ChinaMu is the largest sequence-indexed Mutator (Mu) transposon insertional library in maize (Zea mays). In this study, we made significant improvements to the size and quality of the ChinaMu library. We developed a new Mu-tag isolation method Mu-Tn5-seq (MuT-seq). Compared to the previous method used by ChinaMu, MuT-seq recovered 1/3 more germinal insertions, while requiring only about 1/14 of the sequencing volume and 1/5 of the experimental time. Using MuT-seq, we identified 113,879 germinal insertions from 3,168 Mu-active F1 families. We also assembled a high-quality genome for the Mu-active line Mu-starter, which harbors the initial active MuDR element and was used as the pollen donor for the mutation population. Using the Mu-starter genome, we recovered 33,662 (15.6%) additional germinal insertions in 3,244 (7.4%) genes in the Mu-starter line. The Mu-starter genome also improved the assignment of 117,689 (54.5%) germinal insertions. The newly upgraded ChinaMu dataset currently contains 215,889 high-quality germinal insertions. These insertions cover 32,224 pan-genes in the Mu-starter and B73Ref5 genomes, including 23,006 (80.4%) core genes shared by the two genomes. As a test model, we investigated Mu insertions in the pentatricopeptide repeat (PPR) superfamily, discovering insertions for 92% (449/487) of PPR genes in ChinaMu, demonstrating the usefulness of ChinaMu as a functional genomics resource for maize.
Due to its tropical origins, rice (Oryza sativa) is susceptible to cold stress, which poses severe threats to production. OsNAC5, a NAC-type transcription factor, participates in the cold stress response of rice, but the detailed mechanisms remain poorly understood. Here, we demonstrate that OsNAC5 positively regulates cold tolerance at germination and in seedlings by directly activating the expression of ABSCISIC ACID INSENSITIVE 5 (OsABI5). Haplotype analysis indicated that single nucleotide polymorphisms in a NAC-binding site in the OsABI5 promoter are strongly associated with cold tolerance. OsNAC5 also enhanced OsABI5 stability, thus regulating the expression of cold-responsive (COR) genes, enabling fine-tuned control of OsABI5 action for rapid, precise plant responses to cold stress. DNA affinity purification sequencing coupled with transcriptome deep sequencing identified several OsABI5 target genes involved in COR expression, including DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR 1A (OsDREB1A), OsMYB20, and PEROXIDASE 70 (OsPRX70). In vivo and in vitro analyses suggested that OsABI5 positively regulates COR gene transcription, with marked COR upregulation in OsNAC5-overexpressing lines and downregulation in osnac5 and/or osabi5 knockout mutants. This study extends our understanding of cold tolerance regulation via OsNAC5 through the OsABI5-CORs transcription module, which may be used to ameliorate cold tolerance in rice via advanced breeding.
Drought is a major threat to alfalfa (Medicago sativa L.) production. The discovery of important alfalfa genes regulating drought response will facilitate breeding for drought-resistant alfalfa cultivars. Here, we report a genome-wide association study of drought resistance in alfalfa. We identified and functionally characterized an MYB-like transcription factor gene (MsMYBH), which increases the drought resistance in alfalfa. Compared with the wild-types, the biomass and forage quality were enhanced in MsMYBH overexpressed plants. Combined RNA-seq, proteomics and chromatin immunoprecipitation analysis showed that MsMYBH can directly bind to the promoters of MsMCP1, MsMCP2, MsPRX1A and MsCARCAB to improve their expression. The outcomes of such interactions include better water balance, high photosynthetic efficiency and scavenge excess H2O2 in response to drought. Furthermore, an E3 ubiquitin ligase (MsWAV3) was found to induce MsMYBH degradation under long-term drought, via the 26S proteasome pathway. Furthermore, variable-number tandem repeats in MsMYBH promoter were characterized among a collection of germplasms, and the variation is associated with promoter activity. Collectively, our findings shed light on the functions of MsMYBH and provide a pivotal gene that could be leveraged for breeding drought-resistant alfalfa. This discovery also offers new insights into the mechanisms of drought resistance in alfalfa.
The high-affinity potassium transporters (HKTs), selectively permeable to either Na+ alone or Na+/K+, play pivotal roles in maintaining plant Na+/K+ homeostasis. Although their involvement in salt tolerance is widely reported, the molecular underpinnings of Oryza sativa HKTs remain elusive. In this study, we elucidate the structures of OsHKT1;1 and OsHKT2;1, representing two distinct classes of rice HKTs. The dimeric assembled OsHKTs can be structurally divided into four domains. At the dimer interface, a half-helix or a loop in the third domain is coordinated by the C-terminal region of the opposite subunit. Additionally, we present the structures of OsHKT1;5 salt-tolerant and salt-sensitive variants, a key quantitative trait locus associated with salt tolerance. The salt-tolerant variant of OsHKT1;5 exhibits enhanced Na+ transport capability and displays a more flexible conformation. These findings shed light on the molecular basis of rice HKTs and provide insights into their role in salt tolerance.
