Phytochrome control of the accumulation and rate of synthesis of ascorbate oxidase in mustard cotyledons

Plant Science, 64 (1989)79-90

79

Elsevier Scientific Publishers Ireland Ltd.

PHYTOCHROME CONTROL OF THE ACCUMULATION AND RATE OF SYNTHESIS OF ASCORBATE OXIDASE IN MUSTARD COTYLEDONS

L. LEAPER and H.J. NEWBURY*

School of Biological Sciences, University of Birmingham, Birmingham B15 2TT (U.K.) (Received December 5th, 1988} (Revision received May 3rd, 1989) (Accepted May 10th, 1989)

Mustard seedlings were grown in darkness or exposed to far red light. Investigations into the enzyme ascorbate oxidase (AO) in extracts from cotyledons exposed to 36 h far red light and from dark controls revealed: (1) the activity of AO increases up to 7-fold upon light treatment. (2) The amount of AO decreases after 36 h light treatment compared to dark controls; this has been measured by ELISA using a horseradish peroxidase conjugate employing either a colorimetric or an enhanced ebemfluminescent assay; the latter was applied beth to samples in microtitre plates and on nitrocellulose filters. (3) The rate of A0 synthesis, as measured by incorporation of [uS]methioulne into AO, appears to be lower in extracts of cotyledons exposed to 36 h light than in dark controls; this has been monitored by employing nitrocellulose filters coated with AO-specific antibodies followed by autoradiography. The results are inconsistent with a hypothesis that far red light leads to an increase in the rate of synthesis of this enzyme; rather they suggest post-translational control of AO activity by phytochrome.

Key words: Ascorbate oxidase; phytoehrome; mustard; ELISA, enhanced chemiluminescence

Introduction The plant photoreceptor phytochrome has been shown to mediate a large number of lightinduced responses in green plants. Among the most heavily studied of these are phytochromemediated changes in the activities of certain enzymes [1]. In many cases it has been shown that a light-induced increase in enzyme activity can be related to an increase in the rate of synthesis of enzyme protein and there has been much work on the phytochrome control of gene expression. In spite of these findings, the mechanism by *To whom correspondence should be sent. Abbreviations: AO, ascorbate oxidase; cAO, Cucurbita ascorbate oxidase; DEAE, diethylaminoethyl; ELISA, enzyme-linked immunosorbent assay; FR, far red; HRP, horseradish peroxidase; OPD, orthophenylene diamine; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; RubisCo, ribulose bis-phosphate carbexylase/oxygenase; SDS, sodium dodecyl sulphate; TBS, tris-buffered saline.

which phytochrome regulates the activity of other enzymes is not clear. Ascorbate oxidase (A0) was one of the earliest proteins used to study the effect of phytochrome on enzyme activity. Van Poucke et al. [2] demonstrated that the extractable activity of A0 in darkgrown mustard seedlings was increased if they were exposed to a short period of red light, and that this red light-induced effect was abolished if followed immediately by a short exposure to far red (FR) light. Drumm et al. [3] showed that A0 activity in dark-grown mustard cotyledons was increased in response to continuous high intensity FR light; this phenomenon is shared by many other phytochrome-mediated responses and has been called the high irradiance response [4]. In order to gain information on the mechanism by which AO activity is increased in mustard cotyledons during exposure to FR light, two groups of workers have independently used a protocol involving the density labelling of AO [5,6]. Cotyledons were allowed to take up

