Planta (Berl.) 126, 111--117 (1975) 9 by Springer-Verlag 1975
Control of Chlorophyll Synthesis by Phytochrome I. The Effect of P h y t o c h r o m e on the F o r m a t i o n of 5-aminolevulinate in Mustard Seedlings* M. Masoner and H. Kasemir BiologicM Institute II, University of Freiburg i. Br., SchanzlestraBe 9-11, D-78 Freiburg i. Br., Federal Republic of Germany Received 20 May; accepted 1 July 1975 Summary. Treatment of mustard (Sinapis alba L.) seedlings with levulinate leads to the inhibition of chlorophyll synthesis and causes the accumulation of 5-aminolevulinate which is only formed in light. A stoichiometric relationship exists between the extent of inhibition of chlorophyll synthesis and 5-aminolevulinate accumulation. The formation of 5-aminolevulinate in continuous white light is increased by pre-irradiation. The effect of the preirradiation can be fully attributed to phytochrome. Under various light conditions the rate of 5-aminolevulinate formation in lcvulinate-treated seedlings is similar to the rate of chlorophyll accumulation in seedlings not treated with levulinate. This result supports the hypothesis that the phytochrome-controlled chlorophyll accumulation is regulated at the level of the formation of 5-aminolevulinate.
Introduction The light mediated chlorophyll (Chl) accumulation in mustard seedlings is controlled b y physiologically active phytochrome (Pfr) in two ways [15]. Pfr eliminates the lag phase and increases the steady-state rate of Chl accumulation. While the effect on the lag phase is a relatively fast process (occurring within less than 3 h) the effect on the rate requires at least 12 h to manifest itself. J a b b e n et al. [13] have shown t h a t the increase in the rate of Chl accumulation is due to a corresponding increase in the rate of protochlorophyll(ide) (PChl) regeneration. The question remains how does Pfr control the formation of PChl ? I t is tempting to assume t h a t Pfr acts through an increase of the activity of the rate-limiting enzyme(s) in the biosynthetic sequence leading to PChl. The k e y reaction in the regulation of Chl synthesis seems to be the formation of 5-aminolevulinate (ALA), the first specific step in the Chl biosynthetic pathway. This idea is based on inhibitor experiments and on results obtained by feeding ALA to plant tissues [6, 8, 9, 20, 29]. The source of ALA in plants is presently unknown. I n animal tissues, bacteria and yeast ALA is formed b y the condensation of sueeinyl CoA and glycine catalysed by ALA synthetase [14]. I n greening plants, however, there is no unequivocal evidence for the presence of ALA synthetase. Recently alternative routes of ALA biosynthesis have been suggested by Hedley and Stoddart [12], Gassman et al. [10] and Beale and Castelfraneo [4]. * Abbreviations: Chl=ehlorophyll(ide) a; PChl=protochlorophyll(ide); Ffr=far-red absorbing form of the phytochrome system; ALA=5-aminolevulinate.
112
l~. l~Iasoner and H. Ka~emir
Irrespective of the n a t u r e of the origin of A L A its f o r m a t i o n can be m e a s u r e d b y feeding the p l a n t s l e v u l i n a t e (LA), a competitive i n h i b i t o r of the A L A m e t a b olizing e n z y m e A L A d e h y d r a t a s e [21]. A p p l i c a t i o n of this c o m p o u n d to the growth m e d i u m of Chlorella a n d Euglena [1, 2, 23] a n d to higher p l a n t s [3, 4, 11, 26] causes the i n h i b i t i o n of Chl synthesis a n d leads to the a c c u m u l a t i o n of ALA. Some authors [1, 3, 11, 26] have shown a v e r y similar time course of the a c c u m u l a t i o n of Chl i n etiolated p l a n t s w i t h o u t L A a n d of f o r m a t i o n of A L A after L A application. However, so far no a t t e m p t has been m a d e to d e t e r m i n e w h e t h e r s u c h a relationship also exists u n d e r the influence of different light pretreatments. I n the present s t u d y the influence of Pfr on A L A f o r m a t i o n i n m u s t a r d seedlings has been investigated. W e w a n t e d to know (a) whether Pfr induces a n increase i n A L A f o r m a t i o n i n seedlings t r e a t e d with LA to the same e x t e n t as it increases Chl a c c u m u l a t i o n i n seedlings w i t h o u t LA a n d (b) whether Pfr acts as quickly u p o n the A L A forming system as it does u p o n Chl a c c u m u l a t i o n . The d a t a reported establish a close relationship b e t w e e n the Pfr m e d i a t e d regulation of A L A f o r m a t i o n a n d Chl a c c u m u l a t i o n .
