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ISSN 0582-9879                                        ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(6): 703-706                                     CN 31-1300/Q

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)

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