The coenocytic and cellularization phases during endosperm development require interplay among networks of transcription factors, cell cycle-related genes and phytohormones. miRNAs play a central role in these complex processes by precisely regulating target genes involved in nuclear/cell division and phytohormone homeostasis ( Figure 2A ). The spatiotemporal regulatory networks of miRNAs across species provide multidimensional perspectives for deciphering the mechanisms of endosperm development.

Nuclear/cell division is a direct determinant of endosperm development and seed size. In rice, the miR156-Squamosa Promoter Binding Protein-like (SPL) module plays a central role in governing agronomic traits, including grain size, through downregulating the SPL transcription factors. OsSPL13 and OsSPL16 control grain shape through regulating cell division or expansion by altering the expression of Small and Round Seed 5 (SRS5) and Grain Weight 7 (GW7), respectively (Xie et al., 2006; Wang et al., 2015; Si et al., 2016). The miR159-Myeloblastosis Transcription Factor (MYB) module also acts as a regulating hub in endosperm cell division. In maize, miR159 promotes endosperm cell division and proliferation by targeting ZmMYB74 and ZmMYB138, thereby alleviating their repression of cell cycle-related genes and enhancing grain size and weight (Wang et al., 2023). Notably, miR159 regulation extends beyond maternal tissues. In Arabidopsis, sperm-delivered miR159 silences maternally derived MYB33 and MYB65 transcripts in the central cell, lifting their repression of endosperm nuclear division. Paternal miR159 deficiency results in abnormal accumulation of MYB33/MYB65 mRNAs and proteins post-fertilization, stalling endosperm nuclear division and finally causing seed abortion (Zhao et al., 2018b).

miRNAs regulate phytohormone concentration and signaling to control endosperm development. Auxin is one phytohormone attracting attention within seeds. In maize, miR169o prolongs the endosperm cell cycle by negatively regulating the transcription factor Nuclear Factor Y Subunit A13 (ZmNF-YA13), thereby suppressing its transcriptional activation of the auxin biosynthesis gene YUCCA1 (ZmYUC1). This reduction in auxin level and the auxin/CK ratio ultimately increases endosperm cell numbers and expands seed size (Zhang et al., 2022). In castor bean, miR396b suppresses seed auxin levels by inhibiting the transcription factor Growth Regulating Factor 4 (RcGRF4), which in turn reduces the activation of the auxin biosynthesis gene RcYUC6. Paradoxically, this study revealed that lower auxin content is associated with reduced capacity for endosperm cell proliferation and expansion, and exogenous application of indole-3-acetic acid (IAA) enlarges seeds (Wang et al., 2024a). This difference may stem from the distinct developmental timing when these two miRNAs regulate auxin levels during endosperm development and/or from the fact that ncRNA-mediated regulation of auxin homeostasis in seeds is not fully conserved across species. In addition to auxin content, miRNA-mediated auxin signaling also controls endosperm development. In Arabidopsis, maternally derived miR167A is essential for endosperm development. Maternal loss of miR167A fails to repress its target genes Auxin Response Factors 6 (ARF6) and ARF8, which function as key transcription factors for auxin response genes, finally leading to defective endosperm and seed abortion (Yao et al., 2019).

Other phytohormones also participate in regulating seed development. Rice miR397 regulates seed development through the BR pathway. Overexpression of miR397 downregulates its target gene Laccase (OsLAC), thereby promoting BR signaling, leading to increases in grain size and panicle branching (Zhang et al., 2013). In contrast to miR396’s auxin-mediated regulation of seed size in castor bean, rice studies demonstrate that the miR396-GRF4 network controls grain size by activating BR responses or elevating CK levels (Che et al., 2015; Duan et al., 2015; Sun et al., 2016). These findings highlight species-specific differences in miR396 function. They also point to miR396’s broader role in modulating multiple phytohormone pathways and their crosstalk during seed development.

