Rubus (raspberries and blackberries) is a large genus of over 700 species well known for its taxonomic challenges. Many of its species hold significant economic value as important edible and medicinal plants. Here, near-complete genomes for four wild diploid raspberry species were assembled, including R. ellipticus, R. niveus, as well as the highly heterozygous diploid red raspberry (R. idaeus), and its closely related species R. sachalinensis. Pan-genome analysis of Rubus identified 10,243 core gene families (64% of total), and highlights expansions of flavonoid/terpenoid pathways in Rubus, correlating with fruit bioactive compound diversity. Our discovery of shared ancestral components between R. idaeus and R. sachalinensis subgenomes provides evidence for their homoploid hybrid origin. The centromere sequence characteristics could serve as markers for subgenome assignment in R. idaeus and R. sachalinensis. Moreover, population genomic studies of 125 accessions from ca. 80 species uncovered widespread genetic introgression, particularly in red raspberries, with centromeric haplotype signatures tracing ancestral contributions to cultivated varieties. By integrating metabolome and transcriptome data, we explore the fruit quality regulatory network of Chinese raspberries. We identified a glutathione S-transferase gene that may inhibit the successful transport of anthocyanins into the vacuole and appears to be a limiting factor for the anthocyanin pigmentation in R. ellipticus fruits. In summary, this research sheds new light on the genetic intricacies of raspberry species and their cultivars, and provides a robust foundation for horticultural improvement and genomic selection in raspberry breeding.

Salt stress is a major abiotic constraint limiting global crop production. Oat (Avena sativa L.), an allohexaploid cereal renowned for robust stress tolerance, remains poorly understood in terms of the molecular mechanisms underlying its response to salt stress. Here, we perform transcriptome profiling across multiple developmental stages and tissues of oat under salt stress, and construct the co-expression regulatory network to identify salt tolerance-associated gene modules. Notably, 10 salt-responsive transcription factor (SRTF) families with dynamic expression patterns are identified as core regulators, showing extensive subgenomic functional divergence, characterized by subgenome-dominant expression, as well as subgenome-specific duplication or loss events. Further integration with a genome-wide association study (GWAS) of the germination rate under salt stress in 225 oat accessions identified a 3-bp InDel variation within the duplicated gene AsWRKY49-D2, which specifically modulates its expression by facilitating binding of the TF AsZAT18, with AsWRKY49-D2 further mediating oat salt tolerance through targeted regulation of AsSOS2 and AsSOS3. Intriguingly, the salt-tolerant allele of AsWRKY49 is scarcely distributed in Chinese oat accessions, highlighting its considerable potential for breeding application. These results shed light on the regulatory mechanisms underlying oat salt tolerance, providing valuable information for exploring salt tolerance genes and breeding new salt-tolerant oat varieties.

Herbivorous insects pose a major threat to crop production, with rice suffering significant yield losses due to infestation by the brown planthopper (BPH). To understand the genetic and metabolic basis of BPH resistance in rice, we conducted metabolomic analysis and performed metabolite-based genome-wide association studies (mGWAS) on a rice population composed of 168 varieties, which exhibit a wide range of resistance to BPH. Metabolomic analysis revealed a trend of increasing metabolic divergence with increasing resistance levels compared with the susceptible group, with resistant groups maintaining greater metabolic stability after BPH infestation. Furthermore, using these metabolic biomarkers, we constructed a prediction model for BPH resistance and found that biomarkers in non-infested rice were sufficient to predict BPH resistance. We identified in total 2,738 single-nucleotide polymorphisms (SNPs) associated with key biomarkers in non-infested rice and 1,605 SNPs in BPH-infested rice. Gene Ontology (GO) enrichment analysis revealed that genes associated with biomarkers were enriched in different pathways between non-infested and BPH-infested rice. Notably, the SNP rs6_191562334 was significantly associated with the biomarker β-damascenone, which correlated positively with rice resistance to BPH and has been shown to inhibit BPH feeding on rice. Knockout of LOC_Os06g17970 increased β-damascenone levels and enhanced BPH resistance in rice. Collectively, this integrated approach provided novel insight into the metabolic and genetic mechanisms underlying BPH resistance and facilitated the development of strategies for sustainable control of BPH.

