Although the frequency of ancient hybridization across the Tree of Life is greater than previously thought, little work has been devoted to uncovering the extent, timeline, and geographic and ecological context of ancient hybridization. Using an expansive new dataset of nuclear and chloroplast DNA sequences, we conducted a multifaceted phylogenomic investigation to identify ancient reticulation in the early evolution of oaks (Quercus). We document extensive nuclear gene tree and cytonuclear discordance among major lineages of Quercus and relatives in Quercoideae. Our analyses recovered clear signatures of gene flow against a backdrop of rampant incomplete lineage sorting, with gene flow most prevalent among major lineages of Quercus and relatives in Quercoideae during their initial radiation, dated to the Early-Middle Eocene. Ancestral reconstructions including fossils suggest ancestors of Castanea + Castanopsis, Lithocarpus, and the Old World oak clade probably co-occurred in North America and Eurasia, while the ancestors of Chrysolepis, Notholithocarpus, and the New World oak clade co-occurred in North America, offering ample opportunity for hybridization in each region. Our study shows that hybridization—perhaps in the form of ancient syngameons like those seen today—has been a common and important process throughout the evolutionary history of oaks and their relatives. Concomitantly, this study provides a methodological framework for detecting ancient hybridization in other groups.

Plants, algae and photosynthetic bacteria convert light into chemical energy by means of photosynthesis, thus providing food and energy for most organisms on Earth. Photosynthetic pigments, including chlorophylls (Chls) and carotenoids, are essential components that absorb the light energy necessary to drive electron transport in photosynthesis. The biosynthesis of Chl shares several steps in common with the biosynthesis of other tetrapyrroles, including siroheme, heme and phycobilins. Given that many tetrapyrrole precursors possess photo-oxidative properties that are deleterious to macromolecules and can lead to cell death, tetrapyrrole biosynthesis (TBS) requires stringent regulation under various developmental and environmental conditions. Thanks to decades of research on model plants and algae, we now have a deeper understanding of the regulatory mechanisms that underlie Chl synthesis, including (i) the many factors that control the activity and stability of TBS enzymes, (ii) the transcriptional and post-translational regulation of the TBS pathway, and (iii) the complex roles of tetrapyrrole-mediated retrograde signaling from chloroplasts to the cytoplasm and the nucleus. Based on these new findings, Chls and their derivatives will find broad applications in synthetic biology and agriculture in the future.

Both the filling and development of grain are key processes determining agriculture production and reproductive growth in rice. The processes of grain filling and endosperm development are crucial for the accumulation of major storage compounds in rice grains. This requires extensive remobilization of carbon reserves from source to sink and the precise regulation of sucrose-to-starch conversion. Both the developmental sequence of the panicle and environmental signals influence the carbon flow between the leaves, leaf sheath, stem, and spikelets during grain filling. This, in turn, affects endosperm development and the production of storage compounds. In this review, we synthesize recent insight into grain development in rice, focusing on the dynamic changes in phytohormones and how their homeostasis integrates developmental and environmental cues to control grain filling in the developing panicle. We also highlight recent advances in the genetic control of carbohydrate remobilization and the transcriptional regulatory networks governing carbohydrate metabolism and grain development in rice. The asynchronous initiation and imbalance in grain filling limit the full yield potential of cereal crops. The “superior/inferior spikelets” serve as a model system for understanding the regulatory mechanisms underlying grain filling and development. Systematic research on carbohydrate flow and phytohormone crosstalk could enhance our understanding of optimizing yield production in cereal crops. Additionally, a thorough analysis of key genetic regulatory mechanisms can offer a genetic foundation and targets for precisely adjusting grain filling traits, ultimately aiding in the development of high-yield crop varieties.

Plants have evolved a remarkable ability to sense and respond to changes in photoperiod, allowing adjustments to their growth and development based on seasonal and environmental cues. The floral transition is a pivotal stage in plant growth and development, signifying a shift from vegetative to reproductive growth. CONSTANS (CO), a central photoperiodic response factor conserved in various plants, mediates day-length signals to control the floral transition, although its mechanisms of action vary among plants with different day-length requirements. In addition, recent studies have uncovered roles for CO in organ development and stress responses. These pleiotropic roles in model plants and crops make CO a potentially fruitful target for molecular breeding aimed at modifying crop agronomic traits. This review systematically traces research on CO, from its discovery and functional studies to the exploration of its regulatory mechanisms and newly discovered functions, providing important insight into the roles of CO and laying a foundation for future research.

