Achieving seedlessness in citrus varieties is one of the important objectives of citrus breeding. Male sterility associated with abnormal pollen development is an important factor in seedlessness. However, our understanding of the regulatory mechanism underlying the seedlessness phenotype in citrus is still limited. Here, we determined that the miR159a-DUO1 module played an important role in regulating pollen development in citrus, which further indirectly modulated seed development and fruit size. Both the overexpression of csi-miR159a and the knocking out of DUO1 in Hong Kong kumquat (Fortunella hindsii) resulted in small and seedless fruit phenotypes. Moreover, pollen was severely aborted in both transgenic lines, with arrested pollen mitotic I and abnormal pollen starch metabolism. Through additional cross-pollination experiments, DUO1 was proven to be the key target gene for miR159a to regulate male sterility in citrus. Based on DNA affinity purification sequencing (DAP-seq), RNA-seq, and verified interaction assays, YUC2/YUC6, SS4 and STP8 were identified as downstream target genes of DUO1, those were all positively regulated by DUO1. In transgenic F. hindsii lines, the miR159a-DUO1 module down-regulated the expression of YUC2/ YUC6, which decreased indoleacetic acid (IAA) levels and modulated auxin signaling to repress pollen mitotic I. The miR159a-DUO1 module reduced the expression of the starch synthesis gene SS4 and sugar transport gene STP8 to disrupt starch metabolism in pollen. Overall, this work reveals a new mechanism by which the miR159a- DUO1 module regulates pollen development and elucidates the molecular regulatory network underlying male sterility in citrus.

In eukaryotes, RNA N⁶-methyladenosine (m⁶A) modification and microRNA (miRNA)-mediated RNA silencing represent two critical epigenetic regulatory mechanisms. The m⁶A methyltransferase complex (MTC) and the microprocessor complex both undergo liquid–liquid phase separation to form nuclear membraneless organelles. Although m⁶A methyltransferase has been shown to positively regulate miRNA biogenesis, a mechanism of reciprocal regulation between the MTC and the microprocessor complex has remained elusive. Here, we demonstrate that the MTC and the microprocessor complex associate with each other through the METHYLTRANSFERASE B (MTB)–SERRATE (SE) interacting module. Knockdown of MTB impaired miRNA biogenesis by diminishing microprocessor complex binding to primary miRNAs (pri-miRNAs) and their respective MIRNA loci. Additionally, loss of SE function led to disruptions in transcriptome-wide m⁶A modification. Further biochemical assays and fluorescence recovery after photobleaching (FRAP) assay indicated that SE enhances the liquid–liquid phase separation and solubility of the MTC. Moreover, the MTC exhibited enhanced retention on chromatin and diminished binding to its RNA substrates in the se mutant background. Collectively, our results reveal the substantial regulatory interplay between RNA m⁶A modification and miRNA biogenesis.

Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) 12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396-abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss-of-function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high-yield soybean.

RNA-binding proteins (RBPs) are components of the post-transcriptional regulatory system, but their regulatory effects on complex traits remain unknown. Using an integrated strategy involving map-based cloning, functional characterizations, and transcriptomic and population genomic analyses, we revealed that RBP-K (LOC_Os08g23120), RBP-A (LOC_Os11g41890), and RBP-J (LOC_Os10g33230) encode proteins that form an RBP-A-J-K complex that negatively regulates rice yield-related traits. Examinations of the RBP-A-J-K complex indicated RBP-K functions as a relatively non-specific RBP chaperone that enables RBP-A and RBP-J to function normally. Additionally, RBP-J most likely affects GA pathways, resulting in considerable increases in grain and panicle lengths, but decreases in grain width and thickness. In contrast, RBP-A negatively regulates the expression of genes most likely involved in auxin-regulated pathways controlling cell wall elongation and carbohydrate transport, with substantial effects on the rice grain filling process as well as grain length and weight. Evolutionarily, RBP-K is relatively ancient and highly conserved, whereas RBP-J and RBP-A are more diverse. Thus, the RBP-A-J-K complex may represent a typical functional model for many RBPs and protein complexes that function at transcriptional and post-transcriptional levels in plants and animals for increased functional consistency, efficiency, and versatility, as well as increased evolutionary potential. Our results clearly demonstrate the importance of RBP-mediated post-transcriptional regulation for the diversity of complex traits. Furthermore, rice grain yield and quality may be enhanced by introducing various complete or partial loss-of-function mutations to specific RBP genes using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology and by exploiting desirable natural tri-genic allelic combinations at the loci encoding the components of the RBP-A-J-K complex through marker-assisted selection.

