Quantitative real-time PCR (qPCR) is an indispensable technique for gene expression analysis in modern molecular biology. The selection and evaluation of suitable reference genes is a prerequisite for accurate gene expression analysis. Schimasuperba is a valuable tree species that is environmentally adaptable and highly fire-resistant. In this study, 12 candidate reference genes were selected to check their stability of gene expression in different tissues under abiotic stresses: cold stress, salt stress, and drought stress by ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder. The results indicated that AP-2 was the most stably expressed overall and for the cold stress and drought stress. eIF-5α gene expression was the most stable under the salt stress treatment, while UBQ expression was the most stable across mature leaves, shoots, stems, and roots. In contrast, UBC20, GAPDH, and TUB were the least stably expressed genes tested. This study delivers valid reference genes in S. superba under the different experimental conditions, providing an important resource for the subsequent elucidation of the abiotic stress adaptation mechanisms and genes with biological importance.

In recent years, the field of biology has witnessed a surge of interest in genomics research due to the advancements in biotechnology. Gene expression pattern analysis plays a crucial role in this research, as it enables us to understand the regulatory mechanism of gene expression and the associated biological processes. Real-time quantitative polymerase chain reaction (q-PCR) is an efficient method to analyze the gene expression patterns, for which accuracy relies on the standardized analysis of reference genes. However, numerous studies have shown that no reference gene is universal in all conditions, so screening a suitable reference gene under certain conditions is of great importance. Cinnamomum burmannii (C. burmannii) is rich in volatile components and has high medicinal and economic value. However, knowledge of the screening of reference genes for the gene expression analysis of C. burmannii is insufficient. Aiming at this problem, we evaluated and screened the reference genes in C. burmannii under different experimental conditions, including different abiotic stresses (Cold-treated, PEG-treated and Nacl-treated), different tissues, leaves at different developmental stages and different chemical types. In this study, different algorithms (∆Ct, geNorm, NormFinder and BestKeeper) were used to evaluate the stability of the candidate reference genes, and RefFinder further merged the output data to screen out the optimum reference gene under various experimental conditions in C. burmannii. The results showed that the optimal reference gene number for gene standardization was 2 under different experimental conditions. RPL27|RPS15 was the most suitable combination under the Nacl-treated and PEG-treated samples. RPL27|APT was the optimum combination under the Cold-treated samples. The optimal combinations of other samples were EF1α|ACT7 for different tissues, eIF-5A|Gllα for different borneol clones in C. burmannii, RPS15|ACT7 for leaves at different developmental stages and RPS15|TATA for all samples. Additionally, two terpenoid synthesis-related genes (CbWRKY4 and CbDXS2) were standardized to verify the feasibility of the selected reference genes under different experimental conditions. This study will be helpful for the subsequent molecular genetic mechanism study of C. burmannii.

The intricate interplay between photosynthetic efficiency and terpenoid biosynthesis in plants remains a pivotal yet underexplored area in secondary metabolism research. This study elucidates the physiological and molecular mechanisms underlying this synergy in Cinnamomum burmanni, a chemically diverse Lauraceae species, through a multi-omics approach. A high-quality chromosome-level genome of C. burmanni (1.14 Gb, scaffold N50: 94.90 Mb) was assembled, and evolutionary analyses revealed species-specific gene family expansions, particularly in mono-, sesqui-, and diterpenoid biosynthesis pathways. Comparative analyses of photosynthetic traits across chemotypes demonstrated that the borneol-type exhibits superior photosynthetic capacity, characterized by elevated net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO₂, carboxylation efficiency and non-photochemical quenching. The phenotype is linked to upregulated chlorophyll metabolism, carotenoid biosynthesis regulators, and enhanced light-harvesting complex and photosystem components, optimizing light energy conversion. Mechanistically, photosynthetic activity modulates precursor flux into terpenoid pathways by regulating rate-limiting enzymes. Additionally, lineage-specific expansions of terpene synthase and isopentenyl diphosphate synthase gene families underpin specialized terpenoid production. A post-transcriptional regulatory network involving 14 miRNAs (e.g., miR396, miR2950) was identified, coordinately targeting 11 key genes in both photosynthesis and terpenoid synthesis, suggesting a dual role in metabolic fine-tuning. This work advances understanding of the evolutionary and physiological integration of photosynthesis and secondary metabolism in aromatic plants, offering a genomic foundation for biotechnological applications in metabolite synthesis and chemotype breeding.