The past decade has witnessed rapid developments in gene discovery, biological big data (BBD), artificial intelligence (AI)-aided technologies, and molecular breeding. These advancements are expected to accelerate crop breeding under the pressure of increasing demands for food. Here, we first summarize current breeding methods and discuss the need for new ways to support breeding efforts. Then, we review how to combine BBD and AI technologies for genetic dissection, exploring functional genes, predicting regulatory elements and functional domains, and phenotypic prediction. Finally, we propose the concept of intelligent precision design breeding (IPDB) driven by AI technology and offer ideas about how to implement IPDB. We hope that IPDB will enhance the predictability, efficiency, and cost of crop breeding compared with current technologies. As an example of IPDB, we explore the possibilities offered by CropGPT, which combines biological techniques, bioinformatics, and breeding art from breeders, and presents an open, shareable, and cooperative breeding system. IPDB provides integrated services and communication platforms for biologists, bioinformatics experts, germplasm resource specialists, breeders, dealers, and farmers, and should be well suited for future breeding.

Land plants use diverse hormones to coordinate their growth, development and responses against biotic and abiotic stresses. Salicylic acid (SA) is an essential hormone in plant immunity, with its levels and signaling tightly regulated to ensure a balanced immune output. Over the past three decades, molecular genetic analyses performed primarily in Arabidopsis have elucidated the biosynthesis and signal transduction pathways of key plant hormones, including abscisic acid, jasmonic acid, ethylene, auxin, cytokinin, brassinosteroids, and gibberellin. Crosstalk between different hormones has become a major focus in plant biology with the goal of obtaining a full picture of the plant hormone signaling network. This review highlights the roles of SA in plant immunity and summarizes our current understanding of the pairwise interactions of SA with other major plant hormones. The complexity of these interactions is discussed, with the hope of stimulating research to address existing knowledge gaps in hormone crosstalk, particularly in the context of balancing plant growth and defense.

The rhizosphere plays a crucial role in plant growth and resilience to biotic and abiotic stresses, highlighting the complex communication between plants and their dynamic rhizosphere environment. Plants produce a wide range of signaling molecules that facilitate communication with various rhizosphere factors, yet our understanding of these mechanisms remains elusive. In addition to protein-coding genes, increasing evidence underscores the critical role of microRNAs (miRNAs), a class of non-coding single-stranded RNA molecules, in regulating plant growth, development, and responses to rhizosphere stresses under diverse biotic and abiotic factors. In this review, we explore the crosstalk between miRNAs and their target mRNAs, which influence the development of key plant structures shaped by the belowground environment. Moving forward, more focused studies are needed to clarify the functions and expression patterns of miRNAs, to uncover the common regulatory mechanisms that mediate plant tolerance to rhizosphere dynamics. Beyond that, we propose that using artificial miRNAs and manipulating the expression of miRNAs and their targets through overexpression or knockout/knockdown approaches could effectively investigate their roles in plant responses to rhizosphere stresses, offering significant potential for advancing crop engineering.

Small peptides (SPs) are pivotal signaling molecules that play essential roles in the precise regulation of plant growth, development, and stress responses. Recent advancements in sequencing technologies, bioinformatics approaches, and biochemical and molecular techniques have significantly enhanced the accuracy of SP identification, unveiling their diverse biological functions in plants. This review provides a comprehensive overview of the characteristics and methodologies for identifying SPs in plants. It highlights recent discoveries regarding the biological roles and signaling pathways of SPs in regulating plant growth, development, and plant–microbial interactions, as well as their contributions to plant resilience under various environmental stresses, including abiotic stress, nutrient deficiencies, and biotic challenges. Additionally, we discuss current insights into the potential applications of SPs and outline future research directions aimed at leveraging these molecules to enhance plant adaptation to environmental challenges. By integrating recent findings, this review lays a foundation for advancing the understanding and utilization of SPs to improve plant resilience and productivity.

