Plant Science Letters, 4 (1975) 89--96 89 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

LIGHT-STIMULATED ACCUMULATION OF PROTOCHLOROPHYLLIDE IN LEAVES OF DIFFERENT AGES TREATED WITH 5-AMINOLEVULINIC ACID

CHRISTER SUNDQVIST, BRITA ODENGARD and GUDRUN PERSSON

Department of Plant Physiology, University of G~teborg, Carl Skottsbergs Gata 22, S-413 19 G~teborg (Sweden)

(Received July 17th, 1974)

SUMMARY

Chlorophyll biosynthesis during irradiation of dark-grown wheat seedlings, 6, 8 and 12 days old, as well as the regeneration of protochlorophyllide (PChlide) after a short flash, was found to be age-dependent. The same was found for the accumulation of protochlorophyllide during treatment with 8-aminolevulinic acid (ALA). The accumulation of protochlorophyllide was enhanced by irradiation of the dark-grown leaves. A short irradiation for 5 rain was enough to cause the enhancement provided the irradiation was fol- lowed by a period of 5 h in the dark before the treatment with ALA. The enhancement is supposed to be caused by the action of the phytochrome system. The specific activity of 6-aminolevulinate dehydratase (ALAD) showed only a slight variation in leaves of different ages. During irradiation t h e specific activity increased about 1.6 times. This increase was clearly age- dependent and coincided with the most rapid phase in chlorophyll accumu- lation. The increase in activity was significant but cannot account for any control mechanism in chlorophyll biosynthesis.

INTRODUCTION

The formation of chlorophyll in dark-grow ~eedlings has been shown to depend on the age of the plant [1]. Age dependence ought to be reflected in the changes of specific activity of the enzymes in the first part of the biosyn- thetic chain to chlorophyll as well as in the accumulation of the end product, since no intermediates are found to accumulate during chlorophyll formation [4]. Nadler [10] has shown that the main control of chlorophyll biosynthesis

Abbreviations: ALA, 6-aminolevuUnic acid; ALAD, 8-aminolevulinate dehydratue; ALAS, 6-aminolevulinate synthetase; Chlide, chlorophyllide; PBG, porphobilinogen; PChlide, proto- chlorophyllide; TCA, trichloroacetic acid.

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is by the formation of ALAS, which was shown to have a half-time of 1.5 h. The lag phase in chlorophyll formation can be decreased by a preirradiation

of dark-grown leaves [22]. This increase of the rate of chlorophyll formation is especially evident in older leaves. The mechanism behind this action of light on chlorophyll formation is not known. Kasemir [6] has recently sug- gested that the increase in chlorophyll formation is due to phytochrome ac- tion in at least two different ways. It has earlier been suggested that this in- crease is caused by an effect of light on ALAS [10, 13] or by a prevention of photooxidation of the chlorophyll due to formation of screening pigments [22]. The fact that leaves accumulate PChlide when they are treated with ALA provided the possibility of investigating whether the phytochrome ef- fect is on that part of the chlorophyll biosynthetic chain localized between ALA and PChlide.

The enzyme ALAD (5-aminolevulinate hydro-lyase, EC 4.2.1.24) is one of the enzymes between ALA and PChlide. A careful purification and detailed study of the enzyme was performed by Schneider [11, 12]. Only a few re- sults have been published concerning the specific activity of ALAD in crude extracts from plants during their chlorophyll formation. Steer and Gibbs [17] correlated the changes in specific activity of ALAD during irradiation of etiolated beans with their chlorophyll accumulation. Smith [16] has studied the specific activity of ALAD in dark-grown leaves of different ages, and Schneider [11, 13] correlated the activity of ALAD with chlorophyll formation in tissue cultures of Nicotiana.

In the present study the change in specific activity (nmoles per h per mg protein) of ALAD in dark-grown leaves has been followed and this change in activity has been correlated with PChlide synthesis, with and without ALA addition, as well as chlorophyll accumulation in leaves of different ages.

MATERIAL AND METHODS

Plant material and irradiation The experiments were performed with wheat leaves (Triticum aestivum

L. cv. Starke, Weibull), grown in darkness according to Virgin [21]. After 6, 8 and 12 days, counted from the time for imbibition, the plants were irradiated. During the irradiation samples were taken at different hours during two days. The irradiation source was four fluorescent lamps Phflips TL 20W/32 De-Luxe which gave an energy of 6 W/m 2 at the level of the leaves. A water filter, 10 cm thick, was inserted between the lamps and the leaves. The temperature in the illumination chamber was 25 + 1 °.

Enzyme preparation ALAD was prepared according to the method of Steer and Gibbs [17]

with the exception that polyclar was not used. About 1.5 g of wheat leaves was homogenized in a mortar in 10 ml of a mixture containing 2 ml of each

M Tris--HC1 pH 7.2, 0.5 M 2-mercaptoethanol, 0.5 M MgC12, 0.5 M sodium ascorbate, 15 mM O-phenantrolin.

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The homogenate was pressed through 8 layers of cheesecloth (36 meshes per cm). The filtrate was centrifuged at 2000 g for 10 rain. The supernatant was used for the enzyme assay. The pellet showed no activity. The enzyme assay consisted of 0.5 ml 24 mM ALA, 1.5 ml enzyme and 1 ml 0.3 M Tris-- HC1 pH 7.8. Each sample was incubated at 25 ° in a closed flask, shaken on a waterbath for 1 h. The reaction was found to be linear up to 2 h. A control without ALA was incubated similarly. The reaction was stopped with TCA and HgCI2 according to Shemin [15]. The precipitated protein was spun down and the supernatant was used for determination of PBG and porphyrins.

PBG and porphyrin determinations The PBG was determined with the modified Ehrlich reagent 0.1 gp-

dimethylaminobenzaldehyde in 1 ml 70% perchloric acid and 4.2 ml glacial acetic acid [20]. 1 ml of the supernatant was mixed with 1 ml of the reagent. The absorbance was read at 555 nm 15 min after the mixing. The molar ab- sorption 62 • 1.0 -~ M -1 cm -1 was used [15]. For correction for the amount of PBG transformed to other porphyrins, the method of Schneider [11] was used. 1 ml of the sample was mixed with I ml 5 M HC1 and the absorbance was measured at 406 nm, using the molar absorption 530 - 103 M -1 cm -~ [3]. Every sample was made in duplicate and the difference in absorbance between the experimental and a control (minus ALA) was used for the cal- culations. Each point in the figures represents the mean of at least three such determinations. The specific activity for ALAD was calculated as nmoles PBG + porphyrins formed per h per mg protein. Protein was measured by the Folin method according to Lowry et ah [9] and chlorophyll was measured in 80% acetone according to Anderson and Boardman [2]. In vivo measurements of PChlide resynthesis and ALA treatment were performed according to Sund- qvist [18]. The regeneration of PChlide was measured as increase in absor- bance at 650 nm. The accumulated PChlide after treatment with ALA was measured in acetone extracts according to Sundqvist [19].

RESULTS

The age dependence of chlorophyll formation in our material is shown in Fig.1.6-day-old leaves showed a short lag phase of about one hour and'the most rapid phase of chlorophyll formation was found after about 7--9 h of irradiation. In 8- and 12-day-old leaves there was a longer lag phase and the most rapid phase in chlorophyll formation was found after 11--13 (8 days) and 13--15 (12 days) h, resp. The final level of chlorophyll formed was also de- pendent on the age of the leaves. The 6-day-old leaves had a final level of chlorophyll content 12% higher than that of the 8~lay-old leaves and these in turn had 14% more than the 12-day-old leaves.

The age dependence of chlorophyll formation was compared with the age- dependent regeneration of PChlide6s0 after an electronic flash had been given to the dark-grown leaves (Fig.2). This figure shows that the older the leaves the longer the lag phase in PChlide6s0 regeneration.

9 2

800

o, " 600 ÷ c l

>- 400 " I -

EL. O n, ,

q T 200

/

/

/

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12 O• 18 2~4 hours 4~8 Fig.1. Ch lorophy l l f o r m a t i o n in leaves o f d i f ferent age. Dark-grown leaves were irradiated w i th f luorescent l ight for 4 8 h. The c o n t e n t o f c h l o r o p h y l l a and b is s h o w n for leaves o f varying age: o, 6 days old; ~, 8 days old; D, 12 days old.

Z O

c~

T A A = 0 0 4

ii 1111111111111 . 1"1"I""

0 30 60 90 120 min Fig.2. Regenera t ion o f PChl ide in leaves o f d i f f erent age. The regenerat ion o f PChl ide in dark-grown leaves after a short irradiat ion was measured as the increase in absorbance at 6 5 0 nrn. , l eaves 6 days old; - - - , leaves 8 days o ld; . . . . , leaves 12 days old.

