Mar 25, 1992 - of Catharanthus roseus is induced by exposure of seedlings to light and that enzyme ... UV-B light-absorbing photoreceptors (8). In this report ...
Plant Physiol. (1992) 100, 1029-1032 0032-0889/92/100/1029/04/$01.00/0
Received for publication March 25, 1992 Accepted July 6, 1992
Communication
Phytochrome Is Involved in the Light-Regulation of Vindoline Biosynthesis in Catharanthus' Rob J. Aerts2 and Vincenzo De Luca*
Institut de Recherche
en
Biologie Vegetale, Universite de Montreal, 4101 Sherbrooke est, Montreal, Quebec, Canada, H1X 2B2
ABSTRACT The enzyme acetylcoenzyme A:deacetylvindoline 4-0-acetyltransferase (DAT) catalyzes the final step in the biosynthesis of the monoterpenoid indole alkaloid, vindoline. Previous studies have shown that the appearance of DAT activity in etiolated seedlings of Catharanthus roseus is induced by exposure of seedlings to light and that enzyme activity is restricted principally to the cotyledons. Evidence is now presented that phytochrome is involved in the light-mediated induction of DAT activity in Catharanthus
thesis and result in the quantitative conversion of tabersonine and intermediates into vindoline. The aim of our present study was to investigate the light-control of DAT induction in Catharanthus cotyledons in more detail. Higher plants appear to possess several types of photoreceptors for perception of their light environments. These include the red/far red phytochrome photoreceptors, the blue/UV-A light-absorbing cryptochrome, and one or more UV-B light-absorbing photoreceptors (8). In this report, evidence is presented for the participation of phytochrome in the light-mediated induction of DAT activity in Catharanthus roseus seedlings.
cotyledons.
MATERIALS AND METHODS
Catharanthus roseus L. G. Don produces the dimeric indole alkaloids vinblastine and vincristine, two medically important antitumor agents used in the chemical treatment of human cancers (3). Because the plant produces these compounds in very small amounts, many attempts have been made to produce these dimers or their direct monomeric precursors, catharanthine and vindoline, in C roseus tissue cultures (6). Although tissue cultures can accumulate high levels of the monoterpenoid indole alkaloid catharanthine, cultures producing vindoline, vinblastine, and vincristine have yet to be discovered. The biosynthesis and accumulation of vindoline in the intact plant is controlled by tissue-specific, developmentregulated, and light-dependent factors. When Catharanthus seedlings are grown in the dark, they accumulate high levels of the indole alkaloid tabersonine as well as several posttabersonine intermediates and only trace levels of vindoline (5). Treatment of etiolated seedlings with light induces the hydroxylase (4) (2-oxoglutarate-N(1)-methyl-16-
Growth of Seedlings
Small batches of seeds (0.6 g; approximately 480 seeds) of Catharanthus roseus L. G. Don (Vinca dwarf little mixture, Sakata Seed Corp.) were sterilized in 80% ethanol for 30 s, thoroughly rinsed with sterile, distilled water, and allowed to imbibe water for 1 d. Imbibed seeds were placed in sterile Petri plates (9 cm diameter) on paper tissues with 5 mL of sterile, distilled water. The seeds from each batch were distributed over five Petri plates and germinated in the dark. The growth temperature within the Petri plates was 230C and time zero is defined as the moment when seeds were placed on paper tissues (5, 7).
Light Treatments Etiolated seedlings were exposed at day 7 to incandescent cool white fluorescent light (10 or 40 Mmol.m-2.s-'), or to red light, obtained with the white light source (10 smol.m-2.s-') filtered through a Roscolux No. 19 or a Lee Panavision International No. 106 filter. The Roscolux No. 19 filter transmitted only wavelengths longer than 575 nm and transmitted greater than 90% of the irradiance of wavelengths longer
methoxy-2,3-dihydro-3-hydroxytabersonine-4-hydroxylase; EC 1.14.11.-) and the O-acetyltransferase (5, 7) (DAT3; EC 2.3.1.-) that catalyze the last two steps in vindoline biosyn'This work was supported by a fellowship to R.J.A. from the Netherlands Organization for Scientific Research, and by grants to V.D.L. from the National and Engineering Research Council of Canada and from Les Fonds pour la Formation de Chercheurs et l'Aide a la Recherche de Quebec. 2 Present address: Institut fur Pflanzenbiology, Zollikerstrasse 107, CH-8008, Zurich, Switzerland. 3Abbreviation: DAT, acetylcoenzyme A:deacetylvindoline 4-0acetyltransferase.
