Planta

Planta (1988) 174:19-24

9 Springer-Verlag 1988

Phytochrome-controlled ethylene biosynthesis of intact etiolated bean seedlings J. Vangronsveld, H. Clijsters and M. Van Poucke Limburgs UniversitairCentrum, Department SBM, UniversitaireCampus, B-3610 Diepenbeek, Belgium Abstract. Intact etiolated bean (Phaseolus vulgaris L. cv. Limburgse vroege) seedlings were illuminated with red light (10.5 W . m 2) for 10rain. After different time intervals ethylene production, and contents of 1-aminocyclopropane-l-carboxylic acid (ACC) and 1-(malonylamino)cyclopropane-1carboxylic acid were measured. The red-light-induced decrease of ethylene production in 8-d-old intact etiolated bean seedlings was fast, strong and long-lasting ad was mediated through the phytochrome system. This effect appeared to be strictly age-dependent, as it could not be detected in plants younger than 6 d or older than 11 d. The capacity for the conversion of ACC to ethylene was not affected by red light. The inhibitory effect of the light treatment on ethylene production could be related to a reduced free-ACC content. This reduction was a consequence of a temporary non-reversible increase of ACC malonylation and a long-lasting, for a certain time reversible, inhibition of ACC synthesis. The effect of a brief irradiation with red light on the decrease of ethylene production and free-ACC content was completed after about 2 h. Reversibility by far-red, however, persisted for at least 3 h, and was lost between 3 and 6 h.

Introduction

species. Some investigations showed a reduction of ethylene formation by red light (Goeschl et al. 1967, Pisum sativum; Kang and Ray 1969, Phaseolus vulgaris; Imaseki et al. 1971, Oryza sativa; Samimy 1978, Glycine max), but in other cases an increase has also been reported (Biihler et al. 1978, Sinapis alba; Dei 1981, Cucumis sativus; Rohwer and Schierle 1982, Pisum sativum). Janes etal. (1976, Lactuca sativa) found no effect at all. A coherent interpretation of these results is made difficult by differences in plant material and in experimental conditions, e.g. the use of excised plant parts (Kang and Ray 1969; Imaseki et al. 1971 ; Dei 1981) or intact seedlings (Goeschl et al. 1967; Janes 1976; Samimy 1978; Bfihler etal. 1978; Rohwer and Schierle 1982), and the use of different techniques for ethylene determination (closed systems, except Rohwer and Schierle 1982). We have investigated the effect of phytochrome on the endogenous ethylene production of intact bean seedlings using a continuous-flow system for determination of ethylene (De Greef et al. 1976; De Greef and De Proft 1978). This technique excludes various sources of experimental error inherent in work with injured plant parts confined for prolonged periods of time in sealed vessels. To define more closely the step(s) in the pathway of ethylene synthesis where phytochrome might intervene, we have investigated the effect of phytochrome both on the conversion of exogenous 1-aminocyclopropane-l-carboxylic acid (ACC) applied to the hook region and the cotyledons, and on the changes in endogenous contents of ACC and 1-(Malonylamino)cyclopropane-l-carboxylic acid (M-ACC).

An effect of phytochrome on ethylene production has been found in etiolated seedlings of many plant

Material and methods

Abbrevation: ACC=l-aminocyclopropane-l-carboxylic acid; M-ACC= 1-(malonylamino)cyclopropane-l-carboxylicacid.

Plant material. Seeds of Phaseolus vulgaris L. (r Limburgse vroege) were sown on tap-water-moistened vermiculite a n d

Key words: 1-Amino-cyclopropane-l-carboxylic acid - Ethylene biosynthesis - 1-(Malonylamino)cyclopropane-l-carboxylic acid Phaseolus (ethylene) - Phytochrome and ethylene biosynthesis.

20

J. Vangronsveld et al. : Phytochrome-controlled ethylene biosynthesis

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Fig. l. Mean fresh weight (g) and growth stages during development of intact etiolated bean seedlings grown in complete darkness in a growth chamber at 20 ~ C and 80% relative humidity. Light treatment. Red fluorescent light tubes (TL 15 20 W; Philips, Eindhoven, The Netherlands) were used with an emission spectrum between 640 and 690 nm and with a peak near 660 nm. The far-red light source consisted of 100-W spot lights filtered through a 5-cm layer of running tap water and a F R F 700 filter (formerly Westlake Plastics; USA). The light energy fluence rate was 10.5 W - m -2 red light and 7.5 W - m -2 far-red light, respectively. Ethylene measurements. To identify and measure ethylene production in intact seedlings a continuous-air-flow technique (De Greet et al. 1976; De Greet and De Proft 1978) was used. Ten seedlings were transferred very carefully from vermiculite to a 250-ml glass beaker. No visible root damage was observed.