The modern cultivated tomato (Solanum lycopersicum) was domesticated from Solanum pimpinellifolium native to the Andes Mountains of South America through a “two-step domestication” process. It was introduced to Europe in the 16th century and later widely cultivated worldwide. Since the late 19th century, breeders, guided by modern genetics, breeding science, and statistical theory, have improved tomatoes into an important fruit and vegetable crop that serves both fresh consumption and processing needs, satisfying diverse consumer demands. Over the past three decades, advancements in modern crop molecular breeding technologies, represented by molecular marker technology, genome sequencing, and genome editing, have significantly transformed tomato breeding paradigms. This article reviews the research progress in the field of tomato molecular breeding, encompassing genome sequencing of germplasm resources, the identification of functional genes for agronomic traits, and the development of key molecular breeding technologies. Based on these advancements, we also discuss the major challenges and perspectives in this field.

Ethylene treatment promotes orange coloration in the flavedo of Satsuma mandarin (Citrus unshiu Marc.) fruit, but the corresponding regulatory mechanism is still largely unknown. In this study, we identified a C2H2-type zinc-finger transcription factor, CitZAT4, the expression of which was markedly induced by ethylene. CitZAT4 directly binds to the CitPSY promoter and activates its expression, thereby promoting carotenoid biosynthesis. Transient expression in Satsuma mandarin fruit and stable transformation of citrus calli showed that overexpressing of CitZAT4 inhibited CitLCYE expression, thus inhibiting α-branch yellow carotenoid (lutein) biosynthesis. CitZAT4 overexpression also enhanced the transcript levels of CitLCYB, CitHYD, and CitNCED2, promoting β-branch orange carotenoid accumulation. Molecular biochemical assays, including yeast one-hybrid (Y1H), electrophoretic mobility shift (EMSA), chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR), and luciferase (LUC) assays, demonstrated that CitZAT4 directly binds to the promoters of its target genes and regulates their expression. An ethylene response factor, CitERF061, which is induced by ethylene signaling, was found to directly bound to the CitZAT4 promoter and induced its expression, thus positively regulating CitZAT4-mediated orange coloration in citrus fruit. Together, our findings reveal that a CitZAT4-mediated transcriptional cascade is driven by ethylene via CitERF061, linking ethylene signaling to carotenoid metabolism in promoting orange coloration in the flavedo of Satsuma mandarin fruit. The molecular regulatory mechanism revealed here represents a significant step toward developing strategies for improving the quality and economic efficiency of citrus crops.

Fruit taste quality is greatly influenced by the content of soluble sugars, which are predominantly stored in the vacuolar lumen. However, the accumulation and regulation mechanisms of sugars in most fruits remain unclear. Recently, we established the citrus fruit vacuole proteome and discovered the major transporters localized in the vacuole membrane. Here, we demonstrated that the expression of tonoplast sugar transporter 2 (CsTST2) is closely associated with sugar accumulation during sweet orange (Citrus sinensis) ripening. It was further demonstrated that CsTST2 had the function of transporting hexose and sucrose into the vacuole. Overexpression of CsTST2 resulted in an elevation of sugar content in citrus juice sac, calli, and tomato fruit, whereas the downregulation of its expression led to the reduction in sugar levels. CsTST2 was identified as interacting with CsCIPK23, which binds to the upstream calcium signal sensor protein CsCBL1. The phosphorylation of the three serine residues (Ser277, Ser337, and Ser354) in the loop region of CsTST2 by CsCIPK23 is crucial for maintaining the sugar transport activity of CsTST2. Additionally, the expression of CsCIPK23 is positively correlated with sugar content. Genetic evidence further confirmed that calcium and CsCIPK23-mediated increase in sugar accumulation depends on CsTST2 and its phosphorylation level. These findings not only unveil the functional mechanism of CsTST2 in sugar accumulation, but also explore a vital calcium signal regulation module of CsCBL1/CIPK23 for citrus sweetness quality.