Schima superba, commonly known as the Chinese guger tree, is highly adaptable and tolerant of poor soil conditions. It is one of the primary species forming the evergreen broad-leaved forests in southern China. Dirigent proteins (DIRs) play crucial roles in the synthesis of plant lignin and lignans, secondary metabolism, and response to adversity stress. However, research on the DIR gene family in S. superba is currently limited. This study identified 24 SsDIR genes, categorizing them into three subfamilies. These genes are unevenly distributed across 13 chromosomes, with 83% being intronless. Collinearity analysis indicated that tandem duplication played a more significant role in the expansion of the gene family compared to segmental duplication. Additionally, we analyzed the expression patterns of SsDIRs in different tissues of S. superba. The SsDIR genes exhibited distinct expression patterns across various tissues, with most being specifically expressed in the roots. Further screening identified SsDIR genes that may regulate drought stress, with many showing differential expression under drought stress conditions. In the promoter regions of SsDIRs, various cis-regulatory elements involved in developmental regulation, hormone response, and stress response were identified, which may be closely related to their diverse regulatory functions. This study will contribute to the further functional identification of SsDIR genes, providing insights into the biosynthetic pathways of lignin and lignans and the mechanisms of plant stress resistance.

Camphora officinarum Nees constitutes a pivotal tree species within the evergreen broad-leaved forests of East Asia, endowed with significant economic, ornamental, and ecological importance. Nevertheless, previous research has markedly underestimated the genetic diversity of this species, thereby hindering our efforts in conserving resources and enhancing genetic breeding. The current study generated 155 chloroplast genomes from specimens of C. officinarum obtained from six provinces/regions in China. The results reveal the identification of seven distinct clades (I–VII), with Clades II, III, V, and VII exhibiting genome expansions, primarily influenced by lineage-specific elongation of inverted repeats (IRs), whereas Clades I, IV, and VI maintained conserved IR lengths. Despite the structural plasticity, the GC content remained highly conserved. Geographic patterns indicated gene flow between adjacent regions (e.g., Hunan and Hubei with identical IR lengths), but genetic isolation in Fujian. High-polymorphism regions (psba-matK, ycf1, ycf2, and ndhF) were identified as superior phylogenetic markers, enhancing intraspecies-level resolution. Simple sequence repeats (SSRs) varied significantly among clades, dominated by A/T-rich mononucleotide repeats. These repeats, along with divergent repeat types (e.g., absence of reverse repeats in Clades V/VI), serve as robust tools for resource identification and evolutionary trajectory inference. Phylogenetically, samples from Fujian formed a distinct lineage, while samples from other regions, especially Guangdong, were mixed, with this finding probably being a reflection of historical cultivation and anthropogenic translocation. This study offers a framework for the genetic breeding and investigation of the evolutionary history of C. officinarum.

Cinnamomum burmannii, renowned for its high essential oil content in leaves, is a pivotal species utilized for aromatic medicinal and industrial materials. This study focused on the functional identification of key regulatory enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) for terpenoid biosynthesis in the leaves of C. burmannii. A comprehensive approach, integrating terpenoid profiling assay and synthesis pathway construction, correlation analysis of terpenoid content and gene expression, and genome-wide analysis of DXS family across four developmental stages of leaves in three accessions of C. burmannii, led to the identification of CbDXS1 as a candidate regulatory gene for terpenoid biosynthesis. Overexpressing CbDXS1 in Arabidopsis enhanced plant growth, DXS enzyme activity, and chlorophyll content, and elevated transcriptional levels of terpenoid biosynthesis-related genes, leading to significant increases in terpenoid metabolites in transgenic leaves. Additionally, alterations in metabolite contents in the pathways of glycolysis, the tricarboxylic acid cycle, oxidative pentose phosphate, the Calvin cycle, oxidative phosphorylation, amino acid metabolism, and phytohormone biosynthesis suggested a potential redirection of carbon flux from primary metabolism to terpenoid biosynthesis and changes in endogenous phytohormone contents, the diterpene biosynthesis pathway, and amino acid metabolism, which may collectively contribute to terpenoid accumulation and phenotypic improvement in transgenic Arabidopsis. Our findings elucidated the multifaceted roles of CbDXS1 in modulating carbon flux and phytohormone biosynthesis for terpenoid production and plant development, offering potential strategies for engineering essential oil accumulation in the leaves of C. burmannii.