Only the roots were submersed in 100 ml aerated tap water. For ethylene measurements, the beaker was placed in a cylindrical glass chamber, aerated with a synthetic air mixture, N 2 : 0 2 (80: 20), containing 300 gl. 1- t CO2 but free of ethylene. The flow rate was 101.h ~. The gas inlet ended in the water of the beaker; the outlet was located at the top of the chamber. In this way aeration of the roots and gas flow within the atmosphere of the vessel were ensured. The system equilibrated for at least 6 h to avoid stress-ethylene production arising from mechanical stimulation during sealing of the chamber; in most cases, ethylene production was already stabilized after 4 h. To trap the ethylene produced, the gas outlet of the chamber was connected with a cooled ( - 9 5 ~ precolumn (6.35 mm in diameter) packed with 0.35-0.40 g Porapak-S (50-80 mesh, Waters Ass., Milford, Mass, USA). After 10 rain, this column was inserted into the carrier gas stream of a gas chromatograph. The ethylene was totally released and swept onto the analytical column with the carrier gas by heating the precolnmn to 100 ~ C in boiling water. Ethylene determination was carried out on an Intersmat IGC 125 gas chromatograph (Pavillon-Sous-Bois, France) equipped with a Porapak-R column (50-80 mesh) and a flame-ionisation detector. Measurements o f A CC and M - A CC. Whole seedlings were frozen in liquid nitrogen and powdered in a mixer (type A10, 20000 rpm; Janke & Kunkel, Staufen i.Br., FRG). Three g of this powder were extracted four times with a total volume of 50 ml 80% methanol. The organic phase was vacuum-evaporated at 40~ (Biichi Rotavapor R; Flawil, Switzerland). The water fraction was purified by passing through an activated Sep-pak C 18 column (Waters Ass.). The extraction efficiency and the extent of ACC losses during the purification step were tested by measuring the recovery of ACC added to the powdered tissue. Using 3 g of powder, 200 txl of a 10 6 M-ACC solution were allowed to soak in during 15 rain before the extraction procedure. Apart from the measurable endogenous ACC content of the sample, 91% of the added amount of ACC was recovered. The ACC content in the aqueous extract was determined by chemical conversion to ethylene according to Lizada and Yang (1979), with an efficiency of 89 95%. Quanti-

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Fig. 2. a Ethylene production (pl-h-1-plant-1) during development of intact etiolated bean seedlings 6 h after a J0-min red-light treatment. The data represent mean_+SD of at least 10 measurements, b. Ethylene production (pl-h -1-plant -1) following a 10-min red-light (R) irradiation of 8-d-old bean seedlings. The data represent mean_+SD of eight measurements, u - - n - d a r k control; n - - r ~ = red-irradiated

J. Vangronsveld et al. : Phytochrome-controlled ethylene biosynthesis fication of the M-ACC was carried out by hydrolysing an aliquot of the aqueous extract in 6 N HCI at 100 ~ C for 1 h. Under these conditions, M - A C C was completely hydrolysed without any detectable deterioration of ACC. Therefore, the ACC concentration in this boiled extract, measured according to L~zada and Yang (1979) (yields 88-96%), is its total ACC content. The amount of M - A C C was calculated by substracting the ACC content before hydrolysis from that after hydrolysis. A p p l i c a t i o n o f A C C . An aliquot of 150 g l ' o f an aqueous ACC solution (10 -3 M) was applied to the cotyledons and hook region of the seedlings. It was spread using a small brush moistened with the same solution.

21

Table 1. Reversibility of ethylene production (pl. h - i. p l a n t - 1)

of intact bean seedlings 6 h after light treatment. F R = far-red light; R = red-light

4dold 8dold 14d old

Dark control

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255+_14 489+10 390+_54

260• 484+_27 381_+61

** Significant difference for P = 0 . 9 9 ; Students' t-test; n = 10

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Results

Ethylene production; the effect of red light. The ethylene production of intact etiolated bean seedlings from 4 to 14 d old was measured. The stages of development and the increase in fresh weight are presented in Fig. 1. A peak in ethylene production was measured on day 8 after sowing (Fig. 2a). A single exposure of seedlings of this age to 10 min red light caused a decrease in ethylene production (Fig. 2b), measurable within 30 min after the onset of irradiation. Sixty percent of the control production in continuous darkness remained after about 2 h and this level was then maintained for at least 24 h. In younger seedlings (4-6 d after sowing), ethylene production in darkness was only about 50% of that on day 8. These seedlings were insensitive to red light with respect to their ethylene-production capacity (Fig. 2 a). In older seedlings, ethylene production in darkness diminished and the effect of red light on it seemed to shift from a strong inhibition to a slight stimulation. In 11- and 12-d-old plants, ethylene production in darkness was about 50 and 70%, respectively, of the earlier maximum and was insensitive to red light. In 14-d-old plants, the dark ethylene production was only about 65% of the maximum and a brief irradiation with red light caused a slight but not significant increase in the hours that followed (Fig. 2 a).

Reversibility of the red-light effect on ethylene production. Far-red light (10 min), given immediately after red light, fully reversed the inhibition of ethylene production as measured 6 h after light treatment. This indicates the involvement of the phytochrome system (Table 1). Reversibility was still observed when far-red was given with dark intervals of 15 rain to 3 h after the red-light irradiation (Fig. 3a). The timecourse of changes in ethylene production for a dark interval of 3 h between red and far-red irradiation is presented in detail (Fig. 3b). Three hours after the red-light treat-

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time after irradiation (hours) Fig. 3. a Effect of dark periods intervening between red and far-red light on the reversibility of ethylene production by 8-dold etiolated bean seedlings. Measurements were taken 2.5 h after the far-red irradiation. Each point represents the mean of at least eight measurements, b Ethylene production (pl. h - 1. plant -1) of 8-d-old etiolated bean seedlings following 10 min red light (R) and 10 min far-red light (FR) given at the moments indicated by arrows' . The data represent mean + SD of four measurements, i - - m = dark control; n - - n = red irradiated

ment, ethylene production was already stabilized at its minimum level. Yet it took only 2 h after the far-red irradiation to re-establish the initial dark rate of ethylene production. A second redlight treatment, given then, again induced a

22

J. Vangronsveld et al. : Phytochrome-controlled ethylene biosynthesis

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