Starch biosynthesis is a critical factor in wheat (Triticum aestivum L.) quality and yield. However, the full scope of its regulation is not fully understood. Here we report that TaDL interacts with TaB3 and TaNF-YB1 to synergistically regulate starch biosynthesis and quality in wheat. Genome-edited tadl mutant lines had smaller and lighter grains with lower total starch and amylose contents compared to wild type (WT). Correspondingly, the transcript levels of starch biosynthesis-related genes, including TaSUS1, TaSUS2, TaAGPL2, TaSBEIIa, TaGBSSII, and TaSWEET2a, were markedly lower at 15 d after flowering (DAF) in tadl mutants. TaDL physically interacted with TaB3 and TaNF-YB1 and activated the transcription of TaSUS2 and TaAGPL2 through direct binding to their promoter regions. A null mutant of TaB3 also affected grain filling, with phenotypes similar to those of tadl mutants, whereas overexpression of TaNF-YB1 promoted grain filling. Our study demonstrated that TaDL plays an essential role in starch biosynthesis and identified an elite allele (TaDL-BI) associated with starch content, providing insights into the underlying molecular mechanism of wheat grain filling, which may be useful in breeding of high-yielding wheat and quality improvement.

The publication of several high-quality genomes has contributed greatly to clarifying the evolution of citrus. However, due to their complex genetic backgrounds, the origins and evolution of many citrus species remain unclear. We assembled de novo the 294-Mbp chromosome-level genome of a more than 200-year-old primitive papeda (DYC002). Comparison between the two sets of homologous chromosomes of the haplotype-resolved genome revealed 1.2% intragenomic variations, including 1.75 million SNPs, 149,471 insertions and 154,215 deletions. Using this genome as a reference, we resequenced and performed population and phylogenetic analyses of 378 representative citrus accessions. Our study confirmed that the primary origin center of core Citrus species is in South China, particularly in the Himalaya–Hengduan Mountains. Papeda species are an ancient Citrus type compared with C. ichangensis. We found that the evolution of the Citrus genus followed two radiations through two routes (to East China and Southeast Asia) along river systems. Evidence for the origin and evolution of some individual citrus species was provided. Papeda probably played an important role in the origins of Australian finger lime, citrons, Honghe papeda and pummelos; Ichang papeda originated from Yuanjiang city of Yunnan Province, China, and C. mangshanensis has a close relationship with kumquat and Ichang papeda. Moreover, the Hunan and Guangdong Provinces of China are predicted to be the origin center of mandarin, sweet orange and sour orange. Additionally, our study revealed that fruit bitterness was significantly selected against during citrus domestication. Taken together, this study provides new insight into the origin and evolution of citrus species and may serve as a valuable genomic resource for citrus breeding and improvement.

Autophagy is a highly conserved cellular program in eukaryotic cells which mediates the degradation of cytoplasmic components through the lysosome, also named the vacuole in plants. However, the molecular mechanisms underlying the fusion of autophagosomes with the vacuole remain unclear. Here, we report the functional characterization of a rice (Oryza sativa) mutant with defects in storage protein transport in endosperm cells and accumulation of numerous autophagosomes in root cells. Cytological and immunocytochemical experiments showed that this mutant exhibits a defect in the fusion between autophagosomes and vacuoles. The mutant harbors a loss-of-function mutation in the rice homolog of Arabidopsis thaliana MONENSIN SENSITIVITY1 (MON1). Biochemical and genetic evidence revealed a synergistic interaction between rice MON1 and AUTOPHAGY-RELATED 8a in maintaining normal growth and development. In addition, the rice mon1 mutant disrupted storage protein sorting to protein storage vacuoles. Furthermore, quantitative proteomics verified that the loss of MON1 function influenced diverse biological pathways including autophagy and vacuolar transport, thus decreasing the transport of autophagic and vacuolar cargoes to vacuoles. Together, our findings establish a molecular link between autophagy and vacuolar protein transport, and offer insights into the dual functions of the MON1–CCZ1 (CAFFEINE ZINC SENSITIVITY1) complex in plants.

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.