Lysine malonylation (K_mal), an evolutionarily conserved post-translational modification, serves as a critical regulator of cellular processes including transcriptional control, metabolic coordination, and enzyme activation. While K_mal sites have been mapped in rice (Oryza sativa L.) seeds, their dynamic regulation in rice responses to biotic stresses remains poorly characterized. Here, we reported a global profiling of lysine-malonylated proteins in rice leaf sheaths, and the changes in these proteins under herbivore (Nilaparvata lugens/Chilo suppressalis) or viral (rice stripe virus/rice black-streaked dwarf virus) stresses. Using affinity enrichment and proteomics, we identified 3,113 K_mal sites across 1,324 proteins in wild-type rice leaf sheaths, these data demonstrated that lysine-malonylated proteins are involved in diverse biological processes. K_mal levels were significantly upregulated following herbivore infestation or viral infection, with two herbivores inducing more pronounced changes than viruses infection, revealing stress-specific malonylation landscapes. K_mal preferentially targeted highly expressed proteins in energy metabolism (e.g., glycolysis and the tricarboxylic acid cycle) and photosynthesis, exhibiting an inverse correlation between hypermalonylation and protein abundance. Cleavage under targets and tagmentation analyses revealed K_mal-mediated chromatin remodeling through promoter occupancy at defense-related genes. In addition, the histone deacetylases OsHDA702-704, OsHDA711-713 were functionally characterized as key regulators mediating the erasure of specific malonylation marks in rice plants, with OsHDA711 knockout lines exhibiting enhanced resistance against both herbivore and virus infection. Our work establishes K_mal as a regulator in rice biotic immunity, uncovering novel insights into K_mal-mediated plant defense responses against herbivorous pests and viral pathogens. These findings identify potential genetic targets for developing rice varieties with broad-spectrum immunity to biotic stresses, enhancing crop resilience.

The raw sequencing of RNA-seq from uninfected control check rice (Oryza sativa L.) plants (CK), N. lugens-infested rice plants (Nl), C. suppressalis-infested rice plants (Cs), RSV-infected rice plants (RSV), and RBSDV-infected rice plants (RSV) have been deposited in the Genome Sequence Archive in the National Genomics Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences (GSA: CRA030923), which is publicly accessible at https://ngdc.cncb.ac.cn/gsa. All the other data supporting the conclusions in this study are available in Supporting Information Figures S8, S9, S11, S14, S15, and Tables S2, S3, S5–S9.

Dendrocalamus brandisii (Munro) Kurz is a sympodial bamboo species with inimitable taste and flavorful shoots. Its rapid growth and use as high-quality material make this bamboo species highly valued for both food processing and wood applications. However, genome information for D. brandisii is lacking, primarily due to its polyploidy and large genome size. Here, we assembled a high-quality genome for hexaploid D. brandisii, which comprises 70 chromosomes with a total size of 2,756 Mb, using long-read HiFi sequencing. Furthermore, we accurately separated the genome into its three constituent subgenomes. We used Oxford Nanopore Technologies long reads to construct a transcriptomic dataset covering 15 tissues for gene annotation to complement our genome assembly, revealing differential gene expression and post-transcriptional regulation. By integrating metabolome analysis, we unveiled that well-balanced lignin formation, as well as abundant flavonoid and fructose contents, contribute to the superior quality of D. brandisii shoots. Integrating genomic, transcriptomic, and metabolomic datasets provided a solid foundation for enhancing bamboo shoot quality and developing efficient gene-editing techniques. This study should facilitate research on D. brandisii and enhance its use as a food source and wood material by providing crucial genomic resources.

Recent studies have documented plant responses to climate change extensively, particularly to single-stress exposures. However, critical factors for stress survival, such as sexual differentiation, are not often considered. The dioicous Marchantia polymorpha stands as an evolutionary milestone, potentially preserving ancestral traits from the early colonizers. In this study, we employed proteomic analyses complemented with physiological monitoring to investigate combined heat and drought responses in Tak-1 (male) and Tak-2 (female) accessions of this liverwort. Additionally, targeted transcriptomics was conducted using different natural populations from contrasting environments. Our findings revealed sex-biased dynamics among natural accessions, particularly evident under control conditions and during early stress responses. Although Tak-2 exhibited greater diversity than Tak-1 under control conditions, male accession demonstrated distinct and more rapid stress sensing and signaling. These differences in stress response appeared to be strongly related to sex-specific plasticity influenced by geoclimatic origin. Furthermore, we established distinct protein gene ages and genomic distribution trends, underscoring the importance of protein diversification over time. This study provides an evolutionary perspective on sexual divergence and stress emergence employing a systems biology approach, which allowed for the establishment of global and sex-specific interaction networks in the stress response.

