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Transient
Expression of Strictosidine Synthase in Tobacco Leaves by Vacuum Infiltration
WANG
Miao*, LI Qiu-Rong1
(
Institute of Applied Ecology, the Chinese Academy of Sciences, Shenyang 110016,
China;
1General
Hospital in Shenyang Military District, Shenyang 110016,
China )
Abstract Strictosidine synthase (STR) is the key enzyme
involved in early steps of biosynthesis of monoterpenoid indole alkaloids. STR
catalyzes the condensation of tryptamine with secologanin into strictosidine.
The gene encoding STR targeted to different subcellular compartments was
transiently expressed in the tobacco leaves. In vitro STR enzymatic
activity was measured by the depletion of tryptamine indicated by fluorescence.
The results showed that the recombinant STR was effectively expressed as
soluble protein in three subcellular compartments—chloroplast, vacuole and endoplasmic reticulum in the
leaves of tobacco by Western blot analysis and STR enzymatic assay.
Key
words strictosidine synthase;
vacuum infiltration; subcellular compartments; Nicotiana tabacum
Catharanthus
roseus (L.) G. Don, a member of the Apocynaceae
family, produces terpenoid indole alkaloids (TIA). Some of these alkaloids are
powerful anti-tumor drugs such as vindoline and vincristine. However, there is
a critical shortage of these agents since they accumulate only to trace amount
level in C.roseus. Because of their high therapeutical and commercial
value, extensive research has been devoted to the TIA biosynthetic pathway as
well as the improvement of vinblastine production in cell cultures of C.
roseus[1]. Two key enzymes, tryptophan decarboxylase (TDC, EC
4.1.1.28) and strictosidine synthase (STR, EC 4.3.3.2), catalyze the early
steps of the TIA pathway (Fig.1) and involved in the regulatory mechanism[2].
Considerable progress has been made in the isolation of genes encoding the key
enzymes in TIA biosynthesis[3]. A number of cDNA clones encoding
strictosidine synthase were isolated from Rauvolfia serpentina and C.roseus[4,
5] and they were effectively expressed in the cell cultures of C.roseus[6,
7]. The gene encoding tryptophan decarboxylase has been expressed in
tobacco plants, which resulted in increased tryptamine level[8, 9].
However, there was no report on the expression of recombinant STR enzyme in
different subcellular compartments of plants. This study is to identify a novel
tool for engineering plant secondary metabolism based on the expression and
targeting of the recombinant STR enzyme in the selected subcellular
compartments. In this paper we report the agrobacterium-mediated
transient expression of strictosidine synthase in the leaves of tobacco plants
by vacuum infiltration.
Fig.1 Early steps of biosynthesis of
terpenoid indole alkaloids in Catharanthus roseus
1
Materials and Methods
1.1
Materials
1.1.1 Plant and bacteria Nicotiana tabacum L. cv. Petite Havana
SR1; Agrobacterium-tume-faciens GV3101 (pMP90RK, gmR,kmR,
rifR ).
1.1.2 Reagents
The 9E10 anti c-myc antibody was a gift from Dr. Arjen Schots
(Landbow University, Wageningen, The Netherlands); the anti KDEL antibody and
the goat anti-mouse IgG heavy+light chain alkaline phosphatase-conjugated
(GAM-H+L-AP) antibody were from Jackson ImmunoResearch in West Grove in PA USA.
1.2
Construction of STR targeting expression cassettes
The
expression cassettes containing STR encoding genes were generated by PCR and
standard cloning techniques[10]. Construction of the STR targeting
expression cassettes was designed as reported before[11]. The enzyme
was tagged with signal peptides which targeted the soluble proteins to the
chloroplast, cytosol, vacuole and ER. In order to detect the expression of the
recombinant STR protein, the c-myc tag was cloned at the C- termini of
the cassettes for cytosol, vacuole and chloroplast[12], and the KDEL
epitope was cloned with the C-terminal KDEL sequence (Lys-Asp-Glu-Leu) and used
as detection tag for the ER cassette[13]. The constructs were
subcloned as EcoRI/XbaI fragment into the pSS plant expression
vectors[14] and located between the strong constitutive cauliflower
mosaic virus (CaMV) 35S enhanced promoter and CaMV terminator sequence.