This age dependence can also be found in the activity of ALAD as shown in Fig.3. The results showed that ALAD had a certain activity in dark-grown leaves and that there was a small difference in the activity between the leaves of different ages. During the greening period, however, an activation of the enzyme took place. This activation was age~lependent with an opt imum for 6-day-old leaves after 6 h of irradiation. For the 8~lay-old leaves this optimal activity was reached after 8 h, but for the 12-day-old leaves not until after 12 h of irradiation. These figures should be compared with the most rapid phase of chlorophyll formation, which can be seen from the results presented in Fig.1.

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7

Fig.3. Specific activity of ALAD during irradiation of dark-grown leaves. The specific activity of ALAD was measured during irradiation of dark-grown leaves for 48 h: o, 6 days old; 4, 8 days old; Q, 12 days old. Inset: Specific activity of ALAD in shortly irradiated leaves. Leaves, 8 days old, were irradiated for only 30 rain and then placed in the dark. The activity of ALAD during the following 24 h in the dark was followed.

The increase in the activity of ALAD persisted approximately for as long as the Chl accumulation took place but was then followed by a slight decrease. The activity decreased most slowly in the ll-day-old leaves. The totalchange in activity was approximately 1.6 times the initial activity of ALAD in dark- grown leaves. The activity of ALAD in the fully greened leaves was only slightly lower.

Leaves treated with ALA accumulated PChlide in excess of that originally present. This accumulation was age<lependent as shown in Table I. When leaves had been irradiated for 10 rain with fluorescent light and were then treated with ALA for 3 h the 6-day-old leaves accumulated about 10 times the amount accumulated by 12~lay-old leaves treated in the same way. The accumulation of PChlide in partly greened leaves was increased as compared to dark-grown leaves irradiated for 10 rain only. Thus, as seen in Table I, the younger leaves irradiated for 13 h accumulated PChlide about 1.8 times faster than the leaves irradiated for only 10 rain. Leaves, 12 days old, show a more pronounced acceleration in PChlide accumulatzon. The leaves irradiated for 13 h accumulated PChlide about 10 times as fast as those irradiated for only 10 rain. The high rate of PChlide accumulation persisted even for leaves irradiated for 48 h.

It is shown in Table II that itwas not necessary with a continuous irradiation to achieve the increase in PChlide accumulation. In fact a short light impulse of 5 rain was enough. However, a dark period of about 5 h must pass between the light impulse and the ALA treatment to achieve any increase in accumu- lated PChlide. Under these circumstances the amount accumulated in 8<lay- old leaves was more than doubled after the 5-h dark period as compared to leaves ALA-treated immediately after the short light glimpse. In leaves, 12

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TABLE I

PChlide ACCUMULATED AFTER DIFFERENT PERIODS OF IRRADIATION

Dark-grown leaves of different ages were irradiated for different periods of time. Imme- diately after the irradiation the leaves were kept floating on a 20 mM solution of ALA in a phosphate buffer fo~ 3 h. The PChlide accumulated was estimated and is given in the table as pg PChlide per g fresh matter. Standard errors are indicated.

Age of leaves Time of irradiatio~ 6 days 8 days 12 days

10 min 20.2±4.4 14.0+3.3 1.9+0.9 5 h 29.0+3.3 27.3±4.1 15.0±3.1 13 h 35.2±4.5 26.1+3.2 19.0±3.1 48 h 30.4±3.5 30.9+6.1 16.7±3.1

TABLE II

PChlide ACCUMULATED AFTER A PREIRRADIATION

Dark-grown leaves, 8 or 12 days old, were irradiated for 5 min with white light and then kept in the dark for different periods of time before they were treated with'a solution of 20 mM ALA in phosphate buffer for 3 h. The PChlide accumulated is given as/~g per g fresh matter. Standard errors are indicated.

Time in the dark after irradiation

Age of leaves

8 days 12 days

5 min 15.1+3.0 3.0+0.9 5 h 38.1+2.3 13.2+3.1 13 h 41.2+2.6 13.4+3.2

days old, the a m o u n t a ccumula t ed was a b o u t 4 t imes larger af ter a dark peri- od o f 5 or 13 h as c o m p a r e d to the a m o u n t in leaves t r ea ted wi th A L A direct ly af ter the irradiat ion.

