Leaf color mutants serve as excellent models for investigating the metabolic pathways involved in chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants. This study aimed to elucidate the mechanisms underlying color formation in the yellow–green leaf mutant (YL) of Magnolia sinostellata by employing physiological, cytological and transcriptomic analyses to compare the mutant with control plants (wild type Magnolia sinostellata, WT). Physiological assessments revealed a reduction in chlorophyll content, particularly chlorophyll b, alongside an increase in the flavonoid level in YL relative to WT. Cytological examinations indicated the presence of defective chloroplasts within the mesophyll cells of the mutants. Transcriptomic analysis identified 8205 differentially expressed genes, with 4159 upregulated and 4046 downregulated. Genes associated with chlorophyll metabolism, flavonoid metabolism, photosynthesis, and signaling pathways were found to play crucial roles in leaf yellowing. In conclusion, this study delineated the phenotypic, physiological, cytological, and transcriptomic differences between YL and WT leaves, offering novel insights into the mechanisms driving leaf yellowing in Magnolia sinostellata.

Paper Mulberry (Broussonetia papyrifera) possesses medicinal, economic, and ecological significance and is extensively used for feed production, papermaking, and ecological restoration due to its ease of propagation, rapid growth rate, and strong stress resistance. The recent completion of the sequencing of the Paper Mulberry genome has prompted further research into the genetic breeding and molecular biology of this important species. A highly stable reference gene is essential to enhance the quantitative analysis of functional genes in Paper Mulberry; however, none has been identified. Accordingly, in this study, the leaves, stems, roots, petioles, young fruits, and mature fruits of Paper Mulberry plants were selected as experimental materials, and nine candidate reference genes, namely, α-TUB1, α-TUB2, β-TUB, H2A, ACT, DnaJ, UBQ, CDC2, and TIP41, were identified by RT-qPCR. Their stability was assessed using the geNorm, Normfinder, Delta Ct, BestKeeper, and RefFinder algorithms, identifying ACT and UBQ as showing the greatest stability. The expression of BpMYB090, which regulates the production of trichomes, was examined in the leaves of plants of the wild type (which have more trichomes) and mutant (which have fewer trichomes) at various developmental stages to validate the results of this study. As a result, their identification addresses a critical gap in the field of Paper Mulberry research, providing a solid foundation for future research that will concentrate on the characterization of pertinent functional genes in this economically valuable species.

Liquidambar formosana Hance is renowned for its rich leaf color and possesses notable advantages, such as robust adaptability, strong resistance to diseases and pests, and rapid growth, making it a preferred choice for urban greening and carbon sequestration forest initiatives. The completion of whole-genome sequencing of L. formosana has spurred an increased interest in exploring the molecular mechanisms underlying seasonal changes in leaf color, marking a significant focus in L. formosana breeding research. However, there is currently a lack of stable reference genes suitable for analyzing the expression patterns of functional genes in L. formosana exhibiting varying leaf colors. This study selected five L. formosana varieties with significant differences in leaf colors. Through the RT-qPCR analysis, and evaluation using BestKeeper, geNorm, NormFinder, Delta Ct, and RefFinder, the expression stability of 14 candidate reference genes was examined. Consequently, two reference genes (LifEF1-α and LifACT) with stable expression, suitable for RT-qPCR of L. formosana with diverse leaf colors, were identified. The stability of these selected reference genes was further validated by examining the LifbHLH137 gene, which promoted the biosynthesis of anthocyanins. This advancement facilitated molecular biology and genetic breeding investigations of L. formosana, providing essential data for the precise quantification of functional genes associated with leaf color variation.

Rhododendron lapponicum (L.) Wahlenb., prized for its vibrant and diverse floral displays, holds significant ornamental and ecological value. However, advances in its molecular breeding have been constrained by the absence of reliable tools for accurate gene expression analysis. A fundamental requirement for such studies is the identification of stable reference genes for qRT-PCR. To date, no systematically validated reference genes exist for normalizing gene expression across R. lapponicum cultivars with diverse flower colors, representing a major technical obstacle to elucidating the molecular mechanisms of color formation. This study aimed to fill this gap by systematically identifying and validating optimal reference genes for petal tissues in six distinct R. lapponicum cultivars. We assessed the expression stability of 11 candidate genes using four independent algorithms and integrated the results via RefFinder. Our comprehensive analysis across multiple algorithms consistently identified RlaEF1-α and RlaACT as the most stably expressed reference genes. Their reliability was robustly validated by normalizing the expression of RlaMYB113, a key anthocyanin regulator; the normalized expression levels showed an extremely significant difference between rose-red and white cultivars (p < 0.001) and produced a coherent, phenotype-correlated profile, in contrast to the distorted patterns obtained with unstable references. This study establishes RlaEF1-α and RlaACT as a precise dual-gene internal control for qRT-PCR. By providing a validated normalization framework, our work enables accurate quantification of color-related genes and directly supports molecular breeding efforts aimed at the targeted development and selection of novel R. lapponicum cultivars with desirable and stable flower colors.

Seasonal changes in leaf coloration are a key ecological and ornamental characteristic of Liquidambar formosana. To clarify the molecular basis of this process, we performed an integrated transcriptomic and metabolomic investigation comparing wild-type L. formosana (FX) with the autumn-red cultivar ‘Jinyu’ (JY). Leaves were sampled before and after the color transition. Analyses revealed distinct metabolic pathways driving coloration in each genotype. In JY, the red phenotype was primarily attributed to the activation of anthocyanin biosynthesis, characterized by coordinated upregulation of key structural genes (LfDFR, LfANS, LfBZ1) and significant accumulation of anthocyanins, especially pelargonidin derivatives. Conversely, FX exhibited enhanced flavonol biosynthesis and carotenoid/terpenoid metabolism, leading to greater yellow pigment accumulation and an orange-yellow hue. Weighted gene co-expression network analysis (WGCNA) identified a core module strongly correlated with anthocyanin content in JY, which was significantly enriched with transcription factors from the MYB, bHLH, and WRKY families. These results demonstrate that different L. formosana genotypes employ divergent metabolic strategies for autumn coloration, governed by specific transcriptional regulatory networks. This study provides crucial insights into pigment regulation in woody plants and offers valuable candidate genes for the molecular breeding of ornamental L. formosana cultivars.