Materials and Methods Germination Conditions. Standard techniques for photomorphogenic research with mustard seedlings were used [17]. The mustard seeds (2inapis alba L.) were purchased in 1971 from Asgrow Company (Hamburg, Germany). The seedlings were grown at 25.0 ~=0.2~C in the dark. Preirradiations were performed with a standard far-red source (emission maximum at 740 nm, band width 123 nm, irradiance 3.5 Wm-2) or with monochromatic light (irradiance 7 Wm-~) obtained from a Leitz projector (Prado 500) modified according to Mohr and Schoser [19]. As far as the phytochrome system in the mustard seedling is concerned the standard far-red source is equivalent to the wavelength 718 [22, 25]. This far-red light only leads to traces of Chl [16]. The following filters were used to adapt the Leitz projector: Far-red light--Schott (Mainz, Germany) AL interference filter ('~max756 nm, band width 20nm), Red light-Plexiglas PG 501/3 (l~5hm & Haas, Darmstadt, Fed. Rcp. Germany). Incubation. Under a green safe-light 60-h old mustard seedlings were floated in the germination boxes for 5 rain in 200 ml of the appropriate solutions (LA in 10 mM K-phosphate buffer or only K-phosphate buffer) at pH 7.0. Then the solutions were discarded and after a further 5 min in the dark the seedlings were irradiated with fluorescent white light (Osram tubes, alternating L 40 W/5 and L 40 W/25) at an illuminanee of 7000 lx. LA was purchased from Fluka A.G., Buchs, Switzerland. Extraction and Determination o] A L A and ChI. After various periods of time the cotyledons of 40 seedlings were cut and homogenized in 4 ml ice-cold 0.1n trichloroacetic acid with a Potter-Elvejem Homogenizer (Braun, Melsungen, Germany). The homogenate was centrifuged at 40000 • g for 15 rain. The ALA containing supernatant was mixed with 1 M Na acetate and aeetylacetone (100~ C, 10 min) and the resulting ALA-pyrrole solution was added to the same volume of modified Hg-Ehrlich reagent. After 10 min the optical density was read at 555 mn in a Beckman DB-G spectrophotometer against a control which had been similarily treated but without acetylacetone. In this way disturbances caused by anthocyanin absorption were avoided. The concentration of ALA was calculated using a molar extinction coefficient of 64• 1tool-1 cm-1 [30]. The identity of ALA in the extract was established by paper chromatography. Extraction and determination of Chl have been described previously [15]. The average values given in figures and tables are based on 5-10 independent parallels. The vertical bars in Fig. 2 represent standard errors.
Control of Chlorophyll Synthesis by Phytoehrome. I
113
6.s o
r
o
O
E c,
2.(:
Control of Chlorophyll Synthesis by Phytochrome I. The Effect of P h y t o c h r o m e on the F o r m a t i o n of 5-aminolevulinate in Mustard Seedlings* M. Masoner and H. Kasemir BiologicM Institute II, University of Freiburg i. Br., SchanzlestraBe 9-11, D-78 Freiburg i. Br., Federal Republic of Germany Received 20 May; accepted 1 July 1975 Summary. Treatment of mustard (Sinapis alba L.) seedlings with levulinate leads to the inhibition of chlorophyll synthesis and causes the accumulation of 5-aminolevulinate which is only formed in light. A stoichiometric relationship exists between the extent of inhibition of chlorophyll synthesis and 5-aminolevulinate accumulation. The formation of 5-aminolevulinate in continuous white light is increased by pre-irradiation. The effect of the preirradiation can be fully attributed to phytochrome. Under various light conditions the rate of 5-aminolevulinate formation in lcvulinate-treated seedlings is similar to the rate of chlorophyll accumulation in seedlings not treated with levulinate. This result supports the hypothesis that the phytochrome-controlled chlorophyll accumulation is regulated at the level of the formation of 5-aminolevulinate.
Introduction The light mediated chlorophyll (Chl) accumulation in mustard seedlings is controlled b y physiologically active phytochrome (Pfr) in two ways [15]. Pfr eliminates the lag phase and increases the steady-state rate of Chl accumulation. While the effect on the lag phase is a relatively fast process (occurring within less than 3 h) the effect on the rate requires at least 12 h to manifest itself. J a b b e n et al. [13] have shown t h a t the increase in the rate of Chl accumulation is due to a corresponding increase in the rate of protochlorophyll(ide) (PChl) regeneration. The question remains how does Pfr control the formation of PChl ? I t is tempting to assume t h a t Pfr acts through an increase of the activity of the rate-limiting enzyme(s) in the biosynthetic sequence leading to PChl. The k e y reaction in the regulation of Chl synthesis seems to be the formation of 5-aminolevulinate (ALA), the first specific step in the Chl biosynthetic pathway. This idea is based on inhibitor experiments and on results obtained by feeding ALA to plant tissues [6, 8, 9, 20, 29]. The source of ALA in plants is presently unknown. I n animal tissues, bacteria and yeast ALA is formed b y the condensation of sueeinyl CoA and glycine catalysed by ALA synthetase [14]. I n greening plants, however, there is no unequivocal evidence for the presence of ALA synthetase. Recently alternative routes of ALA biosynthesis have been suggested by Hedley and Stoddart [12], Gassman et al. [10] and Beale and Castelfraneo [4]. * Abbreviations: Chl=ehlorophyll(ide) a; PChl=protochlorophyll(ide); Ffr=far-red absorbing form of the phytochrome system; ALA=5-aminolevulinate.
