To study transient tissue specific linalool emission and to examine its fate, plastid targeted dual linalool/nerolidol synthase (FaNES1) was introduced into Nicotiana benthamiana leaves under LTP1, SUC2, RbcS and CaMV...To study transient tissue specific linalool emission and to examine its fate, plastid targeted dual linalool/nerolidol synthase (FaNES1) was introduced into Nicotiana benthamiana leaves under LTP1, SUC2, RbcS and CaMV35 promoters. Tissue specificity of the promoters was tested with β-glucuronidase (GUS) reporter gene. Promoter::GUS/FaNES1 fusion was confirmed with colony PCR and agro-infiltrated into six weeks old N. benthamiana leaves. Eight days post inoculation (dpi), promoter::GUS constructs were examined with GUS histochemical staining at 1.5 h, 3.5 h, 5.5 h and 16 h incubation times. After 4/8 days, FaNES1 construct agro-inoculated leaves were assessed for linalool emissions and its conjugates using a dynamic headspace system and LC-MS, respectively. There was high affinity of promoters to their respective cell-types although it was not as specific as in stable transformation. This is possibly due to activations of many copies of transiently introduced promotes by few transcription factors of the respective promoter that naturally exist in untargeted cell-types. GUS staining intensity was stronger in leaf veins and injured sites compared to other plant tissues under all heterologous promoters, and it was gradually increased with increase in incubation times that could be explained by promoters wounding responses and/ or GUS leakage to unstained sites. Linalool emission was obtained in the same pattern under all promoters;it was higher at day 4 than day 8. Its concentration declined 44, 12, 4.5 and 4 folds at 8 dpi under LTP1, SUC2, RbcS and CaMV35S promoters, respectively. Conversely, linalool conjugates were significantly increased at day 8. These might be due to T-DNA degradations and/or protein modifications 4 dpi. LTP1 promoter was the least efficient to drive both GUS and FaNES1 possibly due to immature plastids in epidermal cells and/or its weak performance. Hence, to study FaNES1 activity in transient assay it is suggested to use relatively shorter duration and longer inoculation times for linalool and its conjugates, respectively.展开更多
文摘To study transient tissue specific linalool emission and to examine its fate, plastid targeted dual linalool/nerolidol synthase (FaNES1) was introduced into Nicotiana benthamiana leaves under LTP1, SUC2, RbcS and CaMV35 promoters. Tissue specificity of the promoters was tested with β-glucuronidase (GUS) reporter gene. Promoter::GUS/FaNES1 fusion was confirmed with colony PCR and agro-infiltrated into six weeks old N. benthamiana leaves. Eight days post inoculation (dpi), promoter::GUS constructs were examined with GUS histochemical staining at 1.5 h, 3.5 h, 5.5 h and 16 h incubation times. After 4/8 days, FaNES1 construct agro-inoculated leaves were assessed for linalool emissions and its conjugates using a dynamic headspace system and LC-MS, respectively. There was high affinity of promoters to their respective cell-types although it was not as specific as in stable transformation. This is possibly due to activations of many copies of transiently introduced promotes by few transcription factors of the respective promoter that naturally exist in untargeted cell-types. GUS staining intensity was stronger in leaf veins and injured sites compared to other plant tissues under all heterologous promoters, and it was gradually increased with increase in incubation times that could be explained by promoters wounding responses and/ or GUS leakage to unstained sites. Linalool emission was obtained in the same pattern under all promoters;it was higher at day 4 than day 8. Its concentration declined 44, 12, 4.5 and 4 folds at 8 dpi under LTP1, SUC2, RbcS and CaMV35S promoters, respectively. Conversely, linalool conjugates were significantly increased at day 8. These might be due to T-DNA degradations and/or protein modifications 4 dpi. LTP1 promoter was the least efficient to drive both GUS and FaNES1 possibly due to immature plastids in epidermal cells and/or its weak performance. Hence, to study FaNES1 activity in transient assay it is suggested to use relatively shorter duration and longer inoculation times for linalool and its conjugates, respectively.