Soybean (Glycine max) provides vegetable oils and proteins for human consumption. Its production depends on seeds and other production-related agronomic traits. How the seed traits are regulated in soybean remains largely unclear. In this study, we identified a miR172a-ERF416/413 module for the regulation of seed traits. The miR172a can cleave the targets ERF416 and ERF413 to affect the downstream gene expression for the reduction of soybean seed size and weight. Both the MIR172a-overexpressing transgenic soybean plants and the erf416/413 mutants produced smaller seeds than the control. Consistently, the ERF416-overexpressing transgenic soybean plants generated larger seeds. ERF416 and ERF413 were directly targeted to the promoter of GmKIX8-1 and GmSWEET10a to regulate their gene expression for seed size/weight control. Interestingly, the erf416/413 mutants showed higher seed yield per plant and higher total seed fatty acid (FA) content, whereas the MIR172a-transgenic soybean had lower total seed FA content compared with the control cultivar, suggesting that miR172a and ERF416/413 may function in FA accumulation through different pathways. Haplotypes of the ERF416 promoter region were further analyzed and Hap1 was correlated with higher gene expression and higher seed weight, while Hap3 was correlated with higher total seed lipid content. Our study revealed a new module for seed trait control. Manipulation of such alleles should facilitate breeding for high-oil and high-yield soybean cultivars.

Wheat culms, comprising four to six internodes, are critically involved in determining plant height and lodging resistance, essential factors for field performance and regional adaptability. This study revealed the regulatory function of miR319 in common wheat plant height. Repression of tae-miR319 through short tandem target mimics (STTM) caused an increased plant height, while overexpression (OE) of tae-miR319 had the opposite effect. Overexpressing a miR319-resistant target gene TaPCF8 (rTaPCF8), increased plant height. TaPCF8 acted as a transcription repressor of downstream genes TaIAAs, which interact physically with TaSPL14. The significant differences of indole-3-acetic acid (IAA) contents indicate the involvement of auxin pathway in miR319-mediated plant height regulation. Finally, we identified two TaPCF8 haplotypes in global wheat collections. TaPCF8-5A-Hap2, as per association and evolution examinations, was subjected to strong substantial selection throughout wheat breeding. This haplotype, associated with shorter plant height, aligns with global breeding requirements. Consequently, in high-yield wheat breeding, we proposed a potential molecular marker for marker-assisted selection (MAS). Our findings offer fresh perspectives into the molecular mechanisms that underlie the miR319–TaPCF8 module's regulation of plant height by orchestrating auxin signaling and biosynthesis in wheat.

Alternative splicing (AS) is a crucial post-transcriptional mechanism in plants, significantly contributing to the diversification of biological processes and adaptive responses. Distinct splice isoforms are generated by exon skipping (ES), intron retention (IR) and other mechanisms, enabling plants to adapt to a range of biotic stresses, including those posed by bacteria, fungi and viruses. Advances in bioinformatics have greatly improved the detection and characterization of AS events, revealing their critical roles in plant immunity. This review highlights the involvement of AS in regulating RNA interference (RNAi), hormone signaling pathways, and immune responses such as pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). In addition, pathogens exploit AS to produce effectors that subvert plant immunity. Beyond its role in natural immunity, AS also holds promise for pesticide development, offering opportunities to enhance plant disease resistance by targeting pest-associated or immunity-related genes. Future research on AS under biotic stress is expected to uncover novel regulatory mechanisms and provide new strategies for crop improvement and sustainable agriculture.

The development of rapeseed with high resistance against the pathogen Sclerotinia sclerotiorum is impeded by the lack of effective resistance resources within host species. Unraveling the molecular basis of nonhost resistance (NHR) holds substantial value for resistance improvement in crops. In the present study, small RNA sequencing and transcriptome sequencing were carried out between rice (a nonhost species of S. sclerotiorum) and rapeseed during infection, revealing the involvement of rice miRNAs on translation-related processes in both rice and the pathogen. Specifically, rice-specific miRNAs with potential capability for cross-kingdom RNAi against S. sclerotiorum were explored, of which Os-miR169y was selected as a representative case to elucidate its role in resistance to S. sclerotiorum. The silence of Os-miR169y decreased the resistance level of rice to S. sclerotiorum, and heterologous expression of Os-miR169y in Arabidopsis and rapeseed significantly enhanced the host resistance. The dual-luciferase reporter assay indicates that Os-miR169y targets S. sclerotiorum 60S ribosomal protein L19 (SsRPL19). Overexpressing Os-miR169y (OE_ss-miR169y) and RNAi of SsRPL19 (RNAi_ss-RPL19) in S. sclerotiorum significantly impaired the growth and pathogenicity of the pathogen, while overexpressing SsRPL19 exhibited a contrast effect. Yeast-two-hybridization revealed an interlinking role of SsRPL19 with multiple large and small ribosomal subunits, indicating its important role in translation. Proteome sequencing detected a decreased amount of proteins in transformants OE_ss-miR169y and RNAi_ss-RPL19 and significant suppression on key metabolic pathways such as carbon and nitrogen metabolisms. Collectively, this study suggests that rice can secrete specific miRNAs to suppress genes essential for S. sclerotiorum, such as Os-miR169y, which targets and suppresses SsRPL19 and thus impairs protein synthesis in the pathogen. This study sheds light on the intrinsic mechanisms of rice NHR against S. sclerotiorum, and further demonstrates the potential of using nonhost-specific “pathogen-attacking” miRNAs in improving resistance in host species.