Fig. 3, inset, shows tha t the ac t ivat ion o f A L A D can take place af ter on ly a shor t i r radiat ion o f 30 min. In these exper iments a rapid decrease in the act ivi ty t o o k place immedia te ly af ter irradiat ion. This migh t indicate t h a t A L A D to some ex ten t is an e n z y m e the act ivi ty o f which is rapidly increasing with increasing c o n c e n t r a t i o n o f the substrate .

DISCUSSION

The b iosynthes is o f ch lo rophy l l in dark-grown leaves o f higher plants is mainly con t ro l led by the p h o t o r e d u c t i o n o f PChlide t o Chlide and by the

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action of ALAS [10, 13]. Other regulatory mechanisms have also been pro- posed, e.g. reactions governed by phytochrome [6, 14] and by enzymes other than ALAS in the biosynthetic pathway as is especially the case in callus tissues [14].

Chlorophyll formation in greening leaves is an age-dependent process [ 1]. It is known that the regeneration of PChlide after its phototransformation to Chlide is also age-dependent, the amount of PChlide and the different forms of PChlide present varying with age [7]. In which way the aging acts on any other component of this biosynthetic chain to chlorophyll is, however, quite unknown. Studying the changes of plastid RNA and different enzymes in the plastid during the growth of barley leaves in darkness, Smith [16] found an increase in plastid ribosomal RNA and fraction 1 protein, as well as increase in the activity of ALAD during the period of active growth. This investigation was, however, not extended to the aging process.

The length of the lag phase increases with the age of the leaves [ 1 ]. As shown in the present paper the activation of ALAD was also slower in older leaves, but the degree of activation is about the same for all ages. The maxi- mum obtainable increase in the specific activity was about 1.6 times the orig- inal. This value can be compared with the value of 1.2--1.6 times for the ac- tivation of ALAD in Rhodopseudomonas when they are transferred to con- ditions favouring chlorophyll formation [8]. Compared with young leaves (6 days) the oldest leaves (12 days) did not show any appreciably lower activity in the dark, but maximum activity was not reached until after 12 h of irra- diation, whereas for the young leaves maximum activity was reached already after 6 h irradiation. The accumulation of PChlide during t reatment with ALA was also shown to be an age-dependent process. This does not tally with what should be expected as far as the activity of ALAD is concerned as this enzyme showed only a slight decrease in the 12-day-old leaves. This fact might indicate a slower uptake of ALA in the old leaves than in the young ones. It is unlikely that another enzyme in the chlorophyll biosynthetic chain is age-dependent as no sign of accumulation of intermediary porphyrins was found in in vivo spectra of the leaves.

Continuous irradiation of normal leaves increased the chlorophyll forma- tion rate. Also the accumulation of PChlide formed during ALA treatment was increased in partly greened leaves. This increase was age-dependent irj such a manner that the 12-day-old leaves seemed to be most affected by the irradiation. The increase in accumulated PChlide could be caused by activation of at least two different photosensitive systems. One is the phytochrome sys- tem as this system has been shown to have an influence upon chlorophyll formation [22]. The other is the photosynthetic system, as the production of energy rich substances can activate especially the older leaves. An increase of PChlide accumulation due to the action of photosynthetic products should need a continuous irradiation over a longer period, because it takes several hours before the dark-grown leaves will develop a functioning photosynthetic apparatus during the greening period [5], whereas an activation through the

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p h y t o c h r o m e sys tem on ly needs a shor t i r radia t ion [ 20 ] . Th e results pre- sen ted in Table II c o m p a r e d t o t hose in Table I, show tha t , a t least in the old leaves, t he ac t iva t ion t h rough pho tosyn thes i s might be involved. Nevertheless , the main e f f ec t cou ld be a c c o u n t e d for by t he p h y t o c h r o m e sys tem. Th e reason fo r this conc lus ion is the fac t t ha t the re was an increased accu m u la t i o n of PChlide a f t e r a shor t i r radiat ion, b u t this increase was on ly sl ightly l ower in the old leaves if t h e y had been c o n t i n u o u s l y i rradiated. In the y o u n g e r leaves the resul t ob t a ined was reversed. The p ro p o sed p h y t o c h r o m e co n t ro l over PChlide accumula t i on in ALA- t rea ted leaves is u n d e r f u r t h e r investiga- t ion.

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