0168-9452/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

80 2H20, the soluble proteins extracted and subjected to centrifugation in isopycnic gradients, and the buoyant density of enzymically active A 0 was determined. From comparisons of the results obtained using cotyledons held in darkness with those exposed to FR light, both groups concluded that the phytochromemediated increase in A 0 activity was due to de novo synthesis and a consequent increase in the concentration of the enzyme. More recently, Newbury and Smith [7] used rocket immunoelectrophoresis to monitor the level of AO protein in mustard cotyledons. They reported that although there was an increase in the AO activity in cotyledons following transfer from darkness to continuous FR light, the concentration of AO protein remained the same. In addition, unimbibed seeds were shown to contain the same amount of enzyme protein as expanding cotyledons. Two possible interpretations were offered: (1) that inactive enzyme may represent a precursor activated post-translationally through the action of phytochrome or (2) that the increase in activity may be due to de novo synthesis with newly synthesised AO masked by the presence of large amounts of inactive enzyme. The first objective of this investigation was to apply the more sensitive technology of ELISA to this system to monitor the level of AO protein in dark- and FR light-treated mustard cotyledons. The second objective was to determine the rates of synthesis of AO in control and illuminated cotyledons. During the course of the work a technique was developed to allow binding of in vivo labelled AO to A0specific antibodies immobilised on a filter support. This filter could then be used for both the quantitation of total AO protein by using a horseradish peroxidase~onjugated second antibody and an enhanced chemiluminescence assay system, and for the determination of relative rates of synthesis of AO by conventional autoradiography. Materials and Methods

Plant growth and illumination conditions White mustard (Sinapis alba L.) seeds were

obtained from Asmer Seeds, Leicester, U.K. and were grown at 25 _+ 0.5°C on autoclaved 1% agar (Oxoid No. 1} prepared in distilled water. Far red light was delivered by tungsten bulbs and was filtered through red (ICI, 4400) and green (ICI, 6600) perspex filters. The ratio of far red (730 nm) to red (660 nm) light was approximately 25 : 1 when measured as/~E m -~ using a Macam Photometrics Ltd. Spectrc~ radiometer calibrated with a spectroadiometer used by the phytochrome group of Prof. H. Smith at Leicester University.

Assay for ascorbate oxidase activity Cotyledons (1 g fresh wt.) were homogenised with quartz sand in a pre-cbilled mortar in 4 ml of 0.15 M citrate-phosphate buffer (pH 5.5) and the homogenate centrifuged for 2 min at 10 000 × g. The supernatant was then centrifuged at 120 000 × g for 45 min and the pellet discarded. Samples of these crude extracts were then desalted using Sephadex G25 columns with extraction buffer for elution. Aliquots of each desalted sample were added to 2.5 ml of extraction buffer along with 30 ~1 of 5 mM L-ascorbic acid. Enzyme activity was measured spectrc~ photometrically at 25°C by recording the decrease of Am; a decrease of one absorbance unit is equivalent to the loss of 175 nmol of ascorbic acid.

Antiserum production Purified AO from Cucurbita sp. (cAO) was obtained from Boehringer Mannheim. Sheepderived antibodies were produced and purified as previously described [7]. Antiserum was also raised in New Zealand White rabbits. In both cases, following precipitation of protein by addition of ammonium sulphate (final concentration 5 0 ) and dialysis into 10 mM phosphate buffer (pH 8.0), the samples were subjected to DEAE cellulose chromatography to elute the IgG fractions as single peaks. Both rabbit and sheep IgG fractions were prepared at 10 mg m1-1. In experiments involving the use of antibody-coated nitrocellulose filters, antiCucurbita AO serum that had been raised in a sheep and subjected to affinity chromatography using a cAO-sepharose column (adsorbed

81 antiserum; see Ref. 7) was used as a second AOspecific antibody. Double diffusion gels were washed, pressed and stained using the techniques described by Axelsen et al. [8].