All data and code generated during this study are available in the GitHub repository (https://github.com/Valledor/HxD_Marpol_Proteomics). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2022) with the dataset identifier PXD047874 (https://www.ebi.ac.uk/pride/archive/projects/PXD047874).

The roots of legume plant play a crucial role in nitrogen fixation. However, the transcriptomes of different cell types of legume root and their functions remain largely unknown. Here, we performed single‐cell RNA sequencing and profiled more than 22,000 single cells from root tips of Lotus japonicus, a model species of legume. We identified seven clusters corresponding to seven major cell types, which were validated by in situ hybridization. Further analysis revealed regulatory programs including phytohormone and nodulation associated with specific cell types, and revealed conserved and diverged features for the cell types. Our results represent the first single‐cell resolution transcriptome for legume root tips and a valuable resource for studying the developmental and physiological functions of various cell types in legumes.

Plants accumulate a vast array of secondary metabolites, which constitute a natural resource for pharmaceuticals. Oldenlandia corymbosa belongs to the Rubiaceae family, and has been used in traditional medicine to treat different diseases, including cancer. However, the active metabolites of the plant, their biosynthetic pathway and mode of action in cancer are unknown. To fill these gaps, we exposed this plant to eight different stress conditions and combined different omics data capturing gene expression, metabolic profiles, and anti-cancer activity. Our results show that O. corymbosa extracts are active against breast cancer cell lines and that ursolic acid is responsible for this activity. Moreover, we assembled a high-quality genome and uncovered two genes involved in the biosynthesis of ursolic acid. Finally, we also revealed that ursolic acid causes mitotic catastrophe in cancer cells and identified three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking. Altogether, these results constitute a valuable resource to further characterize the biosynthesis of active metabolites in the Oldenlandia group, while the mode of action of ursolic acid will allow us to further develop this valuable compound.

All the data generated or analyzed in this study are included in this article. The genome assembly, its annotation, and the sequencing data (DNA and RNA) have been deposited in the ENA (European Nucleotide Archive) under the accession number PRJEB50026.

Although root nodules are essential for biological nitrogen fixation in legumes, the cell types and molecular regulatory mechanisms contributing to nodule development and nitrogen fixation in determinate nodule legumes, such as soybean (Glycine max), remain incompletely understood. Here, we generated a single-nucleus resolution transcriptomic atlas of soybean roots and nodules at 14 days post inoculation (dpi) and annotated 17 major cell types, including six that are specific to nodules. We identified the specific cell types responsible for each step in the ureides synthesis pathway, which enables spatial compartmentalization of biochemical reactions during soybean nitrogen fixation. By utilizing RNA velocity analysis, we reconstructed the differentiation dynamics of soybean nodules, which differs from those of indeterminate nodules in Medicago truncatula. Moreover, we identified several putative regulators of soybean nodulation and two of these genes, GmbHLH93 and GmSCL1, were as-yet uncharacterized in soybean. Overexpression of each gene in soybean hairy root systems validated their respective roles in nodulation. Notably, enrichment for cytokinin-related genes in soybean nodules led to identification of the cytokinin receptor, GmCRE1, as a prominent component of the nodulation pathway. GmCRE1 knockout in soybean resulted in a striking nodule phenotype with decreased nitrogen fixation zone and depletion of leghemoglobins, accompanied by downregulation of nodule-specific gene expression, as well as almost complete abrogation of biological nitrogen fixation. In summary, this study provides a comprehensive perspective of the cellular landscape during soybean nodulation, shedding light on the underlying metabolic and developmental mechanisms of soybean nodule formation.

All sequencing data and process data have been deposited to the National Genomics Data Center (https://ngdc.cncb.ac.cn/), Beijing Institute of Genomics, Chinese Academy of Sciences, under the BioProject number PRJCA015369. Codes to analyze the data and generate figures are available at GitHub (https://github.com/Yuwang-art/snRNA_seq_in_soybean).