than 620 nm. The Lee Panavision No. 106 filter transmitted only wavelengths longer than 590 nm and transmitted greater than 90% of the irradiance at wavelengths longer than 650 nm. These characteristics produced red light with about the same photon fluence rate as the red component of the original white light source. Other batches of seedlings were exposed at day 6 or 7 to alternate treatments with red (10 ,umol.ms-i white light filtered through Roscolux No. 19 filter) and 1029
Plant Physiol. Vol. 100, 1992
AERTS AND DE LUCA
1 030
than in continuous light (Fig. 1, arrows and closed circles). After 48 h of light treatment, the increase in DAT activity started to level off, whereas after 72 h it began to decrease (not shown; ref. 7). Seedlings grown in the dark showed baseline levels of DAT activity (Fig. 1, dashed arrows).
far red light (10 ,umol m-2 s- white light filtered through Roscolux No. 19, No. 83, and No. 89 filters, which transmitted only wavelengths longer than 710 nm). Photon fluence rates in the 400- to 700-nm range were measured with a LiCor Quantum sensor. Seedlings were harvested at appropriate time points for direct processing or were conserved at -800C.
The Involvement of Phytochrome
To investigate the possible role of phytochrome in the induction of DAT activity, the influence of red light on the process was investigated (see 'Materials and Methods'). Figure 2 shows that the induction of DAT activity in etiolated seedlings proceeded as efficiently in red light as in white light. This result indicates that red light alone is sufficient for the induction process. Analysis of monoterpenoid indole alkaloids by TLC showed that vindoline also accumulated as rapidly in red light as in white light (not shown). After treatment of etiolated seedlings with a 30-min red light pulse, they were either returned to dark growth or subsequently illuminated with a 30-min far red light pulse and returned to dark growth. Analysis after 24 h showed that the red light-induced increase in DAT activity in cotyledons was diminished and could be partially reversed by the far red light treatment (Fig. 3, R and R/FR). The effect of far red light could be eliminated by a second 30-min pulse of red light (Fig. 3, R/FR/R). The photoreversibility of DAT induction by red and far red light indicates the involvement of phytochrome in the induction process. During the first stages of the deetiolation process and the induction of DAT activity in the cotyledons, they were still covered by their brownish seed coats (Fig. 1). When the seed coats were analyzed by spectrophotometry for their light transmission spectrum (Fig. 2, inset), they were found to transmit mostly the red component of the spectrum, which appears to be responsible for the induction of DAT activity.
Determination of DAT Activity
Enzyme was extracted from pairs of cotyledons (25-30) in 1.3 mL of 0.2 M Tris-HCl, pH 7.5, 3 mi EDTA, 5 mm DTT, 30 g/L of PVP, and extracts were desalted as described previously (7). Enzyme assays contained 27.5 Mm deacetylvindoline, [1-'4C]acetyl-CoA (25 nCi; 60 mCi/mmol), and enzyme in 50 mm Tris-HCl, pH 7.5, 28 mm 2-mercaptoethanol in a final volume of 150 AL. Enzyme assays were performed as described previously (7) and control assays either lacked enzyme or were stopped immediately after initiating the reaction. All enzyme assays were performed in duplicate. RESULTS Kinetics of Induction of DAT Activity
Catharanthus seedlings were germinated in the dark, and were exposed to 10 or 40 Amol m-2.s-1 of continuous white light (see 'Materials and Methods'). The light treatments induced an immediate increase in DAT activity in cotyledons (Fig. 1, open circles). After 24 h of continuous illumination, DAT activity increased more rapidly in cotyledons exposed to the higher light intensity (Fig. 1B) and this could be due to the earlier loss of seed coats in these seedlings (see top of Fig. 1). When seedlings were retumed to the dark after different periods of light treatment, DAT activity continued to increase gradually, but at lower rates at day 7 they
dKdKnKa< YK A 40 W:10 jinol m-2s-l
-.1-
B
lW40 lmoL r-2 s1*
-
0
0.
0o L.
0.
n
I.Q
0
00
Q 0