1.3
Transient transformation of tobacco
The
plant expression cassettes were transformed into electrocompetent cells of Agrobacterium
tumefaciens GV3101. The agrobacteria were selected on YEB-agar plates with
rifampicin, kanamycin and carbenicillin. Recombinant agrobacteria were
infiltrated into the young tobacco leaves according to Kapila et al.[15].
The tobacco plants were kept in dark for 1 h and four leaves for each construct
were collected and put into the bacterial suspension, then infiltrated by
vacuum at 60～80
mbar for 20 min. After the release of vacuum, the leaves were washed with tap
water and incubated on wet Whatman paper in a plastic tray. The tray was sealed
with Saran wrap and kept at 22 ℃
under a 16 h photoperiod for 2 days. Wild type of SR1 tobacco leaves were
infiltrated with non-recombinant agrobacteria and used as negative control. At
the end of the incubation, the leaves were weighed, frozen in liquid nitrogen
and stored at -70 ℃
before analysis.
1.4
Detection of STR transient expression
1.4.1 Western blot analysis The vacuum infiltrated leaves were ground in
liquid nitrogen and further ground in the extracting buffer containing 100 mmol/L
Na2HPO4/ NaH2PO4 (pH 7.5), 2 mmol/L
EDTA, 4 mmol/L DTT and 5% polivinyl-pyrrolidone (50 g/L), then centrifuged at
14 000 r/min at 4 ℃
for 20 min and the supernatant was collected for Western blot analysis and
enzymatic assay. The polyacrylamide’s concentration was 10% for SDS-PAGE gels
and the gels were electrobloted onto nitrocellulose membrane. Anti KDEL
antibody was used for detection of STR targeted to ER and 9E10 anti c-myc
antibody was used for STR targeted to the chloroplast, vacuole and cytosol. The
GAM-H+L-AP conjugated antibody was used as the secondary antibody and the
blotted membranes were developed in the solution of nitro-blue tetrazolium /
BCIP and the membranes were scanned.
1.4.2 STR enzymatic activity assay This assay is based on the condensation of
tryptamine with secologanin into strictosidine, catalyzed by STR enzyme. STR
enzymatic activity was measured indirectly by fluorimetrically detecting the
depletion of tryptamine in the reaction mixture[16, 17]. Before
tryptamine and secologanin were added into the reaction mixture, the samples
acting as negative control were boiled for 4 min to destroy the enzymatic
activity. 20 mL
of the leaf crude extracts were mixed with the buffer (0.1 mol/L NaH2PO4/NaOH buffer, 1.0
mmol/L tryptamine, 5 mmol/L secologanin and 3 mmol/L dithiothreitol, pH 6.3) to
a final volume of 1 mL and incubated at 30 ℃
for 1 h. 2 mL of 4 mol/L NaOH were added. 5 mL of ethyl acetate were emulsified
to the buffer solution by vortexing for 30 s. In order to facilitate the
separation of the organic solvent from the buffer, centrifugation was carried
out at 1 500 g for 5 min at room temperature. The organic phase was
subjected to fluorimetric detection using an Aminco Bowman AB2
luminescencespectro-meter. Tryptamine was detected at 280 nm excitation and 340
nm emission wavelengths. For each sample, integrated values of the tryptamine
emission scan were recorded in triplicate. The level of tryptamine in the crude
leaf extracts was expressed as the average of the total integral of the
emission scan area.
2 Results
2.1
Western blot analysis of transient expression
STR
was transiently expressed in three subcellular compartments—ER,
chloroplast and vacuole as indicated by Western blot in Fig.2. Expression level
of the soluble cytosolic STR was very low and was hardly detected[Fig.2(C),
lane 1-4].
Fig.2 Transient expression of STR in the
subcellular compartments in four infiltrated tobacco leaves
(A) ER, (B) chloroplast, (C) cytosol and
vacuole. (A) and (B): M, pre-stained protein marker; 1-4, crude extract of four
leaves infiltrated with recombinant agrobacteria; 5, uninfiltrated leaf; 6,
leaf infiltrated with non-recombinant agrobacteria; 7, positive control[the
KDEL epitope (A) or 65 kD protein engineered with a c-myc tag (B)]. (C):
M, pre-stained protein marker; 1-4, crude extract of four leaves infiltrated
with agrobacteria with the cytosolic expression cassette; 5-8, the vacuolar
expression cassette; 9, uninfiltrated leaf. The recombinant proteins were
detected by using the 9E10 anti c-myc antibody [(A) and (C)] and an
antibody against the KDEL epitope (B).
