Real-time quantitative PCR (RT-qPCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a fast-growing timber species with strong resistance. However, thus far, reliable reference gene identifications have not been reported in S. superba. In this study, 19 candidate reference genes were selected and evaluated for their expression stability in different tissues of S. superba. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results show that SsuACT was the most stable reference gene and that SsuACT + SsuRIB was the best reference gene combination for different tissues. Finally, the stable and less stable reference genes were verified using SsuSND1 expression in different tissues. To our knowledge, this is the first report to verify appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate the future elucidation of gene regulations in this species and useful references for relative species.

The firmness of grape berries is a critical factor influencing their commercial feasibility and is highly valued by both breeders and consumers. However, grape berry firmness is a complex quantitative trait governed by multiple genes, and our understanding of its genetic regulatory network remains incomplete. To elucidate the genetic mechanisms underlying grape berry firmness, this study employed genome-wide association studies (GWAS) to identify potential candidate genes associated with fruit firmness and cellulose content and to explore the gene regulatory network that controls their variation. The comprehensive GWAS results identified CesA as a candidate gene potentially regulating fruit firmness through its involvement in cellulose biosynthesis. To validate these findings, whole-genome gene family identification analysis was conducted. Furthermore, the key gene VvCslD5 was selected for functional validation, which included overexpression studies and subcellular localization. This study provides valuable insights into the regulation of biosynthesis and transcriptional signaling pathways that govern the structure of grape cell walls as well as the mechanisms underlying variations in grape firmness. These findings establish a solid foundation for future functional analyses of grape traits and will enhance breeding practices aimed at improving grape quality.

Background: Real-time quantitative PCR (RT-qPCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results: Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions: This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

Rhododendron, a globally popular ornamental flower, is nevertheless limited in our understanding of the mechanisms underlying its fragrance formation. Notably, terpenoids are the most prevalent volatile metabolite produced by plants. In this study, gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS) and transcriptomics sequencing were conducted to analyze the synthesis mechanisms of terpenoid fragrance compounds of petals in fragrant R. fortunei Lindl. (YJ) and non-fragrant R. “Nova Zembla” (NW). The results identified that (-)-myrtenol, linalool, pinene, myrtenyl acetate, and terpineol were key floral aroma substances in YJ. Furthermore, an analysis of KEGG enrichment and differentially expressed genes (DEGs) revealed that the bud and decay stages exhibited the highest number of enriched DEGs among different aroma types, indicating these as critical stages for the synthesis of terpenoid floral compounds. In this study, a structural gene, denoted as RfFDPS, was identified as a negative regulatory gene for monoterpene accumulation and a positive regulatory gene for sesquiterpene accumulation in YJ. Utilizing subcellular localization technology, we determined that RfFDPS proteins are located in the cytoplasm. A functional analysis through transient expression and gene silencing of RfFDPS demonstrated its ability to regulate the accumulation of monoterpenes and sesquiterpenes. The overexpression of RfFDPS led to an increase in the expression of structural genes related to terpenoid synthesis, resulting in a decrease in monoterpenes and an increase in sesquiterpenes. Conversely, gene silencing had the opposite effect. In conclusion, RfFDPS plays a pivotal role in the synthesis and release of terpenoid volatile compounds in YJ petals, laying a solid theoretical foundation for the cultivation and enhancement of aromatic R. species.

Grapes are prone to softening, which limits their shelf life and suitability for long-distance transport. This study explored the molecular mechanisms underlying the effects of the chemical preservatives gibberellin (GA₃) and the nitric oxide donor sodium nitroprusside (SNP) on grape firmness. Enhancing grape quality, prolonging shelf life, and extending market supply were key objectives. Using transcriptomic and physicochemical analyses, the study found that treatments with 3 mmol/L GA₃ and 20 μmol/L SNP significantly increased the firmness of ‘Yinhong’ grapes, thereby improving overall quality. The mechanisms of action, however, differed between the two treatments. GA₃ inhibited pectin degradation and promoted cellulose accumulation, whereas SNP enhanced lignin and cellulose accumulation, by modulating cell wall metabolism. Furthermore, transcription factors such as CYPs, NAC043, and WRKY33 were identified as key regulators working in concert with target genes to influence berry firmness. These findings highlight the critical roles of GA₃ and SNP in improving grape quality and extending storage potential.

Berry color is a critical determinant of grape quality and market value. While the genetic basis of skin color has been extensively studied, the regulatory network controlling flesh coloration remains largely uncharacterized. To systematically dissect the independent genetic architectures underlying these traits, we performed a genome-wide association study (GWAS) on 130 grape accessions, integrated with spatiotemporal expression profiling, subcellular localization, and functional validation. Our analysis revealed distinct genetic loci for skin and flesh color, confirming their independent regulation. For skin color, GWAS robustly validated VvMYBA2 as a major locus, explaining up to 51.5% of the phenotypic variance. More importantly, for flesh color, we identified and prioritized VvF3′M (Flavonoid 3′-monooxygenase) as a key candidate gene. Heterologous overexpression of VvF3′M in tobacco resulted in a profound 13.5-fold increase in anthocyanin content, suggesting its potential role as a rate-limiting enzyme in flesh pigmentation. Intriguingly, VvF3′M-overexpressing plants also exhibited a significant increase in flower number, suggesting a novel role beyond pigment biosynthesis. This study provides a comprehensive genetic and functional framework for understanding berry coloration, identifies VvF3′M as a prime target for breeding red-fleshed grapes, and reveals unexpected crosstalk between color metabolism and reproductive development.