Influence of Light and Phytohormones on Alkaloid Production in Transformed Root Cultures of Hyoscyamus albus

J. PlantPhysiol. Vol. 140.pp. 147-152(1992)

Influence of Light and Phytohormones on Alkaloid Production in Transformed Root Cultures of Hyoscyamus a/bus 1

MARTINA SAUERWEIN ,3, MICHAEL WINK\ 1

2

3

and KOICHIRO SHIMOMURA2

Ruprecht-Karls-Universitat Heidelberg, Institut fur Pharmazeutische Biologie, 1m Neuenheimer Feld 364, W-6900 Heidelberg, F.R.G. Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences, 1 Hachimandai, Tsukuba, Ibaraki, 305,Japan Address for correspondence

Received December 27, 1991 . Accepted February 18, 1992

Summary

In vitro culture~ of Hyoscyamus albus were transformed with three different strains of Agrobacterium rhizogenes and subjected to various culture conditions and phytohormone concentrations. The production of 5 tropane alkaloids in the transformed as well as in the normal root cultures was examined by HPLC. Hyoscyamine was the main constituent produced by all of the different root cultures. The highest content of total tropane alkaloids was detected in hairy roots transformed with A. rhizogenes strain MAFF03-01724 cultured on WP solid medium containing 1 mg/L kinetin in combination with 1 mg/L IAA cultured in the dark. Littorine was especially abundant in these cultures. On the other hand the accumulation of the oxygenated alkaloids, such as 6~-hydroxyhyoscyamine, 7 ~-hydroxyhyoscyamine and scopolamine was enhanced in the roots cultured in the light. Depending on the strain of Agrobacterium rhizogenes used for transformation, hairy roots responded differently to exogenous phytohormones. Key words: Hyoscyamus albus; Agrobacterium rhizogenes; adventitious root culture; hairy root culture; tropane alkaloid production. Introduction

In vitro cultures of Hyoscyamus species have been examined for their tropane alkaloid production by various researchers [e.g. Hashimoto et aI., 1986; Parr et al., 1990]. The influence of phytohormones and light on the production of tropane alkaloids in transformed roots of Hyoscyamus has not been reported in detail. In hairy roots of Duboisia myoporoides a small effect of IAA on tropane alkaloid production was observed [Deno et aI., 1987]. In our current studies on the formation of tropane alkaloids we established adventitious root cultures of Hyoscyamus albus and hairy roots transformed with different strains of Agrobacterium rhizogenes [Shimomura et aI., 1991]. Their alkaloid content and alkaloid pattern under various culture conditions was determined by HPLC. In this report we describe the influence © 1992 by Gustav Fischer Verlag, Stuttgart

of light and phytohormones on the production of five main tropane alkaloids, the medicinally important hyoscyamine and scopolamine as well as their biogenetic intermediates 6~­ hydroxyhyoscyamine, 7 ~-hydroxyhyoscyamine and littorine.

Materials and Methods

Plant material Adventitious root cultures were established from the axenic shoot cultures of R albus on MS solid medium. Hairy roots were induced from leaf discs by co-culture with Agrobacterium rhizogenes strains 15843, A4 and MAFF03-01724 grown on YEB agar medium [Kamada et aI., 1989]. The bacteria were eliminated on MS solid medium containing 0.5 giL Claforan®. The axenic hairy roots thus ob-

148

MARTINA SAUERWEIN, MICHAEL WINK, and KOICHIRO SHIMOMURA

tained were subcultured in hormone-free WP liquid medium [Lloyd and McCown, 1980]. To prove the transformation, the opines (agropine, mannopine and mikimopine) were extracted, separated by paper electrophoresis and detected with AgN03 (agropine and mannopine) [Petit et al., 1983] or Pauly's reagents (mikimopine) [Kamada et al., 1989]. The root cultures were maintained on a rotary shaker at 100 rpm in the dark or under continuous light (4000 lux) at 25°. All media contained 3 % sucrose and the pH was adjusted to 5.7 before autoclaving. For the culture experiments 2 root tips (ca. 15 mg fresh weight each) were inoculated onto WP medium solidified with 0.2 % Gelrite® containing the phytohormones. Three petridishes were used for each experiment. After 30 days the cultures of 2 plates were harvested and the alkaloid content of the tissues was determined by HPLC. The third culture was further subcultured onto 3 petridishes as described above. Each experiment and culture cycle was repeated twice. Voucher specimens of all cultures were deposited at the Tsukuba Medicinal Plant Research Station.