Multicellular organisms such as plants contain various cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances our understanding of organization and function at the organismal level. Guard cells (GCs) are specialized epidermal cells that regulate the movement of the stomata and gaseous exchange, and provide a model genetic system for analyzing cell fate, signaling, and function. Several proteomics analyses of GC are available, but these are limited in depth. Here we used enzymatic isolation and flow cytometry to enrich GC and mesophyll cell protoplasts and perform in-depth proteomics in these two major cell types in Arabidopsis leaves. We identified approximately 3,000 proteins not previously found in the GC proteome and more than 600 proteins that may be specific to GC. The depth of our proteomics enabled us to uncover a guard cell-specific kinase cascade whereby Raf15 and Snf1-related kinase2.6 (SnRK2.6)/OST1(open stomata 1) mediate abscisic acid (ABA)-induced stomatal closure. RAF15 directly phosphorylated SnRK2.6/OST1 at the conserved Ser175 residue in its activation loop and was sufficient to reactivate the inactive form of SnRK2.6/OST1. ABA-triggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants. We also showed enrichment of enzymes and flavone metabolism in GC, and consistent, dramatic accumulation of flavone metabolites. Our study answers the long-standing question of how ABA activates SnRK2.6/OST1 in GCs and represents a resource potentially providing further insights into the molecular basis of GC and mesophyll cell development, metabolism, structure, and function.

The proteomics data were deposited to the PXD039858/PXD041345 for ProteomeXchange and JPST002022 and JPST002118 for jPOST (https://repository.jpostdb.org/preview/130037734563ddfd8fae6f9, access key: 4097; https://repository.jpostdb.org/preview/1014894330642d434879571, access key: 2721). The metabolomics data were deposited to the MetaboLights study: MTBLS3997 (www.ebi.ac.uk/metabolights/MTBLS3997).

Diterpenoid alkaloids (DAs) have been often utilized in clinical practice due to their analgesic and anti-inflammatory properties. Natural DAs are prevalent in the family Ranunculaceae, notably in the Aconitum genus. Nevertheless, the evolutionary origin of the biosynthesis pathway responsible for DA production remains unknown. In this study, we successfully assembled a high-quality, pseudochromosome-level genome of the DA-rich species Aconitum vilmorinianum (A. vilmorinianum) (5.76 Gb). An A. vilmorinianum-specific whole-genome duplication event was discovered using comparative genomic analysis, which may aid in the evolution of the DA biosynthesis pathway. We identified several genes involved in DA biosynthesis via integrated genomic, transcriptomic, and metabolomic analyses. These genes included enzymes encoding target ent-kaurene oxidases and aminotransferases, which facilitated the activation of diterpenes and insertion of nitrogen atoms into diterpene skeletons, thereby mediating the transformation of diterpenes into DAs. The divergence periods of these genes in A. vilmorinianum were further assessed, and it was shown that two major types of genes were involved in the establishment of the DA biosynthesis pathway. Our integrated analysis offers fresh insights into the evolutionary origin of DAs in A. vilmorinianum as well as suggestions for engineering the biosynthetic pathways to obtain desired DAs.

All data needed to evaluate the conclusions in the paper are presented in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors, and RNA-seq and genome data are available at the National Genomics Data Center under BioProject ID PRJCA014109, and partial data and analysis codes were available at GitHub (https://github.com/ccbio/GA4Aconitum). Reference genome data and raw sequencing reads are available at the BioProject ID PRJCA014109. Genome assembly and annotation are available under accession number GWH: GWHBRAL00000000, genome sequencing data are available under accession number GSA: CRA009577, and transcriptome sequence reads are available under accession number GSA: CRA009538.

