Abstract Trifolium pratense L. is a multifunctional crop of agronomic importance for forage, horticulture, and ecological restoration. However, the lack of a high-quality genome assembly and the limited representation of genetic diversity by a single reference have impeded its genetic research and molecular breeding. Here, we present the first telomere-to-telomere (T2T) gap-free genome for the diploid (2n = 2x = 14) cultivar T. pratense cv. ‘Zhongtian No. 5’ (TpraZt5), assembled through an integrated sequencing strategy. The 390.94 Mb assembly demonstrates high quality, with a base accuracy > 98.5%, 98.1% BUSCO completeness, a long terminal repeat assembly index of 25.65, and a contig N50 of 52.95 Mb. We annotated 35 971 protein-coding genes and found repeat sequences accounting for 59.6% of the genome. The assembly resolved all seven centromeres and 14 telomeres, providing unprecedented insight into these complex genomic regions. We further constructed a 480.76 Mb pan-genome by integrating two additional accessions, which classified genes into core (70.2%), dispensable (25.3%), and private (4.5%) sets. Comparative genomic analyses identified 606 species-specific genes in TpraZt5 and uncovered extensive structural variations. Functional investigations revealed four species-specific genes and six contracted genes associated with isoflavonoid biosynthesis, two expanded chlorophyll a-b binding proteins, and seven expanded auxin-related genes that may contribute to the high productivity of TpraZt5. Additionally, 44 Gypsy-type transposons within the zeatin biosynthesis pathway were identified as potential regulators of trifoliate leaf development. These genomic resources substantially improve structural annotation and functional characterization, providing vital tools for gene discovery and enhancing molecular breeding initiatives in red clover.

Abstract Banana, a globally important staple fruit, is naturally deficient in anthocyanins; however, successful engineering of anthocyanin-enriched banana has not been reported to date. Herein, a regulatory-network of five R2R3-MYBs (MusaUP1, MusaUP2, MbaMA2, MusaMA4, and MusaMA8) differentially synchronizing anthocyanin-biosynthesis in banana bract is reported. RNA-seq data of red-bract revealed a web of regulatory and structural-genes fine-tuning anthocyanin accumulation through amalgamation of MYBs and bHLHs activities. Y1H demonstrated differential affinities of these MusaMYBs to banana TT8, CHS, ANR, UFGT, FLS, ANS, and LAR, revealing an intricate pattern of layered-regulation in bract-pigmentation. Functional competence of this MYBs network resulted in intense anthocyanin-accumulation in whitish-onion and restoration of pigmentation in myb90/tt8 Arabidopsis seedlings. Hierarchical regulation in this MYB network stemmed in contrasting control over early and late flavonoid structural-genes as revealed by disparate orange-fluorescence of myb90/tt8 Arabidopsis-seedlings after DPBA-staining. In banana, a distinctive requirement of TT8 for pigmentation was observed for MbaMA2 and MusaMA8, while MusaUP1, MusaUP2, MusaMA4, were self-competent, although co-expression of MusaTT8 augmented the ectopic pigmentation-effect. Transcript abundance of flavonoid structural-genes in transgenic banana is in coherence with Y1H-data, thus catalysing pigmentation upto 500-fold over control. This regulatory MYBs hierarchical framework manifested flux in a spectrum of distinct pigment-metabolites, viz peonidin-3,5-diglucoside in MusaUP1 and MusaUP2, dalbergiodin in MbaMA2/TT8 lines (FLS-mediated pathway), leucodelphinidin and leucopelargonidin in MusaMA4 lines (DFR to ANS flux), and prodelphinidin B4 in MusaMA8 lines. This study will be a step forward towards metabolic-engineering for bio-fortification of banana and development of functional foods, as evident by strong antioxidant activities of these MYB lines.

Abstract Sucrose synthase (SUS) is a pivotal enzyme bridging primary carbon metabolism and secondary biosynthesis in plants. In Panax notoginseng, we demonstrate that sucrose synthase 1 (PnSUS1) serves as a metabolic bottleneck for saponin glycosylation by supplying UDP-glucose. PnWRKY38 was identified as a WRKY transcription factor whose expression correlated with both PnSUS1 and saponin accumulation. Overexpression of PnWRKY38 could up-regulate PnSUS1 expression by 3.5-fold, increase SUS enzyme activity by 2.8-fold and elevate UDP-glucose pools by 68%. Consequently, the total content of ginsenosides Rg1, Rb1 and Rd rose by 2.1–2.4-fold. Conversely, PnSUS1 or PnWRKY38 suppression reduced UDP-glucose available and saponin biosynthesis by > 50%. Y1H and luciferase assays indicated that PnWRKY38 directly activated PnSUS1 expression by binding to W-box motifs in its promoter. These results not only illustrate the specific function that PnSUS1 executes in UDPG biosynthesis but also reveal a new WRKY transcriptional regulatory module regulating notoginsenosides production.

