To obtain stable Chinese olive reference genes, eight genes (RPN2B, PIP1.4, NIFS1, RPS16, At5g12110, HSC-2, ABCG44, LOS1) exhibiting stable expression were identified as candidate reference genes from the transcriptome. The expression stability of these genes was evaluated across 33 Chinese olive fruit samples from different varieties and seven developmental stages. The most stable reference genes were determined through comparisons using ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder. Analysis revealed that RPN2B and NIFS1 were consistently ranked among the most stable genes across the different algorithms and exhibited stable expression. Therefore, they are recommended as suitable reference genes for gene expression studies in Chinese olive fruits across different varieties and developmental stages. The four different methods of reference gene stability analysis were used to identify the most stable reference genes in different varieties and developmental stages of Chinese olive fruits, which can be used as a reference for the selection of reference genes in the subsequent gene expression studies of Chinese olive fruits.

Floral volatile terpenoids are known to play important roles in plant pollination biology by attracting animal pollinators and repelling antagonists, as well as enhancing resistance to potential microbial pathogens. Terpenoid blend emitted by a flower is usually plant-lineage specific and mostly determined by a set of versatile terpene synthases (TPSs) which catalyze the final step of diverse terpenoid synthesis. The strongly scented flower of Jasminum sambac emits linalool and α-farnesene dominating the nocturnal floral VOC, yet the corresponding TPSs have not been identified. Here, we show that four TPS enzymes are responsible for the synthesis of the mixtures of volatile terpenoids in the flower, based on their highly-correlated and almost-exclusive expression in the petal, and on their enzymatic characterizations in vitro and in Nicotiana benthamiana. JsTPS01 (TPS-a) acts as a sesquiterpene synthase, producing τ-cadinol in yeast at levels that mirror its rhythmic expression in petals. JsTPS02 (TPS-b) carries a plastid-transit peptide, localizes to chloroplasts/plastids, and converts GPP to linalool with high affinity (Km = 28.2 ± 3.4 µM). JsTPS03 is a TPS-b clade member that is able to convert FPP to farnesol with a Km of 14.4 ± 5.9 μM in an in vitro assay using isolated yeast vehicles. JsTPS04 (TPS-e/f) exhibits dual targeting—cytosolic in Arabidopsis protoplasts but plastidic in J. sambac petals—and functions as a bifunctional mono-/sesqui-TPS, forming linalool from GPP (Km = 2.5 ± 0.3 µM) and trans-nerolidol from FPP (Km = 7.6 ± 0.6 µM). Transient expression in Nicotiana benthamiana leaves further confirmed its in-planta linalool production. collectively, we identified four preferentially expressed terpene synthases contribute to linalool, τ-cadinol, tans-nerolidol, and farnesol in Jasminum sambac.

Floral volatile terpenoids are known to play important roles in plant pollination biology by attracting animal pollinators, repelling antagonists, and enhancing resistance to potential microbial pathogens. The terpenoid blend emitted by a flower is usually plant-lineage specific and is primarily determined by a set of versatile terpene synthases (TPSs), which catalyze the final step of diverse terpenoid synthesis. The strongly scented flower of Jasminum sambac (L.) Aiton emits linalool and α-farnesene, which dominate the nocturnal floral VOCs, yet the corresponding TPSs have not been identified. Here, we show that four TPS enzymes are responsible for the synthesis of a mixture of volatile terpenoids in the flower, based on their highly correlated and almost exclusive expression in the petal, as well as their enzymatic characterizations in vitro and in Nicotiana benthamiana Domin. JsTPS01 (TPS-a) acts as a sesquiterpene synthase, producing τ-cadinol in yeast at levels that mirror its rhythmic expression in petals. JsTPS02 (TPS-b) carries a plastid-targeting transit peptide, localizes to chloroplasts/plastids, and converts geranyl diphosphate (GPP) to linalool with high affinity (K_m = 28.2 ± 3.4 µM). JsTPS03 is a TPS-b clade member that can convert farnesyl diphosphate (FPP) to farnesol with a K_m of 14.4 ± 5.9 μM in an in vitro assay using isolated yeast vehicles. JsTPS04 (TPS-e/f) exhibits dual targeting—cytosolic in protoplasts of Arabidopsis thaliana (L.) Heynh, but plastidic in J. sambac petals—and functions as a bifunctional mono-/sesqui-TPS, forming linalool from GPP (K_m = 2.5 ± 0.3 µM) and trans-nerolidol from FPP (K_m = 7.6 ± 0.6 µM). Transient expression in N. benthamiana leaves further confirmed its in-planta linalool production. Collectively, we identified four preferentially expressed terpene synthases that contribute to the production of linalool, τ-cadinol, trans-nerolidol, and farnesol in J. sambac.

Flower color is a core ornamental trait. Jasminum sambac (L.) Ait., a famous raw material for jasmine tea and fragrance, produces white flowers with extremely low seed-setting, which limits its hybrid breeding. Jasmine petals naturally accumulate pigments under biotic/abiotic stress or senescence, while relevant studies on its regulatory mechanism are limited. Here, we established a stable artificial system to reproduce petal coloration, defined four petal developmental stages, and identified cyanidin 3-rutinoside as the main pigment. At 0 h, petal abscisic acid (ABA) was at a relatively high level, and salicylic acid (SA) and ethylene (ETH) peaked at 12 h. Cyanidin 3-rutinoside initiated accumulation at 24 h and peaked at 36 h. By integrating transcriptomic and metabolomic datasets, we uncovered the regulatory TF-gene-metabolite network linked to pigment formation and pinpointed two key structural genes, dihydroflavonol 4-reductase gene (DFR) and anthocyanidin synthase gene (ANS), whose expression surged over five-fold and tightly correlated with pigment accumulation, highlighting their core functions in anthocyanin biosynthesis. JsWRKY70 and two MYBs were further identified as potential regulators of flavonoid and anthocyanin pathways. These findings deepen the understanding of jasmine petal coloration at metabolic and transcriptional levels, and reveal the molecular basis of its flavonoid and anthocyanin biosynthesis.