Sample preparation and HPLC analysis Two cultures were harvested and the fresh weight and the dry weight (after lyophilization) of the tissues were determined individually. Ca. 50 mg of each sample was extracted with 5 mL CHCh/ MeOH/NH4 0H (15: 5: 1) using sonication (10 min). The further sample preparation was the same as described previously [Shimomura et aI., 1991; Sauerwein and Shimomura, 1991]. The alkaloid extracts were dissolved in MeOH and analyzed by HPLC. A TOSOH ODS-120A column (4.6I.D.x250mm) was

HO

tEro~

Results and Discussion

Alkaloid pattern in phytohormonefree media Leaf-disks of in vitro cultures of Hyoscyamus albus were transformed with A. rhizogenes strain 15843, strain A4 and strain MAFF03-01724 as described previously [Shimomura et al., 1991; Sauerwein and Shimomura, 1991]. The successful transformation of the axenic hairy roots was confirmed by the extraction, isolation and detection of the opines produced using paper electrophoresis (agropine and mannopine for strains A4 and 15843; mikimopine for strain MAFF0301724) [Petit et al., 1983]. The hairy roots thus obtained and the normal roots were subcultured onto hormone-free Woody Plant solid medium with 3 % sucrose (WP) in the dark or in the light. After 30 days of culture the content of 7,6-hydroxyhyoscyamine, 6,6-hydroxyhyoscyamine, scopolamine, hyoscyamine and littorine in the roots was determined by HPLC (Fig. 1). The alkaloid content of hairy roots transformed with A. rhizogenes strain 15843 and of normal roots cultured

I ;N~ ro~

oH VHO/

1: 76-hydroxyhyoscyamlne

used, kept at 40° and eluted isocratically with CH 3 CN/I0 mM SDS (PH 3.3, adjusted with 1 % H 3P04) 2: 3 [He et aI., 1990]. The flow rate was 1.1 mL/min throughout. The effluent was monitored by a photo diode array detector. For quantitative analysis the system was calibrated with the authentic samples at 215 nm.

oH

U

2: 66-hydroxyhyoscyamlne

60~

50~

4: hyoscyamine

5: llttorlne

40 :J

a:

3

30

E

5

2

20

5

10

Time

15

(m in. )

20

25

Fig. 1: Separation of alkaloids from Hyoscyamus albus normal root culture by HPLC. Column: TSK-ODS 120A, eluent: acetonitrile/SDS (2/3), pH 3.3, 40°C.

Alkaloid Production in Root Cultures of Hyoscyamus albus light

dark •

711-hydroxyhyoscyamine

o

scopolamine

Ii

6.0

149

611-hydroxyhyoscyaminc

~ hyoscyamine

!§l Iittorine

'"

"0

'is

-;

2.0

..>I

-;

Fig. 2: Influence of light on alkaloid production by normal roots and different hairy roots of H. albus cultured on WP solid medium. Bars indicate the standard error.

A4

under light conditions was much higher than those cultured in the dark (Fig. 2). In contrast, the alkaloid production in the transformants with A. rhizogenes strain A 4 and MAFF03-01724 was only slightly increased under light conditions. Especially the proportion of the oxygenated tropane derivative scopolamine was enhanced in all light grown root cultures. Its maximal amount (4.21 g' kg-! drywt.) was detected in the normal root cultures. In addition, the content of the biogenetic intermediates 7(j-hydroxyhyoscyamine and 6(j-hydroxyhyoscyamine was elevated to some degree under light conditions. Whereas the content of hyoscyamine only slightly increased and littorine showed a tendency to decrease in the light. Thus the oxygenating step in the biosynthesis of the tropine moiety seems to be enhanced by the light, but light is not strictly required [Leete, 1990]. In consequence the contents of scopolamine in the roots cultured under the light were up to 4-fold (normal roots) compared to the dark grown roots. On the other hand the main alkaloid in all cultures was hyoscyamine. Its maximal yield was detected in the hairy roots transformed with A. rhizogenes A4 cultured in the dark (5.42 g' kg-! drywt.) as well as in the normal roots cultured in the light (5.72 g' kg-! drywt.) (Fig.2).