Cotton (Gossypium hirsutum) fibers are elongated single cells that rapidly accumulate cellulose during secondary cell wall (SCW) thickening, which requires cellulose synthase complex (CSC) activity. Here, we describe the CSC-interacting factor CASPARIAN STRIP MEMBRANE DOMAIN-LIKE1 (GhCASPL1), which contributes to SCW thickening by influencing CSC stability on the plasma membrane. GhCASPL1 is preferentially expressed in fiber cells during SCW biosynthesis and encodes a MARVEL domain protein. The ghcaspl1 ghcaspl2 mutant exhibited reduced plant height and produced mature fibers with fewer natural twists, lower tensile strength, and a thinner SCW compared to the wild type. Similarly, the Arabidopsis (Arabidopsis thaliana) caspl1 caspl2 double mutant showed a lower cellulose content and thinner cell walls in the stem vasculature than the wild type but normal plant morphology. Introducing the cotton gene GhCASPL1 successfully restored the reduced cellulose content of the Arabidopsis caspl1 caspl2 mutant. Detergent treatments, ultracentrifugation assays, and enzymatic assays showed that the CSC in the ghcaspl1 ghcaspl2 double mutant showed reduced membrane binding and decreased enzyme activity compared to the wild type. GhCASPL1 binds strongly to phosphatidic acid (PA), which is present in much higher amounts in thickening fiber cells compared to ovules and leaves. Mutating the PA-binding site in GhCASPL1 resulted in the loss of its colocalization with GhCesA8, and it failed to localize to the plasma membrane. PA may alter membrane structure to facilitate protein–protein interactions, suggesting that GhCASPL1 and PA collaboratively stabilize the CSC. Our findings shed light on CASPL functions and the molecular machinery behind SCW biosynthesis in cotton fibers.

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.

Plants need to fine-tune defense responses to maintain a robust but flexible host barrier to various pathogens. Helix-loop-helix/basic helix-loop-helix (HLH/bHLH) complexes play important roles in fine-tuning plant development. However, the function of these genes in plant immunity and how they are regulated remain obscure. Here, we identified an atypical bHLH transcription factor, Oryza sativa (Os)HLH46, that interacts with rice receptor-like cytoplasmic kinase (RLCK) Os BRASSINOSTEROID-SIGNALING KINASE1-2 (OsBSK1-2), which plays a key role in rice blast resistance. OsBSK1-2 stabilized OsHLH46 both in vivo and in vitro. In addition, OsHLH46 positively regulates rice blast resistance, which depends on OsBSK1-2. OsHLH46 has no transcriptional activation activity and interacts with a typical bHLH protein, OsbHLH6, which negatively regulates rice blast resistance. OsbHLH6 binds to the promoter of OsWRKY45 and inhibits its expression, while OsHLH46 suppresses the function of OsbHLH6 by blocking its DNA binding and transcriptional inhibition of OsWRKY45. Consistent with these findings, OsWRKY45 was up-regulated in OsHLH46-overexpressing plants. In addition, the oshlh46 mutant overexpressing OsbHLH6 is more susceptible to Magnaporthe oryzae than is the wild type, suggesting that OsHLH46 suppresses OsbHLH6-mediated rice blast resistance. Our results not only demonstrated that OsBSK1-2 regulates rice blast resistance via the OsHLH46/OsbHLH6 complex, but also uncovered a new mechanism for plants to fine-tune plant immunity by regulating the HLH/bHLH complex via RLCKs.

Sucrose functions as a signaling molecule in several metabolic pathways as well as in various developmental processes. However, the molecular mechanisms by which sucrose regulates these processes remain largely unknown. In the present study, we demonstrate that sucrose promotes flowering by mediating the stability of a regulatory protein that represses flowering in rice. Exogenous application of sucrose promoted flowering by inducing florigen gene expression. Reduction of sucrose levels in the phloem through genetic modifications, such as the overexpression of the vacuolar invertase OsVIN2 or the mutation of OsSUT2, a sucrose transporter, delayed flowering. Analysis of relative transcript levels of floral regulatory genes showed that sucrose activated Ehd1 upstream of the florigen, with no significant effect on the expression of other upstream genes. Examination of protein stability after sucrose treatment of major floral repressors revealed that the Ghd7 protein was specifically degraded. The Ghd7 protein interacted with the E3 ligase IPA INTERACTING PROTEIN1 (IPI1), and sucrose-induced K48-linked polyubiquitination of Ghd7 via IPI1, leading to protein degradation. Mutants defective in IPI1 delayed flowering, confirming its role in modulating proteins involved in flowering. We conclude that sucrose acts as a signaling molecule to induce flowering by promoting Ghd7 degradation via IPI1.