112
l~. l~Iasoner and H. Ka~emir
Irrespective of the n a t u r e of the origin of A L A its f o r m a t i o n can be m e a s u r e d b y feeding the p l a n t s l e v u l i n a t e (LA), a competitive i n h i b i t o r of the A L A m e t a b olizing e n z y m e A L A d e h y d r a t a s e [21]. A p p l i c a t i o n of this c o m p o u n d to the growth m e d i u m of Chlorella a n d Euglena [1, 2, 23] a n d to higher p l a n t s [3, 4, 11, 26] causes the i n h i b i t i o n of Chl synthesis a n d leads to the a c c u m u l a t i o n of ALA. Some authors [1, 3, 11, 26] have shown a v e r y similar time course of the a c c u m u l a t i o n of Chl i n etiolated p l a n t s w i t h o u t L A a n d of f o r m a t i o n of A L A after L A application. However, so far no a t t e m p t has been m a d e to d e t e r m i n e w h e t h e r s u c h a relationship also exists u n d e r the influence of different light pretreatments. I n the present s t u d y the influence of Pfr on A L A f o r m a t i o n i n m u s t a r d seedlings has been investigated. W e w a n t e d to know (a) whether Pfr induces a n increase i n A L A f o r m a t i o n i n seedlings t r e a t e d with LA to the same e x t e n t as it increases Chl a c c u m u l a t i o n i n seedlings w i t h o u t LA a n d (b) whether Pfr acts as quickly u p o n the A L A forming system as it does u p o n Chl a c c u m u l a t i o n . The d a t a reported establish a close relationship b e t w e e n the Pfr m e d i a t e d regulation of A L A f o r m a t i o n a n d Chl a c c u m u l a t i o n .
Materials and Methods Germination Conditions. Standard techniques for photomorphogenic research with mustard seedlings were used [17]. The mustard seeds (2inapis alba L.) were purchased in 1971 from Asgrow Company (Hamburg, Germany). The seedlings were grown at 25.0 ~=0.2~C in the dark. Preirradiations were performed with a standard far-red source (emission maximum at 740 nm, band width 123 nm, irradiance 3.5 Wm-2) or with monochromatic light (irradiance 7 Wm-~) obtained from a Leitz projector (Prado 500) modified according to Mohr and Schoser [19]. As far as the phytochrome system in the mustard seedling is concerned the standard far-red source is equivalent to the wavelength 718 [22, 25]. This far-red light only leads to traces of Chl [16]. The following filters were used to adapt the Leitz projector: Far-red light--Schott (Mainz, Germany) AL interference filter ('~max756 nm, band width 20nm), Red light-Plexiglas PG 501/3 (l~5hm & Haas, Darmstadt, Fed. Rcp. Germany). Incubation. Under a green safe-light 60-h old mustard seedlings were floated in the germination boxes for 5 rain in 200 ml of the appropriate solutions (LA in 10 mM K-phosphate buffer or only K-phosphate buffer) at pH 7.0. Then the solutions were discarded and after a further 5 min in the dark the seedlings were irradiated with fluorescent white light (Osram tubes, alternating L 40 W/5 and L 40 W/25) at an illuminanee of 7000 lx. LA was purchased from Fluka A.G., Buchs, Switzerland. Extraction and Determination o] A L A and ChI. After various periods of time the cotyledons of 40 seedlings were cut and homogenized in 4 ml ice-cold 0.1n trichloroacetic acid with a Potter-Elvejem Homogenizer (Braun, Melsungen, Germany). The homogenate was centrifuged at 40000 • g for 15 rain. The ALA containing supernatant was mixed with 1 M Na acetate and aeetylacetone (100~ C, 10 min) and the resulting ALA-pyrrole solution was added to the same volume of modified Hg-Ehrlich reagent. After 10 min the optical density was read at 555 mn in a Beckman DB-G spectrophotometer against a control which had been similarily treated but without acetylacetone. In this way disturbances caused by anthocyanin absorption were avoided. The concentration of ALA was calculated using a molar extinction coefficient of 64• 1tool-1 cm-1 [30]. The identity of ALA in the extract was established by paper chromatography. Extraction and determination of Chl have been described previously [15]. The average values given in figures and tables are based on 5-10 independent parallels. The vertical bars in Fig. 2 represent standard errors.
Control of Chlorophyll Synthesis by Phytoehrome. I
113
6.s o
r
o
O
E c,
2.(:
