Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; Flowers/genetics* ; Rhododendron/genetics*

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe²⁺ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.
Arabidopsis/metabolism* ; Rhododendron/metabolism* ; Amino Acid Sequence ; Anthocyanins/metabolism* ; Phylogeny ; Flavonoids/metabolism* ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism*

Terpene synthase (TPS) plays important roles in the synthesis of terpenoids which are the main fragrances in Rhododendron flowers. To understand the function of TPS genes in terpenoid metabolism in relation to flower aroma formation, we identified all TPS gene family members in Rhododendron by analyzing its genome database. We then used a transcriptomic approach to analyze the differential gene expression patterns of TPS gene family members in the scented flower Rhododendron fortunei compared to the non-scented flower Rhododendron 'Nova Zembla'. The contents of terpenoid compounds in petals of the above two Rhododendron species at different developmental stages were also measured by using qRT-PCR and head space-solid phase micro-extraction combined with gas chromatography-mass spectrometry. Our results showed that a total of 47 RsTPS members, with individual lengths ranged from 591 to 2 634 bp, were identified in the Rhododendron genome. The number of exons in RsTPS gene ranged from 3 to 12, while the length of each protein encoded ranged from 196 to 877 amino acids. Members of the RsTPS family are mainly distributed in the chloroplast and cytoplasm. Phylogenetic analysis showed that RsTPS genes can be clustered into 5 subgroups. Seven gene family members can be functionally annotated as TPS gene family since they were temporally and spatially expressed as shown in the transcriptome data. Notably, TPS1, TPS10, TPS12 and TPS13 in Rhododendron fortunei were expressed highly in flower buds reached the peak in the full blossoming. Correlation analysis between gene expression levels and terpenoid content indicates that the expression levels of TPS1, TPS4, TPS9, TPS10, TPS12 and TPS13 were positively correlated with the content of terpenoids in the petals of R. fortunei at all flower developmental stages, suggesting that these six genes might be involved in the aroma formation in R. fortunei.
Rhododendron/metabolism* ; Phylogeny ; Terpenes/metabolism* ; Family ; Gene Expression Regulation, Plant