Flowering time is a crucial rice trait that influences its adaptation to various environments, cropping schedules, and agronomic characteristics. Rice breeders have exploited spontaneous mutations in heading date genes to regulate the flowering time. In the present study, we investigated how breeders in Fukui Prefecture regulated days to heading while developing promising rice varieties. Genome-wide association studies (GWAS) identified Hd1, Hd16, and Hd18 as the major genes controlling days to heading in the population. However, we suspected that this highly bred population might exhibit genomic stratification, which could lead to spurious or false correlations in the GWAS. Thus, we also conducted correlation and partial correlation analyses, which uncovered another key heading date gene, Hd17, that GWAS failed to detect because of its linkage disequilibrium with the major effect gene Hd16. Examination of haplotype frequencies across different breeding periods revealed that the early-heading Hd16 (Hd16(E)) and late-heading Hd17 (Hd17(L)) were increasingly co-selected in the Hd1 functional population. Varieties carrying this Hd16(E)/Hd17(L) combination exhibited days to heading in the range of 70–80, which corresponds to the peak temperature and sunshine period and is also optimal for grain quality and yield components in the Fukui environment. The present study highlights that it is imperative to remain vigilant for Type I (false positives) and Type II (false negatives) errors when performing GWAS on highly bred populations and to implement appropriate countermeasures by accounting for gene-by-gene interactions established through the breeding process. We also discuss the effectiveness of Hd16(E), which is not used outside Japan for subtle days to heading control but is widely used in Japan at certain latitudes.

Grain size and grain weight are important determinants for grain yield. In this study, we identify a novel OsMAPK5–OsWRKY72 module that negatively regulates grain length and grain weight in rice. We found that loss-of-function of OsMAPK5 leads to larger cell size of the rice spikelet hulls and a significant increase in both grain length and grain weight in an indica variety Minghui 86 (MH86). OsMAPK5 interacts with OsMAPKK3/4/5 and OsWRKY72 and phosphorylates OsWRKY72 at T86 and S88. Similar to the osmapk5 MH86 mutants, the oswrky72 knockout MH86 mutants exhibited larger size of spikelet hull cells and increased grain length and grain weight, whereas the OsWRKY72-overexpression MH86 plants showed opposite phenotypes. OsWRKY72 targets the W-box motifs in the promoter of OsARF6, an auxin response factor involved in auxin signaling. Dual-luciferase reporter assays demonstrated that OsWRKY72 activates OsARF6 expression. The activation effect of the phosphorylation-mimicking OsWRKY72^T86D/S88D on OsARF6 expression was significantly enhanced, whereas the effects of the OsWRKY72 phosphorylation-null mutants were significantly reduced. In addition, auxin levels in young panicles of the osmapk5 and oswrky72 mutants were significantly higher than that in the wild-type MH86. Collectively, our study uncovered novel connections of the OsMAPKK3/4/5-OsMAPK5-mediated MAPK signaling, OsWRKY72-mediated transcription regulation, and OsARF6-mediated auxin signaling pathways in regulating grain length and grain weight in an indica-type rice, providing promising targets for molecular breeding of rice varieties with high yield and quality.

