Transcript NbSPDS

Investigating the roles of polyamine-spermidine biosynthetic enzyme in root
growth of Nicotiana benthamiana by virus-induced gene silencing
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吳偉誌 、林忠毅 、傅士峰
1Department
of Biology, National Changhua University of Education, Changhua, Taiwan 彰化師範大學生物學系
Abstract
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.
Results
1. Polyamine biosynthesis pathway
4. Accumulation of TRV in roots and suppression of NbSPDS expression
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
polyaminebiosynthetic
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.
5. Silencing of NbSPDS gene decreased the level of root spermidine
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.
2. Virus-induced gene silencing (VIGS) constructs used in this study
Fig. 5 Accumulation of
root spermidine in vector
control
and
NbSPDSsilenced plants
was
analyzed
by
highperformance
liquid
chromatography.
The
experiment was performed
at least three times and one
representative data was
shown.
6. NbSPDS-silenced plants showed abnormal development of fruits
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, selfcleaving ribozyme; N, NOS terminator; CP, coat protein; MCS multiple cloning site.
3. Silencing of NbSPDS gene in N. benthamiana affected root growth
TRV2
Ve
TRV2
NbSPDS
Fig. 3 Silencing of the NbSPDS gene
in N. benthamiana root . The roots of
the control (TRV2-Ve) and NbSPDSsilenced (TRV2-NbSPDS) plants.
The leaves of the N. benthamiana
plants were Agro-infiltrated with the
TRV-derived constructs. The VIGStreated 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.
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.
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