Denaturing gels and blotting technique Proteins were analysed on one dimensional polyacrylamide gels containing SDS using the discontinuous system of Laemmli [9]. Where protein staining was carried out, 0.1% PAGE Blue 83 in 300/0 (v/v) methanol and 10o/0 (v/v) acetic acid was employed. Gels containing radiolabelled proteins were subjected to fluorography using the method of Jen and Thach [10]. Immunoblotting was carried out according to the method of Burnette [11] using nitrocellulose filters (0.2 pm pore, Sartorius). A 1 : 500 dilution of rabbit anti-Cucurbita AO serum was added to blotted filters and incubated at room temperature for 90 rain. Following washing, the sheet was immersed in a 1:1000 dilution of horseradish (HRP)-conjugated swine anti-rabbit IgG serum (Dako) and gently agitated for 1 h at room temperature. Following further washing, staining was carried out using 50 ml 0.060/0 4-chloro-l-naphthol in 0.05 M Tris - HCI (pH 7.4) containing 25 ~i 300/0 H202.

ELISA Initially, checkerboard titrations were carried out using 96 well polystyrene microtitre plates (Nunc) in order to ascertain the appropriate dilutions of antigen, first antibody and HRP-second antibody conjugate to be employed. During the estimation of AO in mustard cotyledon extracts, Cucurbita AO was used to produce a calibration curve for each microtitre plate, cAO was serially diluted to give a range of 0.98-250 ng/ml in coating buffer (0.05 M carbonate buffer, pH 9.6) and 100 ~1 aliquots were applied to duplicate horizontal rows of wells. Dark and far red light-treated mustard cotyledons (1 g) were extracted in 4 ml (final volume) of phosphate buffer (0.1 M, pH 7.0) and diluted extracts were made in the range 1 : 1000 to I : 9000 in coating buffer; 100 ~1 aliquots of each were added to triplicate rows of wells. Plates also included the following controls: (1) wells coated with lO/0 (w/v) bovine

serum albumin; (2) wells coated with the highest concentrations of either cAO (0.25/~g m1-1) or mustard extracts (1 : 1000 dilution) half of which were subsequently treated with nonimmune serum and half with phosphate-buffered saline-Tween (PBS-Tween; 8.1 mM KH2PO 4, 2.7 mM KC1, 140 mM NaC1, 0.05% (v/v) Tween 20, pH 7.4). Coated plates were incubated overnight at 4 °C in humid conditions and then washed three times for 3.5 rain with PBS-Tween with vigorous shaking to remove residual wash solution. Rabbit IgG specific to cAO was diluted to 1 : 1000 in PBS-Tween, 100/A aliquots added to each well (except where non-immune serum or PBS-Tween was used in controls) and the plates left for 1.5 h at 37 °C. HRP-conjugated to swine anti-rabbit IgG serum, diluted to 1:5000 in PBS-Tween, was added in 100 ~1 aliquots to each well and the plates incubated for a further 1.5 h at 37°C. Following further washing (as before) 100 ~l of the HRP substrate OPD (4 mg orthophenylene diamine (Sigma), 5 ~1 H202 (300/0) in 10 ml citrate-phosphate buffer, pH 5.0) was added to each well and the plates incubated at 37 °C. After 20 min the reaction was stopped by the addition of 30 ~1 of 200/0 sulphuric acid and the absorbance at 492 nm measured in each well using a Titertek Multiscan ELISA plate reader (Dynatech Labs. Ltd.}. The amount of AO in test samples was determined using a calibration curve derived from the data obtained using the standard concentrations of cAO. As an alternative to the measurement of bound HRP activity using spectrophotometry, an enhanced chemiluminescence assay was also employed. The ELISA method was identical to that described above except that, instead of using OPD as a substrate, 100 /~1 aliquots of luminescent substrate solution (1.25 mM luminol, 0.136 mM paraiodophenol, 2.7 mM hydrogen peroxide in 100 mM tris buffer, pH 8.5) were added to each well. Light emission from the plates was recorded using a camera luminometer with a 10-30-s exposure of Polaroid type 612 Instant film [12].

In vivo labelling Mustard cotyledons grown in various light

82

regimes were individually painted with a solution containing 250 ~Ci[85S]methionine in 500 ~1 distilled water, containing lO/0 (w/v) Tween 20, 4 h before harvesting. Cotyledons grown in darkness were painted under a green safelight (max. intensity at 660 or 730 nm = 2 × 10 -7/~E cm-2).

A 0 assays using antibody-coated nitrocellulose A dot immunobinding assay was carried out using modifications of the methods of Sampson et al. [13]. A nitrocellulose membrane (0.2 ~m pore, Sartorius) was washed for five minutes in distilled water and allowed to dry at room temperature. The membrane was then immersed in 50 ml of a 1 : 100 dilution of rabbit anti-Cucurbita AO serum in TBS (50 mM tris buffer, 200 mM NaCI, pH 7.4) containing lO/0 (w/v) casein, and agitated gently for 1 h. Following drying at room temperature, the filter was loaded into a Hybridot manifold (Bethesda Research Labs.) and a low vacuum applied. 100-~l aliquots of a dilution series (39.5--10 000 ng m1-1) of Cucurbita AO in PBS-Tween (see above) were applied as duplicate spots. In vivo labelled dark and far red light-treated cotyledons (1 g) were extracted in 4 ml (final volume) of 0.1 M phosphate buffer (pH 7.0); 100-/~l aliquots of a dilution series (1 : 2 0 - 1 : 100) in PBS-Tween were applied as triplicate spots. Following loading, the vacuum was switched off and the membrane allowed to stand for 1 h at room temperature in the manifold before removal and washing by gentle agitation in TBS for 30 min, followed by washing in TBS containing 0.050/0 (w/v) casein for 15 min. The membrane was then immersed and gently agitated in a 1 : 100 dilution of adsorbed sheep anti-Cucurbita AO in TBS for 1 h and then washed as previously described. It was then incubated for 45 min in 50 ml of a 1 : 1000 dilution of H R P conjugated to swine anti-sheep IgG serum (Dako) and washed as before. Following immersion in 20 ml of luminescent substrate (see above) the filter was placed in a transparent holder in a camera luminometer and Polaroid type 612 film exposed to the glowing filter for 2 - 1 0 s. After this non-destructive measurement of the total AO antigen on the filter, it was placed in con-

tact with Kodak AR 5 film for 12--24 days at - 7 0 ° C to allow exposure of areas in contact with radiolabelled AO on the filter. Several controls were performed during experiments involving binding of A 0 to antibody-coated nitrocellulose filters: (1) 100-~! aliquots of 1o/0 bovine serum albumin were used as control antigen; (2) Cucurbita AO or mustard extracts were omitted; (3) the AO-specific antiserum was omitted; (4) a replicate filter was immersed in 50 ml of a 1 : 100 dilution of nonimmune rabbit serum in TBS instead of anti-AO serum. Results

The extractable activity of AO was up to 7fold higher in the cotyledons of mustard seedlings grown in darkness for 36 h and then exposed to FR light for 36 h compared to that found in cotyledons of control seedlings grown in darkness for 72 h (Fig. la). Since it is possible that the differences in activity were due to the presence of inhibitors or activators of AO in the preparations, a mixing experiment was performed. The results (Table I) provide no evidence of interaction between components of preparations extracted from FR light-treated and control cotyledons since the activities measured in the mixed samples are similar to the sums of the activities of the extracts assayed individually. During experiments designed to determine the concentration of AO protein present in control and FR light-treated cotyledons, purified Cucurbita AO (cAO) was used both for construction of calibration curves and the raising of antisera. The cAO used was tested for purity using both polyacrylamide gel electrophoresis under denaturing conditions and isoelectric focussing. In both cases a single protein band was obtained; this was ascribed a molecular weight of 66 kDa which is in agreement with a previous report of the size of the two AO subunits from this source [14]. The IgG-containing fractions of antisera raised against cAO in a sheep and in a rabbit were tested for specificity. When subjected to double-diffusion plate analysis both antisera

83

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7

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Table I. Mustard seedlings were germinated in 36 h darkness, and then exposed to either FR light or further darkness for the times indicated. Cotyledons were harvested and homogenised and centrifuged and equal volumes of certain samples were mixed. All samples were then held at room temperature for 1 h before assay. For the mixed samples ( 7 - 9 ) double the normal volume was assayed; the results expected if mixing has only an additive effect are given in parentheses. Each result is the mean of five replicates.