It has been proposed that cortical fine actin filaments are needed for the morphogenesis of pavement cells (PCs). However, the precise role and regulation mechanisms of actin filaments in PC morphogenesis are not well understood. Here, we found that Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTOR9 (ADF9) is required for the morphogenesis of PC, which is negatively regulated by the R2R3 MYELOBLASTOSIS (MYB) transcription factor MYB52. In adf9 mutants, the lobe number of cotyledon PCs was significantly reduced, while the average lobe length did not differ significantly compared to that of wild type (Col-0), except for the variations in cell area and circularity, whereas the PC shapes in ADF9 overexpression seedlings showed different results. ADF9 decorated actin filaments, and colocalized with plasma membrane. The extent of filament bundling and actin filament bundling activity in adf9 mutant decreased. In addition, MYB52 directly targeted the promoter of ADF9 and negatively regulated its expression. The myb52-2 mutant showed increased lobe number and cell area, reduced cell circularity of PCs, and the PC phenotypes were suppressed when ADF9 was knocked out. Taken together, our data demonstrate that actin filaments play an important role in the morphogenesis of PC and reveal a transcriptional mechanism underlying MYB52 regulation of ADF9-mediated actin filament bundling in PC morphogenesis.

Leaves play a crucial role in the growth and development of rice (Oryza sativa) as sites for the production of photosynthesis. Early leaf senescence leads to substantial drops in rice yields. Whether and how DNA methylation regulates gene expression and affects leaf senescence remains elusive. Here, we demonstrate that mutations in rice ARGONAUTE 2 (OsAGO2) lead to premature leaf senescence, with chloroplasts in Osago2 having lower chlorophyll content and an abnormal thylakoid structure compared with those from wild-type plants. We show that OsAGO2 associates with a 24-nt microRNA and binds to the promoter region of OsNAC300, which causes DNA methylation and suppressed expression of OsNAC300. Overexpressing OsNAC300 causes the similar premature leaf senescence as Osago2 mutants and knocking out OsNAC300 in the Osago2 mutant background suppresses the early senescence of Osago2 mutants. Based on yeast one-hybrid, dual-luciferase, and electrophoresis mobility shift assays, we propose that OsNAC300 directly regulates transcription of the key rice aging gene NAC-like, activated by APETALA3/PISTILLATA (OsNAP) to control leaf senescence. Our results unravel a previously unknown epigenetic regulatory mechanism underlying leaf senescence in which OsAGO2–OsNAC300–OsNAP acts as a key regulatory module of leaf senescence to maintain leaf function.

During their co-evolution with herbivorous insects, plants have developed multiple defense strategies that resist pests, such as releasing a blend of herbivory-induced plant volatiles (HIPVs) that repel pests or recruit their natural enemies. However, the responses of insects to HIPVs in maize (Zea mays L.) are not well understood. Here, we demonstrate that the Asian corn borer (ACB, Ostrinia furnacalis), a major insect pest of maize, shows a preference for maize pre-infested with ACB larvae rather than being repelled by these plants. Through combined transcriptomic and metabolomics analysis of ACB-infested maize seedlings, we identified two substances that explain this behavior: (E)-4,8-dimethylnona-1,3,7-triene (DMNT) and (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). DMNT and TMTT attracted ACB larvae, and knocking out the maize genes responsible for their biosynthesis via gene editing impaired this attraction. External supplementation with DMNT/TMTT hampered the larvae's ability to locate pre-infested maize. These findings uncover a novel role for DMNT and TMTT in driving the behavior of ACB. Genetic modification of maize to make it less detectable by ACB might be an effective strategy for developing maize germplasm resistant to ACB and for managing this pest effectively in the field.

