading to a lethal phenotype (Wood et al., 1999; Basu et al., 2001). Human eIF6 (p27BBP) interacts with 60S ribosome subunits, preventing the assembly of both 40S and 60S subunits in the cytosol (Sanvito et al., 1999). Embryos lacking eIF6 in mice exhibit a lethal phenotype during the preimplantation stage. Additionally, heterozygous mice display insensitivity to insulin and show reduced hepatic and adipose tissue mass, as well as a decrease in protein synthesis (Gandin et al., 2008). The Arabidopsis at-eif6;1 mutant exhibits an embryonic-lethal phenotype, similar to yeast and mouse. One-third of the pale yellow seeds were observed in heterozygotes mutant siliques (Kato et al., 2010). However, little is known about the function of eIF6 in crop plants, despite these studies indicating a critical role for eIF6 in embryogenesis.

Our previous study has shown that OseIF6.1 interacts with OsLa, and it was impossible to isolate a homozygous oseif6.1 mutant, as the plants displayed abnormal floral organs, leading to a lethal phenotype (Guo et al., 2022). However, the function of OseIF6.1 gene has yet to be characterized. Our present study aimed to validate the roles of OseIF6.1 in the growth and development of rice. In this study, we identified and characterized the functions of OseIF6.1 by using RNA interference (RNAi) approach. We found that OseIF6.1 encodes a protein homologous to At-eIF6;1 in Arabidopsis, and OseIF6.1 acts as a positive regulator in grain size and pollen sterility. In addition, OseIF6.1 physically interacts with OsNMD3, which is a nuclear export adaptor for the 60S ribosomal subunit. The OsNMD3-RNAi lines exhibited a phenotype of reduced fertility. Therefore, these findings help to reveal the function of OseIF6.1 in rice grain size and pollen fertility.

Originally purified from wheat germ, eukaryotic initiation factor 6 (eIF6) was discovered to function as a ribosome dissociation factor (Russell and Spremulli, 1980). By binding to the 60S ribosome subunit, eIF6 prevents its association with the 40S ribosome subunit (Russell and Spremulli, 1980). Homologous proteins of wheat eIF6 were subsequently isolated and purified from various sources, including rabbit (Raychaudhuri et al., 1984), calf, human (Si et al., 1997), yeast (Si and Maitra, 1999), and Arabidopsis (Kato et al., 2010). The phylogenetic analysis indicates that the eIF6 protein is conserved among eukaryotes ( Figure 1A ). Structural comparisons of eIF6 proteins have further revealed that OseIF6.1 shares a close relationship with other homologues ( Supplementary Figure 1 ). Several studies on yeast and mammals have demonstrated the crucial role of eIF6 in growth regulation. Depletion of eIF6 is lethal in both yeast and mouse models (Si et al., 1997; Sanvito et al., 1999; Wood et al., 1999; Gandin et al., 2008). Like in yeast and mouse, the null allele of the At-eIF6;1 in Arabidopsis results in an embryonic-lethal phenotype, underscoring the essential role of eIF6 in plant embryogenesis (Kato et al., 2010). Although numerous studies have examined the role of eIF6 proteins in eukaryotes, our previous study demonstrated that oseif6.1 mutant was unable to harvest seeds, which resulted in a lethal phenotype (Guo et al., 2022). Therefore, the functional characterization of the OseIF6.1 gene remains to be fully understood.

Given that the mutant of OseIF6.1 results in embryonic lethality, we conducted an investigation into the function of OseIF6.1 in rice by either knocking down or overexpressing its expression in Nipponbare. In this study, we found that OseIF6.1 plays an important role in the development of plant architecture and grain shape. Both the knockdown and overexpression of OseIF6.1 resulted in a dwarfing phenotype ( Figure 2A ). It has been widely reported that gibberellins (GA) are the primary regulators of plant height, influencing the growth and development of stems and determining the overall stature of the plant (Salas Fernandez et al., 2009). The expression of the genes related to GA biosynthesis in stems of WT and OseIF6.1 transgenic plants was examined. We found that the expression of OsGA20ox1 (Oikawa et al., 2004), OsGA20ox2 (Sasaki et al., 2002; Su et al., 2021), and OsKO2 (Itoh et al., 2004) was suppressed in OseIF6.1-R and OseIF6.1-OE stems ( Supplementary Figure 6 ). These results suggest that altered eIF6.1 expression affects the expression of genes related to GA biosynthesis and ultimately plant height. Furthermore, the knockdown of OseIF6.1 resulted in short, narrow, and thin grains, whereas the overexpression of OseIF6.1 resulted in long grains ( Figures 3A–D ), indicating that OseIF6.1 acts as a positive regulator of grain shape. The conclusion was reinforced through scanning electron microscopy observations, which disclosed that OseIF6.1 has a positive impact on grain size by regulating cell elongation and proliferation ( Figure 4 ). Therefore, OseIF6.1 seems to be a key determinant of plant structure and rice grain morphology. These lines exhibit no difference in seed germination compared to the WT. However, the OseIF6.1-R and OE lines both exhibit a reduced 1000-grain weight compared to the WT ( Figure 3E ), potentially due to changes in OseIF6.1 expression that affect the translation efficiency of mRNAs related to grain development. Despite the OseIF6.1-OE lines having increased translation efficiency, there is a decrease in both grain width and thickness. This could potentially be attributed to compensatory mechanisms or feedback regulation, resulting in a similar 1000-grain weight phenotype to that observed in the OseIF6.1-R lines.