Hybrid rice (Oryza sativa) generally outperforms its inbred parents in yield and stress tolerance, a phenomenon termed heterosis, but the underlying mechanism is not completely understood. Here, we combined transcriptome, proteome, physiological, and heterosis analyses to examine the salt response of super hybrid rice Chaoyou1000 (CY1000). In addition to surpassing the mean values for its two parents (mid-parent heterosis), CY1000 exhibited a higher reactive oxygen species scavenging ability than both its parents (over-parent heterosis or heterobeltiosis). Nonadditive expression and allele-specific gene expression assays showed that the glutathione S-transferase gene OsGSTU26 and the amino acid transporter gene OsAAT30 may have major roles in heterosis for salt tolerance, acting in an overdominant fashion in CY1000. Furthermore, we identified OsWRKY72 as a common transcription factor that binds and regulates OsGSTU26 and OsAAT30. The salt-sensitive phenotypes were associated with the OsWRKY72paternal genotype or the OsAAT30maternal genotype in core rice germplasm varieties. OsWRKY72paternal specifically repressed the expression of OsGSTU26 under salt stress, leading to salinity sensitivity, while OsWRKY72maternal specifically repressed OsAAT30, resulting in salinity tolerance. These results suggest that the OsWRKY72-OsAAT30/OsGSTU26 module may play an important role in heterosis for salt tolerance in an overdominant fashion in CY1000 hybrid rice, providing valuable clues to elucidate the mechanism of heterosis for salinity tolerance in hybrid rice.
Soil salinity has a major impact on rice seed germination, severely limiting rice production. Herein, a rice germination defective mutant under salt stress (gdss) was identified by using chemical mutagenesis. The GDSS gene was detected via MutMap and shown to encode potassium transporter OsHAK9. Phenotypic analysis of complementation and mutant lines demonstrated that OsHAK9 was an essential regulator responsible for seed germination under salt stress. OsHAK9 is highly expressed in germinating seed embryos. Ion contents and non-invasive micro-test technology results showed that OsHAK9 restricted K+ efflux in salt-exposed germinating seeds for the balance of K+/Na+. Disruption of OsHAK9 significantly reduced gibberellin 4 (GA4) levels, and the germination defective phenotype of oshak9a was partly rescued by exogenous GA3 treatment under salt stress. RNA sequencing (RNA-seq) and real-time quantitative polymerase chain reaction analysis demonstrated that the disruption of OsHAK9 improved the GA-deactivated gene OsGA2ox7 expression in germinating seeds under salt stress, and the expression of OsGA2ox7 was significantly inhibited by salt stress. Null mutants of OsGA2ox7 created using clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 approach displayed a dramatically increased seed germination ability under salt stress. Overall, our results highlight that OsHAK9 regulates seed germination performance under salt stress involving preventing GA degradation by mediating OsGA2ox7, which provides a novel clue about the relationship between GA and OsHAKs in rice.
Auxin regulates flower and fruit abscission, but how developmental signals mediate auxin transport in abscission remains unclear. Here, we reveal the role of the transcription factor BEL1-LIKE HOMEODOMAIN11 (SlBEL11) in regulating auxin transport during abscission in tomato (Solanum lycopersicum). SlBEL11 is highly expressed in the fruit abscission zone, and its expression increases during fruit development. Knockdown of SlBEL11 expression by RNA interference (RNAi) caused premature fruit drop at the breaker (Br) and 3 d post-breaker (Br+3) stages of fruit development. Transcriptome and metabolome analysis of SlBEL11-RNAi lines revealed impaired flavonoid biosynthesis and decreased levels of most flavonoids, especially quercetin, which functions as an auxin transport inhibitor. This suggested that SlBEL11 prevents premature fruit abscission by modulating auxin efflux from fruits, which is crucial for the formation of an auxin response gradient. Indeed, quercetin treatment suppressed premature fruit drop in SlBEL11-RNAi plants. DNA affinity purification sequencing (DAP-seq) analysis indicated that SlBEL11 induced expression of the transcription factor gene SlMYB111 by directly binding to its promoter. Chromatin immunoprecipitation-quantitative polymerase chain reaction and electrophoretic mobility shift assay showed that S. lycopersicum MYELOBLASTOSIS VIRAL ONCOGENE HOMOLOG111 (SlMYB111) induces the expression of the core flavonoid biosynthesis genes SlCHS1, SlCHI, SlF3H, and SlFLS by directly binding to their promoters. Our findings suggest that the SlBEL11-SlMYB111 module modulates flavonoid biosynthesis to fine-tune auxin efflux from fruits and thus maintain an auxin response gradient in the pedicel, thereby preventing premature fruit drop.