Grain filling is the key stage of grain development determining final yield and quality. miRNAs are important regulators of grain filling ( Figure 2B ). First, miRNAs affect seed development by regulating photosynthesis or starch synthesis. In rice, overexpressing miR408 positively regulates grain yield by increasing seed number and weight. miR408 inhibits its target Uclacyanin 8 (OsUCL8), which enhances photosynthetic rate by optimizing copper allocation, thus providing sufficient carbohydrates for seed development (Zhang et al., 2017). miR164b directly targets NAM, ATAF1/2, CUC2 Transcription Factor 2 (OsNAC2), and plants overexpressing an OsNAC2 mutant resistant to miR164b cleavage exhibited improved grain number and yield. Further research revealed that these transgenic rice plants have larger flag leaves and more vascular bundles, potentially leading to higher photosynthetic and assimilate transfer rates, ultimately enhancing grain yield (Jiang et al., 2018). In maize, miRNAs show temporal expression during endosperm development, preferentially repressing transcription factors related to starch synthesis genes. Specifically, miR159k-3p targeted ZmMYB138 and ZmMYB115, which regulate the transcriptional activities of key starch synthesis genes (Waxy and Amylose extender 1), thereby influencing starch accumulation in the endosperm (Hu et al., 2021).

Moreover, the miRNA-mediated coordination of phytohormones and metabolic processes is essential for seed endosperm development. In rice, miR156-OsSPL14 orchestrates tiller number and seed weight through regulating the expression of PIN-FORMED 1b (OsPIN1b) and PIN-LIKE6b (PILS6b) to fine-tune auxin transport dynamics (Jiao et al., 2010; Li et al., 2022). During rice endosperm filling, suppression of miR167 expression significantly increases grain weight and yield by enhancing the grain filling rate. Mechanistically, miR167 targeted OsARF12, which directly binds to the promoter of Cyclin-dependent Kinase F;2 (OsCDKF;2) to activate its expression. OsCDKF;2 is involved in auxin and BR signaling and cell cycle processes, and its activation increases grain filling and size. Notably, the expression of miR167 is regulated by miR159, a positive regulator of grain size, which modulates miR167 expression through OsGAMYBL2 (a negative regulator of GA biosynthesis) (Zhao et al., 2023). Additionally, inhibition of miR1432 enhances the grain filling rate by reducing cleavage of its target gene rice Acyl-CoA thioesterase (OsACOT) mRNA, thereby increasing OsACOT expression. OsACOT participates in medium-chain fatty acid biosynthesis, and its altered expression affects fatty acid metabolism as well as the synthesis and signaling of hormones such as IAA and ABA (Zhao et al., 2018a).

In recent years, predictive studies based on small RNA sequencing have provided new perspectives on miRNA regulation in endosperm development. Multiple studies have revealed numerous novel miRNAs in various crops through high-throughput sequencing. The target genes of these miRNAs are predominantly enriched in transcription factors and key metabolic enzymes, with their regulatory networks involving processes such as phytohormone homeostasis, starch synthesis, galactomannan metabolism, seed dormancy, and storage product metabolism. For instance, miRNAs in maize (Huang et al., 2016) and foxtail millet (Setaria italica (L.) P. Beauv.) (Li et al., 2024) target starch synthases and transcription factors to influence starch metabolism; miRNAs in Tartary buckwheat (Fagopyrum tataricum) (Li et al., 2021) and Brassica napus (Huang et al., 2013) participate in seed development and hormone responses; novel miRNAs regulating non-starch galactomannan biosynthesis have been identified in clusterbean (Cyamopsis tetragonoloba) (Tyagi et al., 2018); studies in rice (Panigrahi et al., 2021; Park et al., 2024) and wheat (Meng et al., 2013) have associated miRNAs with seed dormancy and grain filling processes; comparative analyses in rice have revealed differential miRNA expression patterns associated with phytohormone homeostasis and starch accumulation between superior and inferior spikelets, which exhibit distinct grain-filling rates and grain weight (Peng et al., 2011, 2014). However, most current researches are preliminary, primarily relying on differential expression analysis and bioinformatics predictions. Experimental validation through genetic transformation or functional studies remains limited, leaving the hierarchical regulatory mechanisms orchestrated by miRNAs largely unexplored.

The frequent occurrence of hybrid seed abortion following cross-species hybridization poses a persistent challenge in plant breeding, hindering the introduction of advantageous characteristics. This postzygotic hybridization barrier primarily occurs in the endosperm. siRNAs play a critical role in hybrid endosperm rescue, regulating genomic dosage responses, transposon silencing, and epigenetic reprogramming to maintain parental genome balance and determine seed viability (Ng et al., 2012; Bente and Köhler, 2024) ( Figure 2C ).