Heterosis is a fundamental biological phenomenon characterized by the superior performance of hybrids over their parents. Although tremendous progress has been reported in seed crops, the molecular mechanisms underlying heterosis in clonally propagated crops are largely unknown. Potato (Solanum tuberosum L.) is the most important tuber crop and an ongoing revolution is transforming potato from a clonally propagated tetraploid crop into a seed-propagated diploid hybrid potato. In our previous study, we developed the first generation of highly homozygous inbred lines of potato and hybrids with strong heterosis. Here, we integrated transcriptome, metabolome, and DNA methylation data to explore the genetic and molecular basis of potato heterosis at three developmental stages. We found that the initial establishment of heterosis in diploid potato was mainly due to dominant complementation. Flower color, male fertility, and starch and sucrose metabolism showed obvious gene dominant complementation in hybrids, and hybrids devoted more energy to primary metabolism for rapid growth. In addition, we identified ~2 700 allele-specific expression genes at each stage, which likely function in potato heterosis and might be regulated by CHH allele-specific methylation level. Our multi-omics analysis provides insight into heterosis in potato and facilitates the exploitation of heterosis in potato breeding.

Anther development from stamen primordium to pollen dispersal is complex and essential to sexual reproduction. How this highly dynamic and complex developmental process is controlled genetically is not well understood, especially for genes involved in specific key developmental phases. Here we generated RNA sequencing libraries spanning 10 key stages across the entirety of anther development in maize (Zea mays). Global transcriptome analyses revealed distinct phases of cell division and expansion, meiosis, pollen maturation, and mature pollen, for which we detected 50, 245, 42, and 414 phase-specific marker genes, respectively. Phase-specific transcription factor genes were significantly enriched in the phase of meiosis. The phase-specific expression of these marker genes was highly conserved among the maize lines Chang7-2 and W23, indicating they might have important roles in anther development. We explored a desiccation-related protein gene, ZmDRP1, which was exclusively expressed in the tapetum from the tetrad to the uninucleate microspore stage, by generating knockout mutants. Notably, mutants in ZmDRP1 were completely male-sterile, with abnormal Ubisch bodies and defective pollen exine. Our work provides a glimpse into the gene expression dynamics and a valuable resource for exploring the roles of key phase-specific genes that regulate anther development.

Photosynthesis involves a series of redox reactions and is the major source of reactive oxygen species in plant cells. Fluctuating light (FL) levels, which occur commonly in natural environments, affect photosynthesis; however, little is known about the specific effects of FL on the redox regulation of photosynthesis. Here, we performed global quantitative mapping of the Arabidopsis thaliana cysteine thiol redox proteome under constant light and FL conditions. We identified 8857 redox-switched thiols in 4350 proteins, and 1501 proteins that are differentially modified depending on light conditions. Notably, proteins related to photosynthesis, especially photosystem I (PSI), are operational thiol-switching hotspots. Exposure of wild-type A. thaliana to FL resulted in decreased PSI abundance, stability, and activity. Interestingly, in response to PSI photodamage, more of the PSI assembly factor PSA3 dynamically switches to the reduced state. Furthermore, the Cys199 and Cys200 sites in PSA3 are necessary for its full function. Moreover, thioredoxin m (Trx m) proteins play roles in redox switching of PSA3, and are required for PSI activity and photosynthesis. This study thus reveals a mechanism for redox-based regulation of PSI under FL, and provides insight into the dynamic acclimation of photosynthesis in a changing environment.

Fusarium head blight (FHB) caused by Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schw.) Perch) results in large yield losses in annual global wheat production. Although studies have identified a number of wheat FHB resistance genes, a deeper understanding of the mechanisms underlying host plant resistance to F. graminearum is required for the control of FHB. Here, an integrated metabolomics and transcriptomics analysis of infected wheat plants (Triticum aestivum L.) enabled identification of 789 differentially accumulated metabolites, including flavonoids, phenolamides, tryptamine derivatives, and phytohormones, and revealed altered expression of more than 100 genes that function in the biosynthesis or regulation of these pathways. Our data regarding the effects of F. graminearum infection on flavonoids and auxin signaling led to follow‐up experiments that showed that exogenous kaempferide and apigenin application on spikes increased wheat resistance to FHB, while exogenous auxin treatment increased FHB susceptibility. RNAi‐mediated knockdown of the gene encoding the auxin receptor, TaTIR1, increased FHB resistance. Our data supported the use of TaTIR1 knockdown in controlling FHB. Our study provides insights on the wheat response to F. graminearum infection and its FHB resistance mechanisms while illustrating the potential of TaTIR1 knockdown in increasing FHB resistance during crop improvement programs.