The At-eIF6;1 heterozygous plants exhibit a 1:3 ratio of growth defects in their silique seeds, which display a pale yellow coloration (Kato et al., 2010). Interestingly, we have noted that plants with OseIF6.1-R show decreased seed-setting rates alongside diminished pollen fertility ( Figure 2H ; Supplementary Figure 2 ). Subsequent analysis using transmission electron microscopy revealed that the abortion of OseIF6.1-R pollens is associated with the degradation of the tapetum layer ( Figure 5 ). Numerous investigations have identified a range of genes that play a pivotal role in tapetum PCD and the formation of pollen walls. These genes include Udt1 (Jung et al., 2005), Wda1 (Jung et al., 2006), TDR (Zhang et al., 2008), DPW (Shi et al., 2011), EAT1 (Niu et al., 2013), CYP703A3 (Yang et al., 2014), and PTC2 (Uzair et al., 2020). We observed that the expression of CYP703A3, PTC2, TDR, EAT1 and DPW genes was downregulated in OseIF6.1-R young panicles ( Supplementary Figure 3 ). These findings suggest that OseIF6.1 is essential for anther development. Consistent with this, the depletion of eIF6 in yeast causes a reduction in the availability of free 60S ribosomal subunits, resulting in a decrease in translational activity, which is eventually lethal (Wood et al., 1999; Basu et al., 2001). In addition, it has been reported that eIF6-null embryos are lethal at the preimplantation stage in mice, and the quality of hepatic and adipose tissue in the heterozygous eIF6 mice is reduced due to the reduction in cell number and perturbation of the G1/S cell cycle process (Gandin et al., 2008). Together with our results, these data suggest that OseIF6.1 plays a vital role in embryonic development across eukaryotes.

The biogenesis of ribosomes is a complex process that involves various trans-acting factors. eIF6 can bind to immature large ribosomal subunits as well as other trans-acting factors in the nucleolus (Miluzio et al., 2009). NMD3 is a conserved transcriptional factor that encodes the nuclear export adaptor for the 60S ribosomal subunit. This protein is characterized by its N-terminus, which contains Cx2C repeats and a nuclear localization sequence. The C-terminus of NMD3 features a nuclear export sequence, which is essential for maintaining the efficiency of normal protein synthesis (Shi et al., 2014). Overexpression of OsNMD3^ΔNLS , which lacks a nuclear localization site, caused abnormal plant growth and development in rice, including dwarfism and reduced grain size (Shi et al., 2014). In the Arabidopsis AtNMD3ΔNES OE line, pleiotropic phenotypes were observed, such as reduced plant height and stamen size, as well as an obvious curly shape of the rosette leaves (Chen et al., 2012). NMD3 may interact with specific ribosomal proteins, especially those associated with the 60S ribosomal subunit, facilitating their proper assembly and export from the nucleus to the cytoplasm, given the roles of its homologs in other species (Hedges et al., 2006; Sengupta et al., 2010; Chen et al., 2012). It has been reported that OsNMD3 interacts with the 60S subunit through OsRPL10Ac1 (Shi et al., 2014). In this study, we have discovered that OseIF6.1 interacts with OsNMD3 ( Figure 6 ). Interestingly, the OsNMD3-R plants exhibit similar phenotypic traits to those of OseIF6.1-R plants. The OsNMD3-R plants also exhibit reduced plant height and pollen fertility ( Figure 7 ; Supplementary Figure 5 ). The expression of the GA biosynthesis-related genes, including OsGA20ox1, OsGA20ox2, and OsKO2 were decreased in stems of OsNMD3-R plants ( Supplementary Figure 7 ). Similar dwarfism and sterility phenomena have also been observed in other eIFs RNAi plants. For example, it has been reported that OseIF3e protein interacts with OseIF6. Notably, RNAi targeting OseIF3e causes significant plant height and pollen maturation defects (Wang et al., 2016). The mutation of OseIF3h has been found to cause abnormal development in rice, particularly affecting plant height and pollen fertility (Huang et al., 2021). Similarly, the T-DNA mutants of AteIF3e exhibited diminished pollen fertility, along with various floral and reproductive defects. Overexpressing AteIF3e led to dwarfism and affected seed formation (Yahalom et al., 2008). The differences in seed setting between the OseIF6.1-R and OsNMD3-R lines suggest that they may regulate rice growth via distinct or partially overlapping pathways. Higher seed setting rates in OseIF6.1-R lines imply that its role might be partly independent of OsNMD3, indicating the existence of additional pathways or partners for OseIF6.1. The more significant effect observed with OsNMD3 reduction could indicate that OsNMD3 operates downstream of OseIF6.1 or in a parallel pathway that is crucial for rice fertility. In future studies, genetic epistasis analysis can elucidate their relationships in rice development.

In conclusion, our findings indicate that OseIF6.1 plays a regulatory role in the expression of sporopollenin precursor biosynthesis genes, as well as genes involved in tapetal PCD, thereby influencing tapetal development and the formation of pollen. Additionally, it modulates the expression of GA biosynthesis-related genes, thus regulating plant height. Furthermore, OseIF6.1 interacts with OsNMD3, which also plays a crucial role in regulating pollen fertility and plant height. By identifying and characterizing OseIF6.1, the research provides insights into the genetic factors that influence rice growth and development. Understanding these genetic components is crucial for breeding and genetic engineering efforts aimed at crop improvement. The specific roles of OseIF6.1 in regulating grain length and fertility highlight it as a potential target for genetic engineering.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

HG: Investigation, Writing – original draft, Writing – review & editing, Data curation. JL: Data curation, Writing – original draft. XS: Investigation, Writing – original draft. LC: Data curation, Investigation, Writing – original draft. JR: Data curation, Writing – original draft. LL: Data curation, Writing – original draft. MR: Writing – original draft. SL: Writing – original draft. MD: Writing – original draft. GR: Writing – original draft, Writing – review & editing. FG: Writing – original draft, Writing – review & editing.