Influence ofphytohormones on root morphology The morphology of the normal and hairy roots changed to callus-like structures with the addition of phytohormones (Table 1). Calli with roots were obtained with the addition of IAA, pure calli in combination of both phytohormones. The growth of the normal root cultures was enhanced when the medium contained IAA (2 or 4 mg/L) in combination with 1 mg/L kinetin. Similarly the growth rate of the hairy roots transformed with A. rhizogenes A 4 cultured in the light was enhanced with the addition of low concentrations of phytohormones, while only friable calli were obtained when 2 or 4 mg of IAA were added to the medium. Growth and morphology of the hairy roots transformed with A. rhizogenes 15834 was similar to that of the normal root cultures. Here again the maximal growth rate was observed in the medium containing both phytohormones at ratio 1: 1. On the other hand the growth of the hairy roots

15834 MAFF hairy rools

normal rool

A4

15834 MAFF hairy rools

normal root

transformed with A. rhizogenes MAFF03-01724 was reduced with the addition of any combination of phytohormones (Table 1).

Influence ofphytohormones on alkaloid production In normal roots cultured in the dark the addition of IAA (up to 4 mg/L) to the culture medium only slightly affected alkaloid production (Fig. 3 a). On the other hand, when the normal roots were cultured under the light, the alkaloid content decreased with the addition of IAA. Any combination of IAA with kinetin rapidly reduced the alkaloid production in the normal roots cultured in the dark as well as in those cultured in the light. The addition of a single phytohormone or their combination to the hairy root cultures transformed with A. rhizogenes A4 reduced the alkaloid formation substantially (Fig. 3 b). The inhibitory effect of phytohormones on alkaloid production was larger when these hairy roots were cultured in the light. In contrast to the transformants with strain A4, in the hairy roots transformed with A. rhizogenes 15834, the addition of IAA enhanced the alkaloid production, when the roots were cultured in the dark (Fig. 3 c). Maximal alkaloid content in the hairy roots was obtained with 2 mg/L IAA (5.7 g tropane alkaloids kg-! dry wt.). On the other hand alkaloid production was slightly depressed in light grown cultures. Kinetin again reduced alkaloid production in the hairy roots. The addition of phytohormones to the root cultures transformed with A. rhizogenes MAFF03-01724 had a somewhat different result (Fig. 3 d). Whereas kinetin inhibited the alkaloid production in all other cultures in this case it enhanced the alkaloid formation. Especially in the dark the addition of 1 mg/L kinetin or 1 mg/L IAA or their combination resulted in higher productivity. The largest content of littorine (3.8 g' kg-! drywt.) was obtained under those culture conditions. Under light conditions, the phytohormones did not strongly affect the alkaloid production, except for the combination of IAA with kinetin (2 + 1 mg/L). Here the content of hyoscyamine was almost doubled. Other combinations of

150

MARTINA SAUERWEIN, MICHAEL WINK, and KOICHIRO SHIMOMURA

Table 1: Morphology and growth of root cultures of H. albus. The roots were cultured for 30 days on WP solid media. The fresh weight is the mean of 4 experiments (8 roots). morphology

phytohormones (kinetin/IAA) [mg/L]

*

normal root

0/0 0/1 0/2 0/4 1/0 1/1 1/2 1/4

hairy root A4

dark fresh weight

morphology

light fresh weight

[g]

*

root only root only root only callus root only root only c + lOr callus

0.82 0.89 0.77 0.66 1.85 3.42 2.09 0.89

root only c+ lOr

0.54 0.98 not determined not determined not determined c+ 5r 1.96 callus 1.31 callus 1.14

0/0 0/1 0/2 0/4 110 1/1 1/2 1/4

root only root only c+ 5r callus c + lOr c + 5r c + 5r callus

0.58 0.11 0.12 0.09 0.42 0.58 0.48 0.18

root only root only

hairy root 15834

0/0 0/1 0/2 0/4 1/0 1/1 1/2 1/4

root only c + lOr c+ 5r callus c+ 5r callus callus callus

1.63 1.19 1.34 0.33 1.32 3.56 1.78 1.19

root only c + lOr

1.44 1.41 not determined not determined not determined c + lOr 1.51 callus 2.07 callus 0.74

hairy root MAFF03-01724

0/0 0/1 0/2 0/4 1/0 1/1 1/2 1/4

root only root only c + 5r callus c + lOr callus callus callus

3.58 0.56 0.61 0.07 1.16 0.78 0.48 0.42

root only c + 5r

culture

[g]

