Lin Zhang

Tung tree (Vernicia fordii) serves as an economically important woody oilseed plant. High-quality protoplasts are essential for applications such as somatic hybridization, studying cell signal transduction and rapid gene function analysis. A reliable protocol for protoplast isolation and transient gene expression was developed using petals, anther-derived callus, and root tips as source materials. Through systematic optimization of key parameters such as enzyme combinations, osmotic pressure, digestion duration, and centrifugation speed, we successfully established tissue-specific isolation protocols. To ensure the availability of widely applicable experimental materials, protoplasts isolated from anther-derived callus were used to optimize transient transformation conditions. Under the optimized parameters (20 µg plasmid, 40% PEG4000, and 30-min incubation), the transformation efficiency reached 44.91%. Using this system, we conducted multiple experimental validations, including subcellular localization of VfWOX1.2 protein, detection of its protein expression, analysis of its transcriptional regulation of phytohormone biosynthetic genes, and assessment of stress-responsive gene expression under various treatment conditions. Collectively, these results confirm the utility of this platform for functional gene studies in tung tree. This study establishes a robust technical foundation, which is helpful for overcoming the limitation posed by the absence of a stable genetic transformation system and will facilitate future research on gene function in woody species.

Camellia oleifera, belonging to the genus Camellia, is an important woody edible oil plant with high ecological and economic values. In this study, we assembled a chromosome-level genome of Camellia brevistyla that represents the closest diploid relative of polyploid C. oleifera based on current research. The genome size of the assembly was 3.10 Gb with a contig N50 size of 88.60 Mb. The BUSCO completeness score was estimated to be 98.88%, indicating high assembly completeness. A total of 2.29 Gb of the sequences were annotated as repetitive elements, accounting for 73.67% of the entire genome assembly. A total of 35,093 protein-coding genes were predicted, with the annotation completeness evaluated to be 97.03% via BUSCO analysis. In conclusion, the high-quality C. brevistyla genome is a pivotal genetic resource within the genus Camellia, which can serve as a vital diploid comparator for evolutionary and genomic studies in the more complex polyploid C. oleifera.

Hippophae gyantsensis Lian is an important native tree species in the “One River, Two Streams” valley of Tibet, valued for its ecological restoration potential and nutrient-rich fruits. However, this species has several limitations, including a long fruiting cycle (3–5 years to flowering and 10–15 years to reach peak fruit production), small fruit size, and numerous branch thorns. These traits hinder large-scale cultivation and mechanized harvesting, creating an urgent need for improved varieties with larger fruit and higher yield. In this study, we established an efficient Agrobacterium-mediated genetic transformation system for H. gyantsensis using hypocotyls as explants. Under optimized conditions (OD₆₀₀ = 0.5, AS = 200 μmol/L, infection time = 15 min), the transformation efficiency reached 36.67% (calculated as the number of PCR-positive plants divided by the total number of explants initially inoculated with Agrobacterium). A rooting rate of 12.5% was achieved using 100 mg/L rooting powder (ABT1) for 40 min, resulting in an overall success rate of approximately 4–5%. Furthermore, we identified and cloned two fruit-size-related genes, Hgfw2.2 and Hgfw3.2, from H. gyantsensis. Heterologous expression of Hgfw2.2 and Hgfw3.2 in tomato decreased and increased fruit size, respectively, consistent with their regulatory roles in fruit development. Given the positive regulatory effect of Hgfw3.2, this gene was further transformed into H. gyantsensis. This study represents the first report of a stable genetic transformation platform for H. gyantsensis, providing a robust technical foundation for future molecular breeding and the development of improved, large-fruited varieties.

The online version contains supplementary material available at 10.1186/s12864-026-12890-w.

Vernicia montana is a dioecious woody tree with a ZW sex determination system, yet the molecular basis of sex determination remains poorly understood. Here, we constructed a chromosome-level, haplotype-resolved female genome assembly using PacBio HiFi sequencing and Hi-C scaffolding, and integrated population-scale coverage-based genome-wide association analysis of 178 natural individuals to identify sex-associated loci. A highly localized sex-associated signal was detected on ChrB02, defining a ~ 61.4-kb W-specific sex-determining region (SDR) that is sharply bounded by conserved syntenic blocks but internally enriched in haplotype-specific insertions, duplications, inversions, and a complex inverted-repeat architecture. PCR assays further validated multiple female-specific segments within this structurally heterogeneous region. Two conserved genes were identified at the SDR boundaries. Notably, VmBASS4.2 displayed pronounced developmental dynamics and broad reproductive expression and is positioned adjacent to major structural rearrangements within the SDR, making it a compelling candidate for functional validation. Heterologous overexpression in Arabidopsis impaired stigma receptivity and increased floral and silique abortion, supporting a dosage-sensitive role in reproductive development. Collectively, our findings suggest that sex determination in V. montana is not driven by the emergence of a novel sex-determining gene, but instead is associated with local inverted-repeat-mediated structural remodeling that reshapes the regulatory landscape of pre-existing boundary genes such as VmBASS4.2. This study proposes an inverted-repeat-mediated SDR evolution model, providing a plausible framework linking local structural architecture to regulatory divergence during the early evolution of homomorphic sex chromosomes in plants.

