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.

Temperature sensitivity and tolerance play a key role in plant survival and production. Perennial ryegrass (Lolium perenne L.), widely cultivated in cool-season for forage supply and turfgrass, is extremely susceptible to high temperatures, therefore serving as an excellent grass for dissecting the genomic and genetic basis of high-temperature adaptation. In this study, expression analysis revealed that LpHsfA2, an important gene associated with high-temperature tolerance in perennial ryegrass, is rapidly and substantially induced under heat stress. Additionally, heat-tolerant varieties consistently display elevated expression levels of LpHsfA2 compared with heat-sensitive ones. Comparative haplotype analysis of the LpHsfA2 promoter indicated an uneven distribution of two haplotypes (HsfA2^Hap1 and HsfA2^Hap2) across varieties with differing heat tolerance. Specifically, the HsfA2^Hap1 allele is predominantly present in heat-tolerant varieties, while the HsfA2^Hap2 allele exhibits the opposite pattern. Overexpression of LpHsfA2 confers enhanced thermotolerance, whereas silencing of LpHsfA2 compromises heat tolerance. Furthermore,LpHsfA2 orchestrates its protective effects by directly binding to the promoters of LpHSP18.2 and LpAPX1 to activate their expression, preventing the non-specific misfolding of intracellular protein and the accumulation of reactive oxygen species in cells. Additionally, LpHsfA4 and LpHsfA5 were shown to engage directly with the promoter of LpHsfA2, upregulating its expression as well as the expression of LpHSP18.2 and LpAPX1, thus contributing to enhanced heat tolerance. Markedly, LpHsfA2 possesses autoregulatory ability by directly binding to its own promoter to modulate the self-transcription. Based on these findings, we propose a model for modulating the thermotolerance of perennial ryegrass by precisely regulating the expression of LpHsfA2. Collectively, these findings provide a scientific basis for the development of thermotolerant perennial ryegrass 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.

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.

ABSCISIC ACID-INSENSITIVE 4 (ABI4) is a pivotal transcription factor which coordinates multiple aspects of plant growth and development as well as plant responses to environmental stresses. ABI4 has been shown to be involved in regulating seedling photomorphogenesis; however, the underlying mechanism remains elusive. Here, we show that the role of ABI4 in regulating photomorphogenesis is generally regulated by sucrose, but ABI4 promotes hypocotyl elongation of Arabidopsis seedlings under blue (B) light under all tested sucrose concentrations. We further show that ABI4 physically interacts with PHYTOCHROME INTERACTING FACTOR 4 (PIF4), a well-characterized growth-promoting transcription factor, and post-translationally promotes PIF4 protein accumulation under B light. Further analyses indicate that ABI4 directly interacts with the B light photoreceptors cryptochromes (CRYs) and inhibits the interactions between CRYs and PIF4, thus relieving CRY-mediated repression of PIF4 protein accumulation. In addition, while ABI4 could directly activate its own expression, CRYs enhance, whereas PIF4 inhibits, ABI4-mediated activation of the ABI4 promoter. Together, our study demonstrates that the ABI4–PIF4 module plays an important role in mediating CRY-induced B light signaling in Arabidopsis.

Cotton fiber length is basically determined by well-coordinated gene expression and phosphatidylinositol phosphates (PIPs) accumulation during fiber elongation but the regulatory mechanism governing PIPs transport remains unknown. Here, we report a MYB transcription factor GhMYB30D04 in Gossypium hirsutum that promotes fiber elongation through modulating the expression of PIP transporter gene GhLTPG1. Knockout of GhMYB30D04 gene in cotton (KO) results in a reduction of GhLTPG1 transcripts with lower accumulation of PIPs, leading to shorter fibers and lower fiber yield. Conversely,GhMYB30D04 overexpression (GhMYB30D04-OE) causes richer PIPs and longer cotton fibers, mimicking the effects of exogenously applying PIPs on the ovules of GhMYB30D04-KO and wild type. Furthermore, GhMYB30D04 interacts with GhHD1, the crucial transcription factor of fiber initiation, to form an activation complex stabilized by PIPs, both of which upregulate GhLTPG1 expression. Comparative omics-analysis revealed that higher and extended expressions of LTPG1 in fiber elongation mainly correlate with the variations of the GhMYB30D04 gene between two cotton allotetraploids, contributing to longer fiber in G. babardense. Our work clarifies a mechanism by which GhHD1–GhMYB30D04 form a regulatory module of fiber elongation to tightly control PIP accumulation. Our work still has an implication that GhMYB30D04–GhHD1 associates with development transition from fiber initiation to elongation.