Eleocharis vivipara, an amphibious sedge in the Cyperaceae family, has several remarkable properties, most notably its alternate use of C₃ photosynthesis underwater and C₄ photosynthesis on land. However, the absence of genomic data has hindered its utility for evolutionary and genetic research. Here, we present a high-quality genome for E. vivipara, representing the first chromosome-level genome for the Eleocharis genus, with an approximate size of 965.22 Mb mainly distributed across 10 chromosomes. Its Hi–C pattern, chromosome clustering results, and one-to-one genome synteny across two subgroups indicates a tetraploid structure with chromosome count 2n = 4x = 20. Phylogenetic analysis suggests that E. vivipara diverged from Cyperus esculentus approximately 32.96 million years ago (Mya), and underwent a whole-genome duplication (WGD) about 3.5 Mya. Numerous fusion and fission events were identified between the chromosomes of E. vivipara and its close relatives. We demonstrate that E. vivipara has holocentromeres, a chromosomal feature which can maintain the stability of such chromosomal rearrangements. Experimental transplantation and cross-section studies showed its terrestrial culms developed C₄ Kranz anatomy with increased number of chloroplasts in the bundle sheath (BS) cells. Gene expression and weighted gene co-expression network analysis (WGCNA) showed overall elevated expression of core genes associated with the C₄ pathway, and significant enrichment of genes related to modified culm anatomy and photosynthesis efficiency. We found evidence of mixed nicotinamide adenine dinucleotide - malic enzyme and phosphoenolpyruvate carboxykinase type C₄ photosynthesis in E. vivipara, and hypothesize that the evolution of C₄ photosynthesis predates the WGD event. The mixed type is dominated by subgenome A and supplemented by subgenome B. Collectively, our findings not only shed light on the evolution of E. vivipara and karyotype within the Cyperaceae family, but also provide valuable insights into the transition between C₃ and C₄ photosynthesis, offering promising avenues for crop improvement and breeding.

Anchorene, identified as an endogenous bioactive carotenoid-derived dialdehyde and diapocarotenoid, affects root development by modulating auxin homeostasis. However, the precise interaction between anchorene and auxin, as well as the mechanisms by which anchorene modulates auxin levels, remain largely elusive. In this study, we conducted a comparative analysis of anchorene's bioactivities alongside auxin and observed that anchorene induces multifaceted auxin-like effects. Through genetic and pharmacological examinations, we revealed that anchorene's auxin-like activities depend on the indole-3-pyruvate-dependent auxin biosynthesis pathway, as well as the auxin inactivation pathway mediated by Group II Gretchen Hagen 3 (GH3) proteins that mainly facilitate the conjugation of indole-3-acetic acid (IAA) to amino acids, leading to the formation of inactivated storage forms. Our measurements indicated that anchorene treatment elevates IAA levels while reducing the quantities of inactivated IAA–amino acid conjugates and oxIAA. RNA sequencing further revealed that anchorene triggers the expression of numerous auxin-responsive genes in a manner reliant on Group II GH3s. Additionally, our in vitro enzymatic assays and biolayer interferometry (BLI) assay demonstrated anchorene's robust suppression of GH3.17-mediated IAA conjugation with glutamate. Collectively, our findings highlight the significant role of carotenoid-derived metabolite anchorene in modulating auxin homeostasis, primarily through the repression of GH3-mediated IAA conjugation and inactivation pathways, offering novel insights into the regulatory mechanisms of plant bioactive apocarotenoids.

The utilization of metabolomics approaches to explore the metabolic mechanisms underlying plant fitness and adaptation to dynamic environments is growing, highlighting the need for an efficient and user-friendly toolkit tailored for analyzing the extensive datasets generated by metabolomics studies. Current protocols for metabolome data analysis often struggle with handling large-scale datasets or require programming skills. To address this, we present MetMiner (https://github.com/ShawnWx2019/MetMiner), a user-friendly, full-functionality pipeline specifically designed for plant metabolomics data analysis. Built on R shiny, MetMiner can be deployed on servers to utilize additional computational resources for processing large-scale datasets. MetMiner ensures transparency, traceability, and reproducibility throughout the analytical process. Its intuitive interface provides robust data interaction and graphical capabilities, enabling users without prior programming skills to engage deeply in data analysis. Additionally, we constructed and integrated a plant-specific mass spectrometry database into the MetMiner pipeline to optimize metabolite annotation. We have also developed MDAtoolkits, which include a complete set of tools for statistical analysis, metabolite classification, and enrichment analysis, to facilitate the mining of biological meaning from the datasets. Moreover, we propose an iterative weighted gene co-expression network analysis strategy for efficient biomarker metabolite screening in large-scale metabolomics data mining. In two case studies, we validated MetMiner's efficiency in data mining and robustness in metabolite annotation. Together, the MetMiner pipeline represents a promising solution for plant metabolomics analysis, providing a valuable tool for the scientific community to use with ease.