Lysine 2‐hydroxyisobutyrylation (K_hib) is a newly identified post‐translational modification (PTM) that plays important roles in transcription and cell proliferation in eukaryotes. However, its function remains unknown in phytopathogenic fungi. Here, we performed a comprehensive assessment of K_hib in the rice false smut fungus Ustilaginoidea virens, using Tandem Mass Tag (TMT)‐based quantitative proteomics approach. A total of 3 426 K_hib sites were identified in 977 proteins, suggesting that K_hib is a common and complex PTM in U. virens. Our data demonstrated that the 2‐hydroxyisobutyrylated proteins are involved in diverse biological processes. Network analysis of the modified proteins revealed a highly interconnected protein network that included many well‐studied virulence factors. We confirmed that the Zn‐binding reduced potassium dependency3‐type histone deacetylase (UvRpd3) is a major enzyme that removes 2‐hydroxyisobutyrylation and acetylation in U. virens. Notably, mutations of K_hib sites in the mitogen‐activated protein kinase (MAPK) UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molecular dynamics simulations demonstrated that 2‐hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent‐accessible surface area and thereby affected binding between the UvSlt2 enzyme and its substrates. Our findings thus establish K_hib as a major post‐translational modification in U. virens and point to an important role for K_hib in the virulence of this phytopathogenic fungus.

The mass spectrometry proteomics data have been deposited into the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository (http://www.ebi.ac.uk/pride/) with the dataset identifier PXD019114.

Circular RNAs (circRNAs) are a recently discovered type of non-coding RNA derived from pre-mRNAs. R-loops consist of a DNA:RNA hybrid and the associated single-stranded DNA. In Arabidopsis thaliana, circRNA:DNA R-loops regulate alternative splicing (AS) of SEPALLATA3 (SEP3). However, the occurrence and functions of circRNAs and R-loops in Populus trichocarpa are largely unexplored. Here, we performed circRNA-enriched sequencing in the stem-differentiating xylem (SDX) of P. trichocarpa and identified 2,742 distinct circRNAs, including circ-CESA4, circ-IRX7, and circ-GUX1, which are generated from genes involved in cellulose, and hemicellulose biosynthesis, respectively. To investigate the roles of circRNAs in modulating alternative splicing (AS), we detected 7,836 AS events using PacBio Iso-Seq and identified 634 circRNAs that overlapped with 699 AS events. Furthermore, using DNA:RNA hybrid immunoprecipitation followed by sequencing (DRIP-seq), we identified 8,932 R-loop peaks that overlapped with 181 circRNAs and 672 AS events. Notably, several SDX-related circRNAs overlapped with R-loop peaks, pointing to their possible roles in modulating AS in SDX. Indeed, overexpressing circ-IRX7 increased the levels of R-loop structures and decreased the frequency of intron retention in linear IRX7 transcripts. This study provides a valuable R-loop atlas resource and uncovers the interplay between circRNAs and AS in SDX of P. trichocarpa.

All clean reads were deposited in the SRA under the following accession numbers: CircRNA (SRR8517644, SRR8517643, and SRR8517642), PacBio Iso-Seq (SRR8447264), strand-specific RNA-Seq libraries (SRR13481183, SRR13481184, SRR13481185), and R-loop (SRR11845458). All R-loop peaks, circRNA coordinates, and AS events can be found at forestry.fafu.edu.cn/db/Ptr_Circular_RNA.

Plants have a hierarchical circadian structure comprising multiple tissue-specific oscillators that operate at different speeds and regulate the expression of distinct sets of genes in different organs. However, the identity of the genes differentially regulated by the circadian clock in different organs, such as roots, and how their oscillations create functional specialization remain unclear. Here, we profiled the diurnal and circadian landscapes of the shoots and roots of Medicago truncatula and identified the conserved regulatory sequences contributing to transcriptome oscillations in each organ. We found that the light-dark cycles strongly affect the global transcriptome oscillation in roots, and many clock genes oscillate only in shoots. Moreover, many key genes involved in nitrogen fixation are regulated by circadian rhythms. Surprisingly, the root clock runs faster than the shoot clock, which is contrary to the hierarchical circadian structure showing a slow-paced root clock in both detached and intact Arabidopsis thaliana (L.) Heynh. roots. Our result provides important clues about the species-specific circadian regulatory mechanism, which is often overlooked, and possibly coordinates the timing between shoots and roots independent of the current prevailing model.