Dissecting the genetic control of apple fruit harvest date (AFHD) into multiple Mendelian factors poses a significant challenge in modern genetics. Here, a quantitative trait locus (QTL) for AFHD was fine-mapped to the NAC transcription factor (TF) MdNAC18 within the interval defined by the overlap of QTLs Z03.5/Z03.6 and F03.2/F03.3. One direct target of MdNAC18 is the ethylene biosynthesis gene MdACO1. The single nucleotide polymorphisms (SNPs) SNP517 and SNP958 in the MdNAC18 coding sequence modulated activation of MdACO1 by MdNAC18. SNP1229 in the MdACO1 promoter destroyed the MdNAC18 binding site and thus abolished MdNAC18 binding. SNP517 and SNP958 also affected MdNAC18 activation of the TF gene MdARF5; MdARF5 activates the ethylene biosynthesis gene MdACS1. SNP517 and SNP958 in MdNAC18, SNP1229 and SNP769 (linked to InDel62) in MdACO1, and InDel162 in MdACS1 constituted a genetic variation network. The genetic effect of this network on AFHD was estimated as 60.3 d, accounting for 52.6% of the phenotype variation of the training population. The joint effects of these polymorphisms increased the accuracy of a genomics-assisted prediction (GAP) model for AFHD (r = 0.7125). Together, our results suggest that genetic variation in MdNAC18 affects AFHD by modulating ethylene biosynthesis and provide an optimized GAP model for apple breeding.

WRKY transcription factors play key roles in plant resistance to various stresses, but their roles in fruit ripening remain largely unknown. Here, we report a WRKY gene PpWRKY14 involved in the regulation of fruit ripening in peach. The expression of PpWRKY14 showed an increasing trend throughout fruit development. PpWRKY14 was a target gene of PpNAC1, a master regulator of peach fruit ripening. PpWRKY14 could directly bind to the promoters of PpACS1 and PpACO1 to induce their expression, and this induction was greatly enhanced when PpWRKY14 formed a dimer with PpNAC1. However, the transcription of PpNAC1 could be directly suppressed by two EIN3/EIL1 genes,PpEIL2 and PpEIL3. The PpEIL2/3 genes were highly expressed at the early stages of fruit development, but their expression was programmed to decrease significantly during the ripening stage, thus derepressing the expression of PpNAC1. These results suggested a PpEIL2/3–PpNAC1–PpWRKY14 module that regulates fruit ripening by modulating ethylene production in peach. Our results provided an insight into the regulatory roles of EIN3/EIL1 and WRKY genes in fruit ripening.

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.

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.

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.

Soybean rust (SBR), caused by an obligate biotrophic pathogen Phakopsora pachyrhizi, is a devastating disease of soybean worldwide. However, the mechanisms underlying plant invasion by P. pachyrhizi are poorly understood, which hinders the development of effective control strategies for SBR. Here we performed detailed histological characterization on the infection cycle of P. pachyrhizi in soybean and conducted a high-resolution transcriptional dissection of P. pachyrhizi during infection. This revealed P. pachyrhizi infection leads to significant changes in gene expression with 10 co-expressed gene modules, representing dramatic transcriptional shifts in metabolism and signal transduction during different stages throughout the infection cycle. Numerous genes encoding secreted protein are biphasic expressed, and are capable of inhibiting programmed cell death triggered by microbial effectors. Notably, three co-expressed P. pachyrhizi apoplastic effectors (PpAE1, PpAE2, and PpAE3) were found to suppress plant immune responses and were essential for P. pachyrhizi infection. Double-stranded RNA coupled with nanomaterials significantly inhibited SBR infection by targeting PpAE1, PpAE2, and PpAE3, and provided long-lasting protection to soybean against P. pachyrhizi. Together, this study revealed prominent changes in gene expression associated with SBR and identified P. pachyrhizi virulence effectors as promising targets of RNA interference-based soybean protection strategy against SBR.

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.