In the chloroplasts/plastids and mitochondria of flowering plants, RNA editing alters hundreds of cytidines to uridines at specific sites mediated by the editosome. Over the past decade, Multiple Organellar RNA Editing Factor (MORF) proteins have emerged as essential regulators that affect the editing efficiency of most editing sites in plastids and mitochondria. In Arabidopsis, the MORF family consists of nine members, each possessing a single conserved MORF-box that is distributed among flowering plants. Accumulating studies have demonstrated that MORF proteins interact with many other factors, including the PPR proteins and enzymes in different biosynthetic pathways, indicating that the MORF proteins play a more extensive role in regulating organellar development than RNA editing. Recent studies reveal that MORF2 and MORF9 possess holdase activity and may act as chaperones and that MORF8 undergoes heat-dependent phase separation to inhibit RNA editing in chloroplasts. In this review, we provide an overview of our current knowledge of the MORF family proteins and discuss the biological and molecular functions of this family in plants.

INOSITOL-REQUIRING ENZYME 1 (IRE1) is conserved in plants and mammals to regulate stress responses. Here, we found that TaIRE1 is involved in the unconventional splicing of cell membrane-localized TabZIP60 messenger RNA (mRNA), which results in a nucleus resident protein form (TabZIP60s), and enhanced heat stress tolerance. Transcriptome analysis together with binding element prediction revealed 121 high-confidence targets of TabZIP60s responsive to heat stress in wheat (Triticum aestivum), including heat shock protein genes. Interestingly, we found that an asparagine to glutamic acid substitution, located next to DNA-binding domain of TabZIP60s, results in reduced binding affinity and transcriptional activity to downstream targets, and this heat stress tolerance inferior allele was positively selected during modern wheat breeding programs in China, possibly due to their negative effects on yield potential. Finally, we showed that TaIRE1 is also responsible for the mis-cleavage of miR172 precursors, and consequently contribute to heat stress tolerance. To the best of our knowledge, this represents the first report showing that, like in mammals, IRE1 also regulates miRNA cleavage in response to heat stress in plants. Together, this coordinate control of two signaling pathways provides new insights into heat stress tolerance regulation in wheat.

Cytoplasmic male sterile (CMS) lines play a crucial role in utilization of heterosis in crop plants. However, the mechanism underlying the manipulation of male sterility in cotton by long non-coding RNA (lncRNA) and brassinosteroids (BRs) remains elusive. Here, using an integrative approach combining lncRNA transcriptomic profiles with virus-induced gene silencing experiments, we identify a flower bud-specific lncRNA in the maintainer line 2074B, lncRNA67, negatively modulating with male sterility in upland cotton (Gossypium hirsutum). lncRNA67 positively regulates cytochrome P274B (GhCYP724B), which acted as an eTM (endogenous target mimic) for miR3367. The suppression of GhCYP724B induced symptoms of BR deficiency and male semi-sterility in upland cotton as well as in tobacco, which resulted from a reduction in the endogenous BR contents. GhCYP724B regulates BRs synthesis by interacting with GhDIM and GhCYP90B, two BRs biosynthesis proteins. Additionally, GhCYP724B suppressed a unique chimeric open reading frame (Aorf27) in 2074A mitochondrial genome. Ectopic expression of Aorf27 in yeast inhibited cellular growth, and over expression of Aorf27 in tobacco showed male sterility. Overall, the results proved that the miR3367–lncRNA67–GhCYP724B module positively regulates male sterility by modulating BRs biosynthesis. The findings uncovered the function of lncRNA67–GhCYP724B in male sterility, providing a new mechanism for understanding male sterility in upland cotton.