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F i g . 1. Mustard seedlings were either grown in darkness for 72 h ( • ) , or were exposed to continuous far red light after 36 h growth in darkness (O). Cotyledons were harvested 48, 60 and 72 h after sowing and soluble protein extracts prepared as described in Materials and Methods. These samples were then used for: (a) a spectrophotometric assay for AO activity, (b) an ELISA assay for AO protein and (c) an assay of total protein. Standard error bars are shown and significant differences between data are indicated by stars: *P < 0.05; ***P < 0.001.

induced the formation of a single precipitin band in tests against cAO, extracts from mustard seeds, or extracts from cotyledons raised in darkness or exposed to FR light (Fig. 2). However, it is clear that there was only partial fusion between the precipitin lines of cAO and

mustard extracts, which suggests that cAO contains some antigenic determinants not shared by AO from mustard. The precipitin lines from different mustard extracts were completely fused, suggesting that the AO from the various samples was immunochemically identical. The sheep and rabbit IgG fractions were separately incorporated into agarose gels and used for rocket immunoelectrophoresis. Crude extracts from dark- and FR light-treated cotyledons again produced only single rocketshaped precipitates (not shown). The specificity of the rabbit IgG fraction was further demonstrated by immunoblotting. Samples of cAO and mustard cotyledon proteins were fractionated by PAGE under denaturing conditions and electrophoretically blotted onto nitrocellulose. Blots were probed with the rabbit IgG fraction, and AO subunit bands visualised after incubation with horseradish peroxidase conju-

84

Fig.2. Ouchterlonydouble diffusionplatesstainedwith Coomassie BrilliantBlue B. The centralwellscontainsera:l~I,nonimmune serum; S, the IgG fractionof an anti-CucurbitaA O serum raisedin a rabbit.Peripheralwells containsamples of Cucurbita AO (C):an extractfrom ungerminated mustard seeds (Se):extractsfrom mustard cotyledonsharvestedfrom seedlingsthathad been grown in continuousdarkness(D),with the number inparenthesesindicatingthe time,in hours,sincesowing:extractsfrom mustard cotyledonsharvestedfrom seedlingsthathad been grown in darknessfor36 h and then exposed to farred light{F),with the number inparenthesesindicatingthe time,in hours,ofexposureto farred light.

gated to swine anti-rabbit antibodies and subsequent addition of a peroxidase substrate. The results (Fig. 3) show that this antiserum specifically recognises a single protein in a population of proteins from mustard cotyledons. In order to quantify the amount of A0 protein in cotyledons the AO-specific rabbit IgG fraction was used in a conventional E L I S A employing microtitre plates. Cucurbita A O was used to prepare a standard calibration curve although, as previously noted, c A O possesses antigenic determinants not shared by A O from mustard. Thus, the measurements of mustard e n z y m e protein ascertained using antiserum raised to c A O and using c A O standards can not be assigned absolute values; they do, however,

represent the relative concentrations of AO in dark- and FR light-treated mustard cotyledons. ELISA tests employing the peroxidase substrate OPD detected progressively smaller amounts of AO protein in extracts from both dark- and FR light-treated cotyledons with time (Fig. lb). Initial data was obtained here in the form of absorbances of a coloured reaction product. After 36 h of F R light there w a s a significant (P <~ 0.001) decrease in the a m o u n t of A O present compared to dark controls. A O protein was also detected in unimbibed mustard seeds in similar quantities to those measured in expanding cotyledons. A n enhanced chemiluminescence procedure was also used; the results (Fig. 4) again showed no apparent differences between the quantities of A O extracted from

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Fig. 3. Tracks 1 - 3 , Cucurbita AO subjected to electrophoresis under denaturing conditions and: stained for protein (track 1); blotted onto nitrocellulose and probed using rabbit anti-Cucurbita AO serum (track 2); blotted onto nitrocellulose and probed using non-immune rabbit serum (track 3). Tracks 4 - 6, sample of cotyledon extract, from seedlings grown in darkness for 36 h and then exposed to far red light for 36 h, subjected to electrophoresis under denaturing conditions and stained for protein (track 4); blotted onto nitrocellulose and probed using rabbit anti-Cucurbita AO serum (track 5~; blotted onto nitrocellulose and probed using non-immune rabbit serum (track 6).

dark- and FR light-treated cotyledons for the first 24 h of treatment; again at 36 h there was a clear decrease in the FR light-treated cotyledon extracts. The amount of total soluble protein extracted from dark- and FR light-treated cotyledons was also shown to decrease with time (Fig. lc). After 36 h FR light treatment there was a significant (P < 0.05) decrease in the amount of total protein extracted compared to dark controls. However, the decrease in concentration of AO protein was not just a consequence of the decline in amount of total protein. A comparison of the linear regression equa-

tions calculated from the total soluble protein and AO quantitation data showed a significant difference (P < 0.001) between the two dark controls and between the two FR light slopes. In order to determine the relative rates of synthesis of AO in control and FR light-treated cotyledons, [~S]methionine was applied to these organs and the level of incorporation into A 0 protein was monitored. It was necessary to use a green safelight to allow application of [3~S]methionine to cotyledons grown in darkness. The suitability of green light for use in photomorphogenetic studies has been questioned since relatively low fluences of green

86 light have, in some instances, been shown to induce the same response as low fluences of red light [15,16]. However, experiments comparing green light treatment of cotyledons with dark controls failed to reveal any change in extracted AO activity. To purify AO from other labelled proteins,

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Discussion

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There is strong evidence that phytochrome acts to control the rate of transcription of a number of genes (e.g. phenylalanine ammonia

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samples of cotyledon extract were applied to a nitrocellulose filter to which had been bound AO-specific antibodies. This procedure employed a dot-blot apparatus and allowed the assessment of both the amount of accumulated AO and the amount of incorporation of [80S]methionine into AO in the same sample of cotyledon extract. Estimation of the concentration of AO was carried out using the enhanced chemiluminescence technique previously applied to samples on microtitre plates. The results (Fig. 5) again show that the 36 h far red light treatment leads to a decrease in the amount of AO protein over controls. The filters were then subjected to autoradiography. The results (Fig. 5) clearly indicate that there is a much lower level of labelled AO protein in the samples extracted from cotyledons exposed to FR light for 36 h than in dark controls. The relative rates of incorporation of [85S]methionine into total soluble cotyledon protein was also assessed. The results (Table II) are not consistent with a more rapid decline in [ssS]methionine incorporation into total soluble protein following treatment with far red light compared to dark controls.

Fig. 4. Photographs of light emission from an enhanced chemiluminescence assay for ascorbate oxidase on microtitre plates. In each of the three blocks: (1) the left-hand vertical row of wells was loaded with 1% bovine serum albumin as a control; (2) the upper two horizontal rows were loaded with a dilution series of Cucurbita AO; (3) the lower six horizontal rows were loaded with a dilution series of extracts from mustard cotyledons (initially 1 g homogenized in a total of 4 ml) harvested from seedlings exposed to the light regimes shown above. Antigen-coated wells were then inoculated first with a 1 : 1000 dilution of a rabbit Cucurbita AO-specific IgG fraction, and then with a I : 5000 dilution of horseradish conjugated with swine anti-rabbit IgG serum. Luminescent substrata solution was added and film exposed to the plates using a camera luminometer (block a exposed for 15 s, and blocks b and c for 10 s).