In Arabidopsis, when paternal genome dosage is doubled during interploidy crosses, excessive easiRNA accumulation interferes with the RdDM pathway, leading to a significant decrease in methylation levels of transposable elements and abnormal activation of paternally expressed imprinted genes, ultimately causing endosperm developmental defects and triploid seed abortion (Martinez et al., 2018; Satyaki and Gehring, 2019). Specifically, paternal easiRNAs are triggered by miR845, and their biogenesis depends on RNA polymerase IV activity. miR845 activates the synthesis of 21–22 nt easiRNAs in a dose-dependent manner by targeting the reverse transcription primer binding site of LTR-retrotransposons in Arabidopsis pollen (Borges et al., 2018). Remarkably, paternal Pol IV deficiency reduces easiRNA production, restoring transposable element methylation and seed viability (Borges et al., 2018; Martinez et al., 2018).

Beyond paternal mechanisms, maternal siRNAs also regulate endosperm development by suppressing parental genome conflicts through targeting the methylation network. In Capsella hybrid endosperm, dose-sensitive maternal sirenRNAs repress the expression of several AGAMOUS-like MADS-box transcription factor genes (AGLs) that target hypomethylated regions and regulates endosperm development. Insufficient sirenRNA dosage causes increased AGL expression and a loss of DNA methylation, triggering seed abortion in response to paternal-excess interspecies and interploidy hybridizations (Dziasek et al., 2024). A similar regulatory mechanism mediated by maternal siRNAs occurs in Arabidopsis seeds (Lu et al., 2012). Interestingly, like paternally derived siRNAs, the production of these maternal siRNAs also requires Pol IV activity. However, unlike paternal loss of Pol IV, maternal lack of Pol IV activity causes defects in chromatin condensation, DNA methylation, and hybrid seed formation (Dziasek et al., 2024). These findings suggest that maternally and paternally derived Pol IV-dependent siRNAs exert divergent regulatory roles during hybrid endosperm development.

Based on the above studies, siRNAs constitute the core molecular mechanism of endosperm hybridization barriers through dose-dependent transposable element silencing, dynamic methylation regulation, and imprinted gene expression balance. Paternally derived siRNAs and maternally derived siRNAs maintain parental genome-epigenetic homeostasis by antagonizing or enhancing the RdDM pathway, respectively. These findings may illuminate strategies for polyploid crop breeding and breaking reproductive isolation.

In summary, sRNAs orchestrate endosperm development through modular regulatory networks integrating hormone homeostasis, cell cycle progression, biomass metabolism and epigenetic elements. Future research combining multi-omics technologies and functional experiments to precisely identify and edit these networks will offer novel strategies for high-yield, high-quality breeding.

Endosperm development governs seed morphogenesis and nutrient accumulation efficiency through tightly orchestrated processes such as syncytium formation, cellularization, differentiation, and nutrient accumulation. Recent studies reveal that lncRNAs dynamically regulate key biological processes in endosperm development—such as cell cycle progression, cytoskeleton remodeling, and starch biosynthesis—forming intricate molecular regulatory networks ( Figure 3 ).

A seminal example is the maternal lncRNA Mis-Shapen Endosperm (MISSEN) discovered in rice ( Figure 3A ). Through a genome-wide screen of lncRNAs during rice sexual reproduction, a T-DNA insertion mutant of MISSEN was identified with low fertility and mis-shapen seed phenotype (Zhang et al., 2014). Further functional analysis revealed that the T-DNA insertion was an activating mutation with higher MISSEN levels, and it produced a similar phenotype to MISSEN overexpressing plants, suggesting that MISSEN negatively regulates endosperm development (Zhou et al., 2021). Mechanistically, MISSEN binds specifically to the helicase family protein HeFP, competitively disrupting HeFP-microtubule interactions. This leads to aberrant microtubule polymerization, nuclear division defects, and delayed endosperm cellularization. Notably, MISSEN knockdown lines exhibit accelerated cellularization and enlarged grain size, highlighting the MISSEN-HeFP pathway as a potential target for improving grain yield. Moreover, the author also found that epigenetic modifications H3K27me3 progressively suppress MISSEN expression post-pollination, unveiling a dosage-dependent regulatory logic in which maternal lncRNAs precisely control endosperm developmental timing. This study provides a paradigm for functional characterization of lncRNAs during seed and endosperm development.