2.2
STR enzymatic activity assay
In
vitro STR enzymatic activity assay in the crude
extracts of transgenic tobacco leaves was shown in Fig.3.
In
all boiled samples the levels of tryptamine were as high as those in the wild
type (boiled or not boiled). Tryptamine was nearly depleted in one hour by STR
enzyme that appeared in chloroplast, vacuole and ER. The level of residue
tryptamine was much higher in the leaves of transiently expressing targeted STR
in the cytosol than those in the chloroplast, ER and vacuole and actually was
as high as those in the boiled samples and in wild type. The STR enzymatic
activity was hardly detected in the cytosol.
3 Discussion
In
recent years there has been a rapidly increasing interest in plant secondary
metabolism and the possibilities of genetic modification opened exciting
perspectives to exploit the biosynthetic capacity of plants. The genes encoding
TDC and STR had been expressed in various plants and plant cells[6-9].
In accordance with the reported results, we found that the recombinant TDC was
effectively expressed in selected subcellular compartments in tobacco plants[11].
The enzyme has been purified from the cell cultures of C.roseus[18].
In order to study the regulation of the enzyme activity of TIA pathway, we are
interested in strictosidine synthase. Because str gene does not exist in
the tobacco genome, endogenous STR activity cannot be detected. Therefore,
depletion of tryptamine in the crude extract of infiltrated leaves represented
direct evidence of in vivo function of strictosidine synthase. The
biosynthesis of terpenoid indole alkaloids in C.roseus requires at least
three compartments, the plastid for the production of the terpenoid moiety and
tryptophan, the cytosol for the decarboxylation of tryptophan and the vacuole
for the coupling of tryptamine with secologanin. Further steps of the alkaloid
biosynthesis occur in the cytosol and even in chloroplasts for certain
alkaloids[19]. Strictosidine synthase locates in the vacuole of
plant cells. To proceed in TIA biosynthesis, strictosidine has to be
transported outside the vacuole and further to the ER, where it can be
hydrolyzed. Better understanding of the pathway in plants may lead to the
development of strategies to modify the flux of the desired type of alkaloids.
Strictosidine synthase is the objective of the present research not only
because it has been fully characterized at the molecular and biochemical level
but also because it represents a general model in metabolic engineering of
plant secondary metabolism.
In
present study STR was transiently expressed in different subcellular
compartments of transgenic tobacco plants in order to study effect of
compartmentation on the in vivo functionality. The vacuolar STR represented
an internal control to compare with the recombinant enzyme targeted to other
subcellular compartments. The results showed that STR was effectively expressed
in three subcellular compartments—chloroplast,
vacuole and ER (Fig.2). In vitro enzymatic assay ascertained the STR
functionally targeted to the same three subcellular compartments (Fig.3).
Transgenic tobacco plants displayed high expressing levels of STR in those
subcellular compartments compared with the wild type. Tryptamine was completely
depleted by STR in the crude extracts of leaves expressing STR in the
chloroplast, vacuole and ER. The results demonstrated that the coordinated
depletion of tryptamine in transgenic tobacco leaves could lead to the
accumulation of strictosidine. The STR enzyme activity detected was consistent
with results of Western blot analysis. The expression level of STR was very low
in leaves expressing STR in the cytosol. The reason for the results was that
STR is a vacuolar enzyme and the stability of recombinant STR was low in the
cytosol or the compartment conditions in cytosol of tobacco plants were not
suitable for the expression of the recombinant STR enzyme was degraded and
misfolded in the cytosol. Much work on the transcription analysis should be
carried out to confirm these propositions.
In
general the stable transformation is used to analyze the expression of gene in
plants, but it is time-consuming in generation of transgenic plants. Recently
the transient expression system in plants has been developed and it is
characterized as the fast and easy method[20]. Transiently
expression of STR in selected subcellular compartments is desirable for
increased flux of TIA production through metabolically engineering TIA
biosynthetic pathway.
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Received：May
6, 2002 Accepted：July
24, 2002
This
work was supported by grants from the Chinese Academy of Sciences
(No.KZCX-406-4) and Science and Technology of Liaoning Province (No.2001101034)
*Corresponding
author: Tel, 86-24-23916282; Fax, 86-24-23843313; e-mail, [email protected]