87

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1:20/1.'101 Fig. 5. The effect of far red light on the accumulation and rate of synthesis of ascorbate oxidase in mustard cotyledons. The left-hand blocks are nitrocellulose filters, coated with a Cucurbita AO-specific rabbit IgG fraction and clamped in a Hybridot manifold, before loading as follows: (1) the left-hand vertical row of wells were loaded with 1% BSA as a control; (2) the upper two horizontal rows of wells were loaded with a dilution series of Cucurbita AO; (3) the lower six horizontal rows of wells were loaded with dilution series of extracts from mustard cotyledons; seedlings were grown in the light regimes indicated and the cotyledons exposed to [~S]methionine for 4 h immediately prior to harvesting. The filters were then incubated first with an adsorbed Cucurbita AO-specific sheep serum and then with horseradish peroxidase conjugated to swine anti-sheep IgG serum before addition of luminescent substrate. Instant film was exposed to the filters for 2 s in a camera luminometer. The filters, from which the upper portion bearing the unlabelled Cucurbita AO had been removed, were then subjected to autoradiography for 28 days and the X-ray films are shown on the right.

88

lyase, the small subunit of Rubisco, the light harvesting chlorophyll a/b binding protein) [17,18]. However, the results reported here provide no evidence to support a model in which far red light increases the activity of AO in mustard cotyledons by increasing the rate of synthesis of this enzyme. AO protein can be detected in extracts from unimbibed mustard seeds (Ref. 7; see Fig. 2) which do not contain measurable AO activity. It is therefore clear that AO may exist in both active and inactive forms in mustard tissues. No immunochemical differences have been detected between these two forms. Only one species of AO protein has been observed following analysis by double diffusion plates, rocket immunoelectrophoresis or Western blotting. Double diffusion plates loaded with mustard extracts exhibiting no AO activity (from dry seeds) and extracts containing high activity (from FR light-treated cotyledons) showed complete fusion of precipitin bands suggesting that AO populations in the two tissues are immunochemically identical. The ELISA measurements presented here therefore appear to represent the total population of AO (active ÷ inactive) in the extracts. Newbury and Smith [7] reported that the concentration of AO protein measured using rocket immunoelectrophoresis was the same in cotyledons held in darkness and those treated with FR light. The results obtained here using the more sensitive ELISA protocol indicate that the total amount of AO protein falls with time in both control and FR light-treated cotyledons, and that samples extracted after illumination with FR light for 36 h contain less AO protein than samples extracted from dark controls. Because 36 h FR leads to an increase in fresh weight of approximately 15% over dark controls, the difference in amounts of AO in dark- and FR-treated samples would appear slightly smaller in Fig. lb if the results were expressed on a 'per cotyledon pair' basis; however, the amount of AO in FR-treated cotyledons would still be significantly lower than in dark controls after the 72-h time point. One possible reason for the FR light-induced

decrease in AO protein concentration is an increase in the rate of degradation of this enzyme in illuminated cotyledons. The rate of decrease of AO protein concentration was faster than the rate of decrease of total soluble proteins in FR light-treated cotyledons, suggesting that an AO-specific protease may be present. There have been other proposals that enzyme-specific proteases exist in plants. For instance, a nitrate reductase inactivator protein [19-21] was found to be a protease. However, inactivators of nitrate reductase in rice [22] and soybean [23] showed no proteolytic activity. It was proposed that the maize inactivator was not specific for the enzyme, but that the susceptibility of nitrate reductase to proteolytic attack had created an illusion of a specific protease [24]. A similar situation may exist in mustard cotyledons with AO either being susceptible to a relatively non-specific protease produced in response to FR light, or FR light inducing a change in AO which renders it more susceptible to a pre-existing protease. A second possible reason for the decline in AO concentration in FR light-treated cotyledons is that the rate of AO synthesis is decreased. This model is in direct opposition to the proposal that the FR light-induced increase in AO activity is due to an increase in the rate of synthesis of the enzyme. The in vivo labelling data are also inconsistent with the hypothesis that the increase in AO activity is due to an increase in the rate of synthesis of AO protein. The autoradiographs show that a 36 h FR light treatment led to a reduction, not an increase in the rate of incorporation of labelled amino acid into AO protein. Comparison of spot intensities at different dilutions suggests that the rate of incorporation is approximately half that encountered in cotyledons held in darkness. Care must be taken over the interpretation of in vivo labelling data because endogenous (unlabelled) methionine will compete with the ~S]methionine applied for incorporation into protein. The relative sizes of endogenous pools of methionine in different samples can therefore infieuence the specific radioactivity of newly-synthesised pro-