Circular RNAs (circRNAs), a type of head-to-tail closed RNA molecules, have been implicated in various aspects of plant development and stress responses through transcriptome sequencing; however, the precise functional roles of circRNAs in plants remain poorly understood. In this study, we identified a highly expressed circular RNA, circZmMED16, derived from exon 8 of the mediator complex subunit 16 (ZmMED16) across different maize (Zea mays L.) inbred lines using circRNA-seq analysis. This circRNA is predominantly expressed in maize tassels and functions in the cytoplasm. Overexpression of circZmMED16 resulted in increased expression of ZmMED16/AtMED16 and delayed flowering in both maize and Arabidopsis thaliana, compared with that in wild-type plants. In contrast, overexpression of the parent gene ZmMED16 did not alter the flowering time of transgenic plants in Arabidopsis, suggesting that circZmMED16 plays a specific role in regulating flowering, distinct from that of linear ZmMED16. To further understand the mechanisms underlying the regulation of flowering time by circZmMED16, we performed RNA pull-down, dual-luciferase, RNA interference (RNAi), and ribonuclease protection assays (RPA). These results indicate that circZmMED16 interacts with small subunit 1 of ADP-glucose pyrophosphorylase (APS1) mRNA in both maize and Arabidopsis. The knockdown of circZmMED16 increased the expression of ZmAPS1, whereas the overexpression of circZmMED16 led to the downregulation of ZmAPS1 RNA and protein. By affecting ZmAPS1 expression, circZmMED16 reduced ADP-glucose pyrophosphorylase (AGPase) activity and led to delayed flowering. These results revealed a novel regulatory mechanism for circRNAs in flowering time and shed light on their functional and regulatory roles in plants.

Root-knot nematodes (RKNs; Meloidogyne spp.) are a serious threat to crop production. The competition between plants and pathogens for assimilates influences the outcome of their interactions. However, the mechanisms by which plants and nematodes compete with each other for assimilates have not been elucidated. In this study, we demonstrated that miR396a plays a negative role in defense against RKNs and a positive role in sugar accumulation in tomato roots. The overexpression of SlGRF8 (Solanum lycopersicum growth-regulating factor 8), the target of miR396a, decreased the sugar content of the roots and the susceptibility to RKNs, whereas the grf8-cr mutation had the opposite effects. Furthermore, we confirmed that SlGRF8 regulated the sugar content in roots by directly activating the transcription of SlSTP10 (Solanum lycopersicum sugar transporter protein 10) in response to RKN stress. Moreover, SlSTP10 was expressed primarily in the tissues surrounding giant cells, and the SlSTP10 knockout increased both the sugar content in the roots and the plant's susceptibility to RKNs. Overall, this study provides important insight into the molecular mechanism through which the miR396a-SlGRF8-SlSTP10 module regulates sugar allocation in roots under RKN stress.

Modifications to RNA have recently been recognized as a pivotal regulator of gene expression in living organisms. More than 170 chemical modifications have been identified in RNAs, with N⁶-methyladenosine (m⁶A) being the most abundant modification in eukaryotic mRNAs. The addition and removal of m⁶A marks are catalyzed by methyltransferases (referred to as “writers”) and demethylases (referred to as “erasers”), respectively. In addition, the m⁶A marks in mRNAs are recognized and interpreted by m⁶A-binding proteins (referred to as “readers”), which regulate the fate of mRNAs, including stability, splicing, transport, and translation. Therefore, exploring the mechanism underlying the m⁶A reader-mediated modulation of RNA metabolism is essential for a much deeper understanding of the epigenetic role of RNA modification in plants. Recent discoveries have improved our understanding of the functions of m⁶A readers in plant growth and development, stress response, and disease resistance. This review highlights the latest developments in m⁶A reader research, emphasizing the diverse RNA-binding domains crucial for m⁶A reader function and the biological and cellular roles of m⁶A readers in the plant response to developmental and environmental signals. Moreover, we propose and discuss the potential future research directions and challenges in identifying novel m⁶A readers and elucidating the cellular and mechanistic role of m⁶A readers in plants.

Disease resistance is often associated with compromised plant growth and yield due to defense-growth tradeoffs. However, key components and mechanisms underlying the defense-growth tradeoffs are rarely explored in maize. In this study, we find that ZmSKI3, a putative subunit of the SUPERKILLER (SKI) complex that mediates the 3′-5′ degradation of RNA, regulates both plant development and disease resistance in maize. The Zmski3 mutants showed retarded plant growth and constitutively activated defense responses, while the ZmSKI3 overexpression lines are more susceptible to Curvularia lunata and Bipolaris maydis. Consistently, the expression of defense-related genes was generally up-regulated, while expressions of growth-related genes were mostly down-regulated in leaves of the Zmski3-1 mutant compared to that of wild type. In addition, 223 differentially expressed genes that are up-regulated in Zmski3-1 mutant but down-regulated in the ZmSKI3 overexpression line are identified as potential target genes of ZmSKI3. Moreover, small interfering RNAs targeting the transcripts of the defense- and growth-related genes are differentially accumulated, likely to combat the increase of defense-related transcripts but decrease of growth-related transcripts in Zmski3-1 mutant. Taken together, our study indicates that plant growth and immunity could be regulated by both ZmSKI3-mediated RNA decay and post-transcriptional gene silencing in maize.