The post-translational modification lysine 2-hydroxyisobutyrylation (K_hib) plays an important role in gene transcription, metabolism, and enzymatic activity. K_hib sites have been identified in rice (Oryza sativa). However, the K_hib status of proteins in rice flowers during pathogen infection remains unclear. Here, we report a comprehensive identification of K_hib-modified proteins in rice flowers, and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens. By using a tandem mass tag-based quantitative proteomics approach, we identified 2,891 K_hib sites on 964 proteins in rice flowers. Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. K_hib levels were substantially reduced upon infection with U. virens. Chromatin immunoprecipitation polymerase chain reaction (PCR) and reverse transcription quantitative PCR analyses revealed that histone K_hib is involved in the expression of disease-resistance genes. More importantly, most quantified sites on core histones H3 were downregulated upon U. virens infection. In addition, the histone deacetylases HDA705, HDA716, SRT1, and SRT2 are involved in the removal of K_hib marks in rice. HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U. virens, Magnaporthe oryzae, and Xanthomonas oryzae pv. oryzae (Xoo). Our data suggest that U. virens could modulate K_hib in rice flowers during infection.

The MS proteomics data produced in this research can be found at the ProteomeXchange (http://www.ebi.ac.uk/pride/) with the accession number PXD019114.

High-throughput single-cell RNA sequencing (scRNA-seq) has advantages over traditional RNA-seq to explore spatiotemporal information on gene dynamic expressions in heterogenous tissues. We performed Drop-seq, a method for the dropwise sequestration of single cells for sequencing, on protoplasts from the differentiating xylem of Populus alba × Populus glandulosa. The scRNA-seq profiled 9,798 cells, which were grouped into 12 clusters. Through characterization of differentially expressed genes in each cluster and RNA in situ hybridizations, we identified vessel cells, fiber cells, ray parenchyma cells and xylem precursor cells. Diffusion pseudotime analyses revealed the differentiating trajectory of vessels, fiber cells and ray parenchyma cells and indicated a different differentiation process between vessels and fiber cells, and a similar differentiation process between fiber cells and ray parenchyma cells. We identified marker genes for each cell type (cluster) and key candidate regulators during developmental stages of xylem cell differentiation. Our study generates a high-resolution expression atlas of wood formation at the single cell level and provides valuable information on wood formation.

RNA-seq and scRNA-seq data have been deposited in GenBank (BioProject ID: PRJNA703312).

Translational control of gene expression, including recruitment of ribosomes to messenger RNA (mRNA), is particularly important during the response to stress. Purification of ribosome-associated mRNAs using translating ribosome affinity purification (TRAP) followed by RNA-sequencing facilitates the study of mRNAs undergoing active transcription and better proxies the translatome, or protein response, to stimuli. To identify plant responses to Magnesium (Mg) deficiency at the translational level, we combined transcriptome and translatome analyses. Excitingly, we found 26 previously unreported Mg-responsive genes that were only regulated at the translational level and not the transcriptional level, during the early response to Mg deficiency. In addition, mutants of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), the H⁺/CATION EXCHANGER 1 and 3 (CAX1 and CAX3), and UBIQUITIN 11 (UBQ11) exhibited early chlorosis phenotype under Mg deficiency, supporting their functional involvement in ion homeostasis. Overall, our study strongly supports that TRAP-seq combined with RNA-seq followed by phenotype screening could facilitate the identification of novel players during stress responses.

Climate change scenarios predict an increase in mean air temperatures and in the frequency, intensity, and length of extreme temperature events in many wine‐growing regions worldwide. Because elevated temperature has detrimental effects on berry growth and composition, it threatens the economic and environmental sustainability of wine production. Using Cabernet Sauvignon fruit‐bearing cuttings, we investigated the effects of high temperature (HT) on grapevine berries through a label‐free shotgun proteomic analysis coupled to a complementary metabolomic study. Among the 2,279 proteins identified, 592 differentially abundant proteins were found in berries exposed to HT. The gene ontology categories “stress,” “protein,” “secondary metabolism,” and “cell wall” were predominantly altered under HT. High temperatures strongly impaired carbohydrate and energy metabolism, and the effects depended on the stage of development and duration of treatment. Transcript amounts correlated poorly with protein expression levels in HT berries, highlighting the value of proteomic studies in the context of heat stress. Furthermore, this work reveals that HT alters key proteins driving berry development and ripening. Finally, we provide a list of differentially abundant proteins that can be considered as potential markers for developing or selecting grape varieties that are better adapted to warmer climates or extreme heat waves.