89 Table II. Cotyledons (1 g) from seedlngs grown in continuous darkness or, after 36 h, exposed to FR light, were painted with a solution containing [~S]methionine 4 h before harvest. They were then homogenised in 0.1 M phosphate buffer (pH 7.0) (final volume 4 ml), centrifuged at 120 000 x g for 45 rain, and samples subjected to precipitation with trichloroacetic acid before counting radioactivity incorporated. The results are derived from two independent experiments. Treatmentafler 36 h darkness

Exp. 1

Exp.2

Dark 12h 24 h 36h

78111 ± 957 103060 ± 3837 107801 ± 5951

80 240 ± 1478 84 614 ± 5269 98412 ± 4240

80 690 ± 1563 85 607 _ 5543 120 136 ± 6564

79 551 ± 740 98 050 _ 4893 113 464 ± 5358

FR light 12 h 24 h 36 h

teins. This pool size could be altered by a FR light-induced change in the rate of amino acid synthesis. Although nitrate reductase and glutamine synthetase, both important enzymes in the pathway leading to amino acid synthesis, have been shown to be under phytochrome control [25,26], it has recently been demonstrated that the rate of amino acid synthesis in mustard cotyledons is identical in darkness and FR light [27,28]. An alternative process that can alter the level of available amino acids in cotyledon tissue is their release following the degradation of storage proteins. The results obtained in this study show that there is a significant hu < 0.05) decrease in the concentration of total soluble cotyledon protein following 36 h FR light treatment compared to dark controls; this suggests that there is a FR light-induced increase in protein degradation, as previously reported by Hacker [29], and hence an increase in the unlabelled amino acid pool. However, in our experiments FR light had no effect on the rate of incorporation of labelled methionine into total protein (Table II) and this is in agreement with the results of Tobin [30]. Hence, there is no evidence that the lower rate of incorporation of labelled precursor into AO observed following exposure of cotyledons to FR light was due to an effect on the endogenous pool of methionine in the cotyledons. The simplest explanation for these results is

that AO is post-translationally activated. However, the existence of post-translational regulation is difficult to prove directly because of the large range of mechanisms by which this could be achieved [31]. Among the many possibilities in this case is control of catalytic activity by a change in the amount of copper present in the enzyme. AO has been reported to contain either 1 0 - 1 2 [32] or 8 [33] atoms of copper per holoenzyme. Changes in the activity of AO have been reported as a result of altered availability of this element in two recent reports. Copperdepleted pea leaves have been shown to contain lower AO activities than controls [34] and AO activity in cultured Cucurbita cells has been shown to be influenced by the amount of copper present in the medium [35]. However, many other possible mechanisms of post-translational regulation exist; at present it is only possible to state that phytochrome does not appear to regulate AO activity by altering its rate of synthesis. References 1 2

3

P. Schopfer, Phytochrome control of enzymes. Annu. Rev. Plant Physiol., 28 (1977) 2 2 3 - 252. M. Van Poucke, F. Barthe and H. Mohr, Phytochromemediated induction of ascorbic acid oxidase in mustard seedlings. Naturwissenschaften, 56 (1969) p. 417. H. Drumm, K. Bruning and H. Mohr, Phytochromemediated induction of ascorbic oxidase in different organs of a dicotyledonous seedling (Sinapis alba L.). Planta, 106 (1972) 259-- 267.

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