Abstract Chilli incurs substantial yield losses due to Thrips parvispinus (Karny) infestation, necessitating sustainable resistance breeding strategies. Understanding biochemical basis of resistance will help in exploring the candidate metabolites for indirect selection. LC-MS and GC-MS profiling of two resistant (IIHR-B-HP-79, IIHR 4550) and two susceptible (IIHR 3455, IIHR 4604) chilli accessions were performed. LC-MS profiling revealed Inositol with higher levels in susceptible accession IIHR 3455 (8.74μg/g & 0.33μg/g; VIP score: 2 & 2.5 under control and infested conditions respectively), indicating its role as a stress-induced metabolite rather than a marker for resistance. Secondary metabolites contribution to resistance was genotype-specific and may possibly be driven by complex interactions among these metabolites. Untargeted leaf volatile profiling revealed Hex-3(Z)-enyl butyrate as a significant volatile compound in resistant accessions IIHR 4550 and IIHR-B-HP-79, in high concentrations suggesting that its production is not strictly species-specific effects as a synthetic volatile. Validation of Hex-3(Z)-enyl butyrate through bioassays and olfactometer studies demonstrated reduced scraping damage percentage at 8 μl-L and 16 μl-L concentrations in leaf dip bioassays. Four-arm olfactometer studies indicated that Hex-3(Z)-enyl butyrate significantly affected T.parvispinus time spent and entries at 16 μl-L. identified metabolites defences can serve as markers for breeding and also can be explored in pest management strategies.

Abstract Ripening Inducing Factor (RIF) is a key NAC transcription factor regulating strawberry fruit ripening. Previous studies using RIF-RNAi and overexpression lines in Fragaria × ananassa and CRISPR knock-out lines in F. vesca have established the role of RIF in controlling ABA biosynthesis and signaling, cell wall remodeling, and secondary metabolism. In this study, we deciphered FaRIF’s transcriptional regulatory network by combining ChIP-seq-based identification of its direct targets with an analysis of FaRIF-RNAi transcriptome data. These analyses revealed FaRIF’s direct role in multiple aspects of strawberry fruit ripening, including the regulation of ripening-related transcription factors, phytohormone content and signaling, primary and secondary metabolism, and cell wall degradation. Additionally, using the TurboID-based proximity labeling approach, we have identified FaRIF interactors, including proteins involved in mRNA and protein homeostasis, as well as several NAC transcription factors. Among these, FaNAC021 and FaNAC034 were found to potentially cooperate with FaRIF to enhance the transcription of shared target genes. This integrative analysis, combining transcriptome analysis, in vivo ChIP-seq, and proximity labeling, broadens our understanding of FaRIF-mediated transcriptional networks and interaction partners, providing valuable insights into the molecular regulation of strawberry fruit ripening by this transcription factor.

Flower color is a key trait influencing insect pollination and ornamental value, yet the molecular mechanisms underlying heterozygous flower color remain unclear. In this study, we identified the creation of a yellow-white chimeric flower (cf) mutation in Brassica napus, characterized as the coexistence of yellow and white colors on petals of the same flower. Genetic analysis revealed that chimeric flower formation is controlled by a completely dominant gene. Map-based cloning, transgenic complementation, and CRISPR/Cas9 experiments consistently confirmed that BnaC05G0385300ZS on chromosome C05 is the causal gene of CF, which encodes a plastid DNA polymerase IB (BnaC05.POLIB). A G-to-A mutation in the seventh exon results in a D742N substitution, which disrupts Mg2+ binding and impairs polymerase activity. This leads to a reduced plastid genome copy number, decreased chromoplast formation, and aberrant carotenoid accumulation, ultimately resulting in the chimeric phenotype in a dosage-dependent manner. These findings reveal a novel role for BnaC05.POLIB in petal color patterning and provide a strategy for breeding ornamental plants with heterozygous flowers.

Abstract Camellia sinensis Fuding Dahaocha, a triploid white tea cultivar widely cultivated in south China, exhibits distinctive traits including dense leaf trichomes, early sprouting, and robust stress resistance. Here, we present the first high-quality chromosome-level genome assembly of this triploid variety, resolved through integrated PacBio long-read sequencing and Hi-C scaffolding. The genome assembly spans 45 chromosomes with a scaffold N50 of 182 Mbp. A total of 149 455 gene models were annotated and mapped to chromosomes, among which 30 568 were identified as protein-coding genes. The genome features high repetitiveness (65.9% TEs), heterozygosity, and three distinct haplotype sets with substantial allelic variation (17 601 tri-allelic genes), with the retained haplotype-specific genes potentially contributing to regulatory complexity through dosage effects. Genome completeness assessment revealed a BUSCO completeness of 99.0% (2303 out of 2326 conserved core genes identified), which included 40 single-copy (1.7%) and 2263 duplicated (97.3%) genes. Evolutionary analyses indicated conserved relationships among the three homologous chromosome sets. We also performed single-nucleus RNA sequencing on a sufficiently large pooled sample of leaf tissues to study trichome development, overcoming technical limitations posed by secondary metabolites and low protoplast isolation efficiency. This yielded a single-cell atlas for woody plants, identifying 35 trichome-specific marker genes and modeling developmental trajectories during epidermal differentiation. Functional validation identified CsCUT1 as a suppressor of trichome branching and CsMYB4 as a negative regulator of trichome initiation. Cell cycle analysis showed G2-phase dominance in developing trichomes. These findings provide a genetic framework for trichome development and offer resources for tea breeding.