Hormones are important signaling molecules regulating developmental processes and responses to environmental stimuli in higher plants. Rice endosperm, the portion of the seed surrounding the embryo, is the main determinant of rice grain shape and yield; however, the dynamics and exact functions of phytohormones in developing endosperm remain elusive. Through a systemic study including transcriptome analysis, hormone measurement, and transgene‐based endosperm‐specific expression of phytohormone biosynthetic enzymes, we demonstrated that dynamic phytohormone levels play crucial roles in the developing rice endosperm, particularly in regard to grain shape and quality. We detected diverse, differential, and dramatically changing expression patterns of genes related to hormone biosynthesis and signaling during endosperm development, especially at early developmental stages. Liquid chromatography measurements confirmed the dynamic accumulation of hormones in developing endosperm. Further transgenic analysis performed on plants expressing hormone biosynthesis genes driven by an endosperm‐specific promoter revealed differential effects of the hormones, especially auxin and brassinosteroids, in regulating grain shape and quality. Our studies help elucidate the distinct roles of hormones in developing endosperm and provide novel and useful tools for influencing crop seed shape and yield.

N⁶‐methyladenosine (m⁶A) is a prevalent modification in messenger RNAs and circular RNAs that play important roles in regulating various aspects of RNA metabolism. However, the occurrence of the m⁶A modification in plant circular RNAs has not been reported. A widely used method to identify m⁶A modifications relies on m⁶A‐specific antibodies followed by next‐generation sequencing of precipitated RNAs (MeRIP‐Seq). However, one limitation of MeRIP‐Seq is that it does not provide the precise location of m⁶A at single‐nucleotide resolution. Although more recent sequencing techniques such as Nanopore‐based direct RNA sequencing (DRS) can overcome such limitations, the technology does not allow sequencing of circular RNAs, as these molecules lack a poly(A) tail. Here, we developed a novel method to detect the precise location of m⁶A modifications in circular RNAs using Nanopore DRS. We first enriched our samples for circular RNAs, which we then fragmented and sequenced on the Nanopore platform with a customized protocol. Using this method, we identified 470 unique circular RNAs from DRS reads based on the back‐spliced junction region. Among exonic circular RNAs, about 10% contained m⁶A sites, which mainly occurred around acceptor and donor splice sites. This study demonstrates the utility of our antibody‐independent method in identifying total and methylated circular RNAs using Nanopore DRS. This method has the additional advantage of providing the exact location of m⁶A sites at single‐base resolution in circular RNAs or linear transcripts from non‐coding RNA without poly(A) tails.

Raw Nanopore DRS data has been submitted to the NCBI Gene Expression Omnibus under accession no PRJNA613867. All locus identifiers and circRNAs are available in http://forestry.fafu.edu.cn/pub/circDRS.

Brassica napus L. (canola) is one of the world's most economically important oilseeds. Despite our growing knowledge of Brassica genetics, we still know little about the genes and gene regulatory networks underlying early seed development. In this work, we use laser microdissection coupled with RNA sequencing to profile gene activity of both the maternal and filial subregions of the globular seed. We find subregions of the chalazal end including the chalazal endosperm, chalazal proliferating tissue, and chalazal seed coat, have unique transcriptome profiles associated with hormone biosynthesis and polysaccharide metabolism. We confirm that the chalazal seed coat is uniquely enriched for sucrose biosynthesis and transport, and that the chalazal endosperm may function as an important regulator of the maternal region through brassinosteroid synthesis. The chalazal proliferating tissue, a poorly understood subregion, was specifically enriched in transcripts associated with megasporogenesis and trehalose biosynthesis, suggesting this ephemeral structure plays an important role in both sporophytic development and carbon nutrient balance, respectively. Finally, compartmentalization of transcription factors and their regulatory circuits has uncovered previously unknown roles for the chalazal pole in early seed development.