1. Abstract Results Polyamines are implicated in a wide array of fundamental processes in plants such as adaptation and tolerance to environmental stresses. However, their roles in root growth have not been fully elucidated. In this study, Nicotiana benthamiana was used as a model plants to investigate the roles of polyamine in root growth. Genes encoding polyamine biosynthesis enzymes were knocking down by using the virus-induced gene silencing (VIGS) strategies. Analysis of root growth was carried out in the spermidine synthase (SPDS)-silenced plants. In NbSPDS-silenced plants, it was observed that the number of lateral roots was decreased as compared to vector-control plants. The effects of VIGS on gene expression level were validated by using reverse transcription-PCR. The expression of genes encoding Tobacco rattle virus (TRV) coat protein was detected in the VIGS-treated root tissues. When compared with the vector-control lines, the expression of NbSPDS gene was significantly decreased. These results indicate successful triggers of VIGS by infectious TRV in roots. High performance liquid chromatography analysis of polyamine content showed that the content of spermidine was decreased in the NbSPDS-silenced plants as compared to the vector-control plants. Silencing of NbSPDS did not affect Nitric Oxide gene expression. In summary, this study suggests that spermidine may play important roles in root growth. 1.Polyamine biosynthesis pathway 4. Accumulation of TRV in roots and suppression of NbSPDS expression 3. Silencing of NbSPDS gene in N. benthamiana affected root growth 5. Silencing of NbSPDS gene decreased the level of root spermidine 6. NbSPDS-silenced plants showed abnormal development of fruits Conclusions and future work 1. These results indicate successful triggers of VIGS by infectious TRV in roots. 2. Spermidine may play important roles in root growth. 3. Future work will focus on the identification of the relationship between of polyamine and nitric oxide 吳偉誌 1 、林忠毅 1 、傅士峰 1 1 Department of Biology, National Changhua University of Education, Changhua, Taiwan 彰化師範大學生物學系 TRV2 Ve TRV2 NbSPDS 2. Virus-induced gene silencing (VIGS) constructs used in this study Fig. 4 Suppression of NbSPDS gene expression in N. benthamiana plants by VIGS. (A) Accumulation of TRV in roots was examined by reverse-transcription PCR using TRV-specific primers spanning the viral coat protein. (B) Analysis of genes encoding polyamine- biosynthetic and NO biosynthestic enzymes in vector-control and NbSPDS-silenced N. benthamiana plants. (C) Quantitative gene expression analysis in vector control and NbSPDS-silenced plants by real-time RT-PCR analysis. EF1 was used as a reference gene. The experiment was performed at least three times and one representative data was shown. Fig. 2 Schematic of TRV1,TRV2, and TRV2-NbSPDS constructs. LB, left border; RB, right border, RdRp, RNA-dependent RNA polymerase; MP, movement protein; 16K, 16 KDa protein; Rz, self- cleaving ribozyme; N, NOS terminator; CP, coat protein; MCS multiple cloning site. Fig. 3 Silencing of the NbSPDS gene in N. benthamiana root. The roots of the control (TRV2-Ve) and NbSPDS- silenced (TRV2-NbSPDS) plants. The leaves of the N. benthamiana plants were Agro-infiltrated with the TRV-derived constructs. The VIGS- treated plants were transfer to a liquid-culture equipped bottle supplemented with nutrients. The phenotypic analysis of the roots from the TRV2-Ve and TRV2-SPDS were performed after 3 week post inoculation. Investigating the roles of polyamine-spermidine biosynthetic enzyme in root growth of Nicotiana benthamiana by virus-induced gene silencing Fig. 1 Arginine catabolism and the coordinate biosynthesis of polyamines and nitric oxide in plants. ADC, Arginine decarboxylase; ODC, Ornithine decarboxylase; SPDS, Spermidine synthase; SPMS, Spermine synthase; SAM, S-adenosylmethionine; SAMDC, SAM decarboxylase; dcSAM, decarboxylated S-adenosylmethionine; NOS, Nitric oxide synthase; NR, Nitrite reductase. Fig. 5 Accumulation of root spermidine in vector control and NbSPDS- silenced plants was analyzed by high- performance liquid chromatography. The experiment was performed at least three times and one representative data was shown. Fig. 6 The effects of NbSPDS silencing on development of fruits and flowers.Ve, vector control; PDS, positive control; ADC, NbADC; ODC, NbODC; ADC:ODC,NbADC:NbODC, SPDS, NbSPDS.