In metabolic pathway regulation, lncRNAs play particularly critical roles in controlling endosperm starch biosynthesis. During seed development, stage-specific lncRNAs are significantly enriched in carbohydrate metabolism, starch synthesis, and transport-related pathways (Zhang et al., 2023d). In line with this, a genome-wide analysis of early panicles in cultivated rice and wild rice revealed that most differentially expressed lncRNAs are downregulated during domestication, with this directional selection primarily influencing the expression of energy metabolism genes related to carbohydrate transport and accumulation (Zheng et al., 2019) ( Figure 3B ). Transgenic overexpression of these three wild rice lncRNA alleles (LncRNA_2308, LncRNA_1267, and LncRNA_1631) in cultivated rice resulted in higher grain chalkiness and lower grain weight compared to controls. Notably, expression of Waxy—encoding granule-bound starch synthase, a key regulator of grain starch content—was significantly reduced in lncRNA-transgenic plants. This study demonstrates that domestication-driven selection for targeting lncRNA suppression represents a critical mechanism for enhancing starch accumulation and seed weight. Consistent with the MISSEN regulatory framework, these lncRNAs likely function as inhibitory regulators during seed development. Investigating the molecular mechanisms underlying lncRNA suppression may therefore uncover novel genes functionally linked to endosperm development.

Endosperm starch contains amylose and amylopectin, with high-amylose starch considered resistant (“healthy”) starch beneficial for preventing diet-related diseases like obesity. In bread wheat endosperm, lncRNA TCONS_00130663 shows strong correlation with SBEIIb (encoding starch branching enzyme) and LYPL (encoding lysophospholipase), indicating that lncRNAs may regulate resistant starch biosynthesis through unknown mechanisms (Madhawan et al., 2020). Storage proteins, another key endosperm component determining grain quality, also show co-expression patterns with specific lncRNAs, which may regulate enzymes involved in storage protein biosynthesis (Zhang et al., 2023d).

Several lncRNAs regulate seed yield traits, though endosperm-specific roles are not always confirmed. For example, the lncRNA leucine-rich repeat receptor kinase antisense intergenic RNA (LAIR) is a positive regulator of seed production and its overexpression increases yield-related traits including grain number and panicle number ( Figure 3C ). Further, it was revealed that LAIR induces epigenetic changes through specifically binding histone modification proteins like Males Absent on the First (OsMOF) and WD Repeat Domain 5 (OsWDR5), and then activates the expression of its neighboring yield-associated gene Leucine-rich Repeat Receptor Kinase (LRK1) (Wang et al., 2018). A recent study found that LAIR has nine alternative splicing isoforms and different compositions of LAIR isoforms could fine-tune LRK1 expression and seed yield under different abiotic stress (Wang et al., 2024b).

Several studies using genome-wide analysis to identify lncRNAs candidates regulating seed development have been carried out in rice, maize, barley (Hordeum vulgare L.), castor bean, rapeseed (Brassica napus), and Jatropha curcus, etc (Shen et al., 2018; Wang et al., 2019; Yan et al., 2020; Zhao et al., 2020; Zou et al., 2020; Pinky et al., 2023; Zheng et al., 2023). They have identified differentially expressed lncRNAs participating in starch/sucrose metabolism, gene expression regulation, and cell growth. While these provide comprehensive catalogs of lncRNAs potentially regulating endosperm development, extensive functional validation is required to elucidate specific molecular mechanisms.

Parental epigenetic asymmetry leads to monoallelic gene expression, a phenomenon termed genomic imprinting. In plants, imprinting primarily manifests in the endosperm, reflecting divergent epigenetic compositions between parental gametes (Muthusamy et al., 2024). Several lncRNAs exhibit imprinting effects, manifesting allelic expression divergence in plants. In hybrid endosperm of castor bean, most allelic lncRNAs displayed dosage insensitivity while conforming to the expected 2:1 maternal-to-paternal expression ratio. However, a small subset of allelic lncRNAs significantly deviated from the expected ratio, predominantly at genomic loci where substantial parental expression divergence was detected. This differential expression of allelic lncRNAs partially reflects parent-of-origin effects or genomic imprinting. Maternally imprinted lncRNAs were consistently clustered with maternally imprinted protein-coding genes, with some demonstrating strong positive correlation with adjacent imprinted protein-coding genes ( Figure 3D ). These findings suggest that imprinted lncRNAs may function within imprinting clusters.

