Floral scent components in Rhododendron fortunei and its regulation by gene expression of S-adenosyl-l-methionine: benzoic acid carboxyl methyl transferase (BAMT) / Componentes de aroma floral em Rhododendron fortunei e sua regulação pela expressão gênica de S-adenosil-L-metionina: ácido benzóico carboxil metil transferase (BAMT)

Rhododendron fortune belongs to a scented Rhododendron species native to China, which produces fragrant flowers of great ornamental and environmental values for landscaping or indoor beautification. However, the scents in Rhododendron fortuneihave not yet been investigated, let alone the mechanism of the formation of these fragrance in the flowers. In this study, we measured the scents in terms of its volatile components and contents (VOC) in Rhododendron fortuneiat four different flowering stages and in different tissues of the plant by headspace solid-phase micro-extraction combined (HS-SPME) with gas chromatography-mass spectrometry (GC-MS). Then the characteristic aromatic values, which reflects the degree of scent perception by human, of each VOC in the plant was calculated according to its respective aromatic thresholds. Results showed that three main VOCs measured from highest to lowest are methyl benzoates, terpenes and fatty acid derivatives. Their content increased after the flower bud opening and reached the highest at half to full blossom. In a flower most VOC contents were measured in petals and only trace amount in other tissues such as stamen, pistil. A small amount of VOCs was determined in leaves as well.All aromatic values were almost corresponded to the contents of three main VOCs, indicating that the flower fragrance arises truly from these VOC components. S-adenosyl-L-methionine: benzoic acid carboxyl methyl transferase (BAMT) catalyzes the final step to form methyl benzoates. To understand the mechanism of the formation of this main type fragrance and its regulation, we firstly isolate a gene of RfBAMT from petal of Rhododendron fortuneiby using homologous cloning and RACE technology. The full length of its cDNA was 1383 bp，with an open reading frame of 1104 bp, encoding a total of 368 amino acids. The phylogenetic tree analysis showed that RfBAMT was the closest to the BSMT of Camellia japonica, belonging to methyltransferases family. Then we measured the expression level of RfBAMT again at four flower developmental stages and in different flower tissues and leaves. The results showed that the expression level of this gene was highly positively correlated with the emitted content of methyl benzoates in the flowering, implying that RfBAMT plays a pivotal role in the formation and regulation of methyl benzoates in Rhododendron fortune.Thisresearchshowed that the RfBAMT was cloned and identified in our study and its expression level was highly positively correlated with the emitted content of methyl benzoates in the flowers and leaves, which indicated this gene may play an important role on regulation of methyl benzoate synthesis in Rhododendron fortunei.

Rhododendron fortunei pertence a uma espécie de rododendro perfumada nativa da China, que produz flores perfumadas de grande valor ornamental e ambiental para paisagismo ou embelezamento de interiores. No entanto, os aromas em Rhododendron fortunei ainda não foram investigados, muito menos o mecanismo de formação dessas fragrâncias nas flores. Neste estudo, medimos os aromas em termos de seus componentes e conteúdos voláteis (VOC) em Rhododendron fortunei em quatro diferentes estágios de floração e em diferentes tecidos da planta por microextração em fase sólida headspace combinada com cromatografia gasosa-espectrometria de massa. Em seguida, foram calculados os valores aromáticos característicos, que refletem o grau de percepção olfativa pelo ser humano, de cada VOC na planta de acordo com seus respectivos limiares aromáticos. Os resultados mostraram que três COVs principais medidos do mais alto ao mais baixo são benzoatos de metila, terpenos e derivados de ácidos graxos. Seu conteúdo aumentou após a abertura do botão floral e atingiu o máximo na metade da floração total. Em uma flor, a maioria dos teores de COV foram medidos em pétalas e apenas traços em outros tecidos, como estame, pistilo. Uma pequena quantidade de COVs foi determinada nas folhas também. Todos os valores aromáticos foram quase correspondentes aos teores de três COVs principais, indicando que a fragrância da flor surge verdadeiramente desses componentes de COV. Para entender o mecanismo de formação deste tipo principal de fragrância e sua regulação, primeiramente isolamos um gene de RfBAMT da pétala de Rhododendron fortunei usando clonagem homóloga e tecnologia RACE. O comprimento total de seu cDNA era de 1383 bp, com um quadro de leitura aberto de 1104 bp, codificando um total de 368 aminoácidos. A análise da árvore filogenética mostrou que RfBAMT foi o mais próximo do BSMT de Camellia japonica, pertencente à família das metiltransferases. Em seguida, medimos o nível de expressão de RfBAMT novamente em quatro estágios de desenvolvimento da flor e em diferentes tecidos e folhas de flores. Os resultados mostraram que o nível de expressão deste gene foi altamente correlacionado positivamente com o conteúdo emitido de benzoatos de metila na floração, implicando que RfBAMT desempenha um papel fundamental na formação e regulação de benzoatos de metila em Rhododendron fortune foi clonado e identificado em nosso estudo e seu nível de expressão foi altamente correlacionado positivamente com o conteúdo emitido de benzoatos de metila nas flores e folhas, o que indicou que este gene pode desempenhar um papel importante na regulação da síntese de benzoato de metila em Rhododendron fortunei.