Panicle exsertion is one of the crucial agronomic traits in rice (Oryza sativa). Shortening of panicle exsertion often leads to panicle enclosure and severely reduces seed production. Gibberellin (GA) plays important roles in regulating panicle exsertion. However, the underlying mechanism and the relative regulatory network remain elusive. Here, we characterized the oswrky78 mutant showing severe panicle enclosure, and found that the defect of oswrky78 is caused by decreased bioactive GA contents. Biochemical analysis demonstrates that OsWRKY78 can directly activate GA biosynthesis and indirectly suppress GA metabolism. Moreover, we found OsWRKY78 can interact with and be phosphorylated by mitogen-activated protein kinase (MAPK) kinase OsMAPK6, and this phosphorylation can enhance OsWRKY78 stability and is necessary for its biological function. Taken together, these results not only reveal the critical function of OsWRKY78, but also reveal its mechanism via mediating crosstalk between MAPK and the GA signaling pathway in regulating panicle exsertion.
The recretohalophyte Limonium bicolor thrives in high-salinity environments because salt glands on the above-ground parts of the plant help to expel excess salt. Here, we characterize a nucleus-localized C3HC4 (RING-HC)-type zinc finger protein of L. bicolor named RING ZINCFINGER PROTEIN 1 (LbRZF1). LbRZF1 was expressed in salt glands and in response to NaCl treatment. LbRZF1 showed no E3 ubiquitin ligase activity. The phenotypes of overexpression and knockout lines for LbRZF1 indicated that LbRZF1 positively regulated salt gland development and salt tolerance in L. bicolor. lbrzf1 mutants had fewer salt glands and secreted less salt than did the wild-type, whereas LbRZF1-overexpressing lines had opposite phenotypes, in keeping with the overall salt tolerance of these plants. A yeast two-hybrid screen revealed that LbRZF1 interacted with LbCATALASE2 (LbCAT2) and the transcription factor LbMYB113, leading to their stabilization. Silencing of LbCAT2 or LbMYB113 decreased salt gland density and salt tolerance. The heterologous expression of LbRZF1 in Arabidopsis thaliana conferred salt tolerance to this non-halophyte. We also identified the transcription factor LbMYB48 as an upstream regulator of LbRZF1 transcription. The study of LbRZF1 in the regulation network of salt gland development also provides a good foundation for transforming crops and improving their salt resistance.
The structural and functional diversity of plant metabolites is largely created via chemical modification of a basic backbone. However, metabolite modifications in plants have still not been thoroughly investigated by metabolomics approaches. In this study, a widely targeted metabolite modificomics (WTMM) strategy was developed based on ultra-high performance liquid chromatography-quadrupole-linear ion trap (UHPLC-Q-Trap) and UHPLC-Q-Exactive-Orbitrap (UHPLC-QE-Orbitrap), which greatly improved the detection sensitivity and the efficiency of identification of modified metabolites. A metabolite modificomics study was carried out using tomato as a model, and over 34,000 signals with MS2 information were obtained from approximately 232 neutral loss transitions. Unbiased metabolite profiling was also performed by utilizing high-resolution mass spectrometry data to annotate a total of 2,118 metabolites with 125 modification types; of these, 165 modified metabolites were identified in this study. Next, the WTMM database was used to assess diseased tomato tissues and 29 biomarkers were analyzed. In summary, the WTMM strategy is not only capable of large-scale detection and quantitative analysis of plant-modified metabolites in plants, but also can be used for plant biomarker development.
Nypa fruticans (Wurmb), a mangrove palm species with origins dating back to the Late Cretaceous period, is a unique species for investigating long-term adaptation strategies to intertidal environments and the early evolution of palms. Here, we present a chromosome-level genome sequence and assembly for N. fruticans. We integrated the genomes of N. fruticans and other palm family members for a comparative genomic analysis, which confirmed that the common ancestor of all palms experienced a whole-genome duplication event around 89 million years ago, shaping the distinctive characteristics observed in this clade. We also inferred a low mutation rate for the N. fruticans genome, which underwent strong purifying selection and evolved slowly, thus contributing to its stability over a long evolutionary period. Moreover, ancient duplicates were preferentially retained, with critical genes having experienced positive selection, enhancing waterlogging tolerance in N. fruticans. Furthermore, we discovered that the pseudogenization of Early Methionine-labelled 1 (EM1) and EM6 in N. fruticans underly its crypto-vivipary characteristics, reflecting its intertidal adaptation. Our study provides valuable genomic insights into the evolutionary history, genome stability, and adaptive evolution of the mangrove palm. Our results also shed light on the long-term adaptation of this species and contribute to our understanding of the evolutionary dynamics in the palm family.