Lilies (Lilium spp.) are globally important ornamental crops which are constrained by their narrow thermal tolerance range. However, tiger lily (Lilium lancifolium), a wild lily species, exhibits remarkable cold tolerance. Based on our previous findings, we proposed that LlR3MYB, an R3-MYB transcription factor (TF), confers cold tolerance via transcriptional regulation of flavonoid metabolism in tiger lily. Here, we revealed that LlR3MYB represents a unique CPC-type R3-MYB TF exhibiting a bifunctional role in flavonoid metabolism. Specifically, LlR3MYB suppresses anthocyanin biosynthesis while promoting non-anthocyanin flavonoid accumulation (i.e. flavonols, flavones, and chalcones) responding to cold stress. Overexpression of LlR3MYB in tobacco and tiger lily increased total flavonoid content but reduced anthocyanin levels, consistent with the upregulation of early biosynthesis genes (e.g. CHS and FLS) and repression of late biosynthesis genes (e.g. DFR and ANS) in the pathway. In contrast, silencing LlR3MYB in tiger lily reduced total flavonoid production, enhanced anthocyanin accumulation, and compromised cold resistance. Mechanistically, LlR3MYB can directly bind to the AC-I element (ACCTACC) and MBSI motif (CAACGGTT) in the LlCHS2 promoter and activating its transcription, with enhanced activation under low temperature conditions. Mutations of critical residues within the C1/C2 repressor motifs may endow LlR3MYB with this transcriptional activation function. Furthermore, LlDREB can directly bind to the DRE motif (ACCGAC) in the LlR3MYB promoter and activating its transcription in a low-temperature-dependent manner. Our findings uncover a branch-specific regulatory mechanism by which MYB TFs fine-tune flavonoid biosynthesis, highlighting their essential role in plant cold stress responses.

Camellia oil, a nutritionally rich edible oil derived from Camellia oleifera seeds, is predominantly stored as triacylglycerol (TAG) during fruit maturation. However, a limited understanding of the genetic and regulatory mechanisms governing Camellia oil accumulation has hindered efforts to optimize its yield. In this study, three C. oleifera diacylglycerol acyltransferase (CoDGAT) genes were identified and characterized, wherein CoDGAT1 emerged as the primary contributor to seed TAG biosynthesis, functioning within the endoplasmic reticulum and increasing seed oil content by 64.4% in transgenic plant. Through weighted gene coexpression network analysis, we identified WRINKLED1 (CoWRI1) as a candidate transcriptional regulator of CoDGAT1. Both genes exhibited significant upregulation during seed maturation. Mechanistically, CoWRI1 activated CoDGAT1 by directly binding to AW-box motifs in its promoter, thereby promoting TAG accumulation. Additionally, CoWRI1 expression was suppressed by salicylic acid, methyl jasmonate, and darkness, suggesting that phytohormones and environmental signals modulate TAG accumulation through the WRI1-DGAT1 pathway. These findings provide an important basis for improving Camellia oil production through biotechnological manipulation of DGAT1 and WRI1 genes.

Vernicia montana is a dioecious plant widely cultivated for high-quality tung oil production and ornamental purposes in the Euphorbiaceae family. The lack of genomic information has severely hindered molecular breeding for genetic improvement and early sex identification in V. montana. Here, we present a chromosome-level reference genome of a male V. montana with a total size of 1.29 Gb and a contig N50 of 3.69 Mb. Genome analysis revealed that different repeat lineages drove the expansion of genome size. The model of chromosome evolution in the Euphorbiaceae family suggests that polyploidization-induced genomic structural variation reshaped the chromosome structure, giving rise to the diverse modern chromosomes. Based on whole-genome resequencing data and analyses of selective sweep and genetic diversity, several genes associated with stress resistance and flavonoid synthesis such as CYP450 genes and members of the LRR–RLK family, were identified and presumed to have been selected during the evolutionary process. Genome-wide association studies were conducted and a putative sex-linked insertion and deletion (InDel) (Chr 2: 102 799 917-102 799 933 bp) was identified and developed as a polymorphic molecular marker capable of effectively detecting the gender of V. montana. This InDel is located in the second intron of VmBASS4, suggesting a possible role of VmBASS4 in sex determination in V. montana. This study sheds light on the genome evolution and sex identification of V. montana, which will facilitate research on the development of agronomically important traits and genomics-assisted breeding.