Effects of melatonin on testicular function in adult male mice under different photoperiods.

Photoperiod is an important environmental factor affecting animal physiological function. Melatonin is an endogenous hormone that plays an important role in circadian and seasonal (or cyclical) rhythms and seasonal reproduction in mammals. To investigate the effects of melatonin on the reproductive performance of adult male mice under different photoperiods, sixty mice were randomly allotted to six groups: control (Light Dark, 12 L:12 D), control plus melatonin (MLD, 12 L:12 D), 24-hour continuous light (LL, 24 L:0 D), 24-hour continuous light plus melatonin (MLL 24 L:0 D), constant darkness (DD, 0 L:24 D), and constant darkness plus melatonin (MDD, 0 L:24 D). Normal saline (100 µL) was injected into the LD, LL, and DD groups at noon each day; the MLD, MLL, and MDD groups were injected with melatonin (1 mg/mL; 2 mg/kg·body weigh). After 24 hours of prolonged light exposure, testis morphology decreased, convoluted seminiferous tubules became sparse, the diameter of convoluted seminiferous tubules decreased, and the level of sex hormones decreased. After the administration of exogenous melatonin, testicular morphology and sex hormone levels decreased in the MLD group under normal light conditions. In the MLL group, the testicular tissue morphology returned to normal, the diameter of convoluted tubules increased, the hormone levels of LH (Luteinizing hormone) and MTL (melatonin) significantly increased (P<0.05), and th0e gene expressions of LHß and Mtnr1A (Melatonin receptors 1A) increased. There was almost no difference in the MDD group under continuous darkness. In conclusion, melatonin can damage the reproductive performance of male mice under normal light conditions, while exogenous melatonin can alleviate and protect the testicular injury of male mice under continuous light conditions.

Effects of melatonin on testicular function in adult male mice under different photoperiods

Photoperiod is an important environmental factor affecting animal physiological function. Melatonin is an endogenous hormone that plays an important role in circadian and seasonal (or cyclical) rhythms and seasonal reproduction in mammals. To investigate the effects of melatonin on the reproductive performance of adult male mice under different photoperiods, sixty mice were randomly allotted to six groups: control (Light Dark, 12 L:12 D), control plus melatonin (MLD, 12 L:12 D), 24-hour continuous light (LL, 24 L:0 D), 24-hour continuous light plus melatonin (MLL 24 L:0 D), constant darkness (DD, 0 L:24 D), and constant darkness plus melatonin (MDD, 0 L:24 D). Normal saline (100 µL) was injected into the LD, LL, and DD groups at noon each day; the MLD, MLL, and MDD groups were injected with melatonin (1 mg/mL; 2 mg/kg·body weigh). After 24 hours of prolonged light exposure, testis morphology decreased, convoluted seminiferous tubules became sparse, the diameter of convoluted seminiferous tubules decreased, and the level of sex hormones decreased. After the administration of exogenous melatonin, testicular morphology and sex hormone levels decreased in the MLD group under normal light conditions. In the MLL group, the testicular tissue morphology returned to normal, the diameter of convoluted tubules increased, the hormone levels of LH (Luteinizing hormone) and MTL (melatonin) significantly increased (P<0.05), and th0e gene expressions of LHß and Mtnr1A (Melatonin receptors 1A) increased. There was almost no difference in the MDD group under continuous darkness. In conclusion, melatonin can damage the reproductive performance of male mice under normal light conditions, while exogenous melatonin can alleviate and protect the testicular injury of male mice under continuous light conditions.(AU)