0.33 0.75 not determined not determined not determined c + lOr 0.58 callus 0.11 callus 0.15

4.63 0.95 not determined not determined not determined c+ 5r 0.51 callus 0.53 callus 0.87

* Morphology is expressed as: roots = more than 20 lateral branches; c + lOr = callus with more than 10 lateral branches per initial root tip; c + 5r = callus with less than 10 lateral branches per initial root tip; callus = no branching was observed. Fig. 3: Influence of light and phytohormones on alkaloid production by H. albus root cultures cultured on WP solid medium. Phytohormones were added either alone or in combination. Bars indicate the standard error.

dark

Fig.3a

light •

-: 6.0

7fi-hyuroxyhyoscyaminc

,. 6n-hydroxyhyoscyamine

o

~

i:'

scopolamine

~ hyoscyamine

'tl

~

~4.0

o'f

littorine

~

.,

'tl

·c

;;

2.0

..:oi

;; 0.0 0/0

Fig. 3 a: Normal roots.

011

0/2

0/4

1/0

1/1

112

1/4

0/0

011

111

112

phytohormones [mg/Ll kinetin/ IAA

Alkaloid Production in Root Cultures of Hyoscyamus

albus

light

dark Fig.3b 6.0

i ....

.

." '01)

..III

•

7B-hydroxyhyoscyamine

o

6B-hydroxyhyoscyamine scopolamine

I

4.0

Ii:l

hyoscyamine

~

littorine

~

'"

."

'0

ii 2.0 ..III ii 0.0 0/0

110

011

Fig. 3 b: Hairy roots transformed with A.

111

114

0/0

011

III

rhizogenes A4.

dark

Fig.3c

112 phytohormones [mg/L] kinetin IIAA

light

•

6.0

7f1.hydroxyhyoscyamine 68·hydroxyhyoscyamine

III

i

0

.....

."

'01)

1/2

4.0

scopolamine

~

hyoscyamine

~

IiUorine

..III

~

'"

."

'0

ii ..III ii

2.0

0.0

0/0

011

0/2

0/4

Fig. 3 c: Hairy roots transformed with

Fig. 3d

110

114

0/0

011

III

112

phytohormones [mg/L] kinetin IIAA

dark

light •

7fi-hydroxyhyoscyamine

o

6R-hydroxyhyoscyamine

I

i ....

..

scopolamine

~ hyoscyamine

."

'01)

112

A. rhizogenes 15834.

6.0

-

III

~

4.0

littorine

~ ~

'"

."

'0 2.0

ii ..III ii

0.0 0/0

011

110

Fig. 3 d: Hairy roots transformed withA.

III

112

1/4

rhizogenes MAFF03-01724.

0/0

011

111

112 phytohormones [mg/LJ kinetin II AA

151

152

MARTINA SAUERWEIN, MICHAEL WINK, and KOICHIRO SHIMOMURA

phytohormones resulted in a suppressed scopolamine production. The biosynthesis of tropane alkaloids is known to be located in the roots of the plants. From there hyposcyamine is transported via the xylem to the aerial parts of the plants, where the transformation into scopolamine takes place [Leete, 1990; Liebisch and Schiitte, 1985J. In this context it is remarkably that in normal roots and hairy roots of H albus cultured in the light the alkaloid content and especially the formation of scopolamine and its intermediates was increased compared to the cultures grown in the dark. On the other hand, in the dark larger amounts of littorine were produced. Littorine is supposed to be the biosynthetic intermediate of the acid moiety of the tropane alkaloids [Bachmann et aI., 1991J. Therefore its increased content in dark grown roots may act as precursor for the biosynthesis of the oxygenated tropanes occurring in the light. The effect of phytohormones on tropane alkaloid production varied according to the strain of A. rhizogenes used for transformation. This might be caused by the different insertion of the Riplasmid or clone [Mano et al., 1986, 1989J. As the Ri-plasmid contains genes coding for phytohormone biosynthesis the content of endogenous phytohormones may influence the response of the hairy roots to exogenous phytohormones. Further investigations to examine the phytohormone content in the different hairy roots will be carried out in our laboratory. Acknowledgments

This work was supported in part by a grant of Deutsche Forschungsgemeinschaft (DFG).

References

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