Consistent with this, genome-wide analysis in rice endosperm identified 16 intergenic imprinted lncRNAs linked to imprinted genes. They were located near imprinted genes, or were transcribed oppositely to adjacent imprinted alleles, implying cis-regulation of neighboring imprinted genes (Yuan et al., 2017). Studies in maize have revealed more complex regulatory patterns: four maternally imprinted intronic lncRNAs are transcribed from paternally expressed genes. For instance, the GRMZM2G477503 locus produces a maternally expressed lncRNA (ZmMNC18) from its third intron, while its exonic regions exhibit paternal-specific expression. Zhang et al. hypothesize that these maternal lncRNAs may recruit silencing complexes to repress maternal alleles, thereby establishing paternal dominance in expression of these genes (Zhang et al., 2011).

Previously mentioned MISSEN is also maternally imprinted. CRISPR-Cas9-edited MISSEN transcripts were expressed in endosperm exclusively when mutant plants served as female parents, confirming maternal-specific expression (Zhou et al., 2021). Collectively, these studies suggest that imprinted lncRNAs may play specific roles in endosperm development.

Despite progress, research on imprinted lncRNAs remains in its infancy, with studies limited to few species. Mechanistic interactions between imprinted lncRNAs and genes require deeper validation, and connections to seed agronomic traits are underexplored.

lncRNAs can interact with sRNAs as precursors or endogenous miRNA target mimics (eTMs). A genome-wide identification in maize revealed >90% of lncRNAs are predicted sRNA precursors (Li et al., 2014). While proportions vary across species/studies (Liu et al., 2012; Palos et al., 2023), experimental evidence confirms that lncRNAs function as sRNA precursors during seed development. For example, rice lncRNA Long Hairpin-structure containing noncoding RNA (LHR) forms a hairpin structure serving as a phasiRNA precursor (Huang et al., 2019) ( Figure 3E ). Intriguingly, LHR-derived phasiRNA biogenesis depends on DCL4—but not RDR2/6—via a miRNA-like pathway. A T-DNA insertion mutant lhr, which blocks hairpin structure formation, causes multiple developmental defects including reduced rice grain size and weight. Degradome analysis suggests LHR-phasiRNAs target thylakoid-related genes to regulate photosynthesis, highlighting the importance of LHR-mediated phasiRNA production in seed development.

Another rice lncRNA, osa-eTM160, counteracts the inhibitory effects of osa-miR160 on osa-ARF18 transcripts through target mimicry, thereby influencing grain size (Wu et al., 2013; Wang et al., 2017b) ( Figure 3F ). Transgenic plants overexpressing osa-eTM160 exhibited reduced osa-miR160 levels, while plants expressing a mutant osa-eTM160 (insufficient miRNA pairing) exhibited no such decrease. Moreover, osa-eTM160 and osa-ARF18 share similar expression profiles, and their overexpression similarly reduces seed setting rate and seed size. This study highlights how lncRNAs act as stage-specific modulators to fine-tune sRNA-mediated gene regulation during restricted developmental windows of seeds.

Studies in other species have also initially revealed the interaction between lncRNAs and miRNAs in endosperm. For example, in wheat grains, lncRNA TCONS_00130663 interacts with miR1128, potentially regulating LYPL for lipid biosynthesis (Madhawan et al., 2020), implying an lncRNA-miRNA-mRNA regulatory axis during endosperm development. In maize seeds, a transcriptomic analysis revealed that four endosperm-specific expressed lncRNAs (lncRNA_71309, lncRNA_02785, lncRNA_86055, and lncRNA_58195) may act as eTMs in lncRNA-miRNA-mRNA co-expression networks enriched for carbon fixation, BR biosynthesis, and lipid metabolism (Zhu et al., 2017).

Taken together, current research has unveiled lncRNAs’ cellular regulation, metabolic intervention, and miRNA interaction mechanisms in endosperm development, providing multidimensional insights into seed development networks. Nevertheless, despite advancing omics screening, cross-species evolutionary conservation analyses and dynamic expression landscapes across entire grain developmental stages of lncRNAs still lack. Moreover, mechanistic studies on specific lncRNAs remain inadequate, constituting key bottlenecks for functional characterization and breeding applications.