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Changes in leaf phosphate status have only small effects on the photochemical apparatus of sugar beet leaves
Plant Science, 50 (1987) 49--55
49
Elsevier Scientific Publishers Ireland Ltd.
CHANGES IN LEAF PHOSPHATE STATUS HAVE ONLY SMALL EFFECTS PHOTOCHEMICAL APPARATUS OF SUGAR BEET LEAVES
ON THE
JAVIER ABADIA*, I. MADHUSUDANA RAO and NORMAN TERRY** D e p a r t m e n t o f Plant and Soil Biology, University o f California, Berkeley, CA 94 720 (U.S.A.)
(Received September 29th, 1986) (Revision received January 3rd, 1987 ) (Accepted January 5th, 1987 )
The effects of orthophosphate (Pi) on the structure and function of thylakoid membranes was investigated by monitoring changes in thylakoid polypeptides, pigment-proteins, cytochromes (Cyt), atrazine-binding sites, leaf fluorescence characteristics, photosynthetic quantum yield and electron transport (including Cyt f dark reduction) in leaves of sugar beet (Beta vulgaris L. cv. F58-554H1 ) plants cultured hydroponically with different levels of Pi supply. Low-P treatment (i.e. low external Pi supply) decreased soluble leaf phosphorus (P) by 78% and total plant dry matter by 77%, and decreased light-saturated photosynthetic CO2 fixation by 32%. The results show that low-P treatment had only minor effects on thylakoid membrane composition and function. There was some evidence that low-P increased the amounts of photosystem (PS) I and light harvesting chlorophyll-protein complexes and of the Cyt f and bs63, relative to the amounts of PS II reaction center complex, the number of atrazine binding sites and Cyt bs59. Low-P treatment had very little effect, if any, on photosynthetic quantum yield and only small effects on chlorophyll fluorescence. Key words: thylakoid membrane; chlorophyll fluorescence; leaf phosphate; sugar beet
Introduction *Recipient of a postdoctoral fellowship from the Spanish Superior Council of Scientific Re,search. Present address: Estacion Experimental de Aula Dei, Apartado 202, Zaragoza, Spain. **To whom correspondence should be sent. Abbreviations: Chl, chlorophyll; CF, coupling factor; Cyt, cytochrome; DCIP, 2,6-dichlorophenol-indophenol; DMBQ, 2,5-dimethyl benzoquinone; FI, emission peak related to photosystem I; FII , lowwavelength emission peak related to photosystem II; F0, initial chlorophyll fluorescence yield; Fro, maximum chlorophyll fluorescence yield; F v = F m -- Fo, variable chlorophyll fluorescence yield; LHCP, light harvesting chlorophyll a/b protein; LIDS, lithium dodecyl sulfate; MV, methyl viologen; PAR, photosynthetically active radiation; Pi, orthophosphate; PS, photosystem; Q, first quinone-type acceptor of photosystem II; TMBZ, 3,3',5,5'-tetramethylbenzidene.
In r e c e n t years there has been m u c h interest in the idea t h a t the level o f o r t h o p h o s p h a t e (Pi) in p l a n t tissues m a y have a role in the r e g u l a t i o n of various aspects of p h o t o synthesis [ 1 - - 7 ] . O n e a p p r o a c h to the s t u d y of the role of Pi in p h o t o s y n t h e s i s in vivo is to v a r y Pi levels in leaves n u t r i t i o n a l l y a n d t h e n m o n i t o r the c h a n g e s which o c c u r in various aspects o f p h o t o s y n t h e s i s [ 5 - - 8 ] . In a s t u d y of this kind, B r o o k s [5] observed that P nutrition affected thylakoid membranes; she f o u n d t h a t low-P t r e a t m e n t diminished p h o t o s y n t h e t i c quantum yield a n d caused changes in 7 7 ° K fluorescence. R a o et al. [8] s h o w e d t h a t low-P t r e a t m e n t
0618-9452/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
50 affected the light scattering and Chl fluorescence characteristics of leaves and that these effects were reversible when the Pi supply was increased. The objective of the present work was to further explore the effects of P nutrition on the structure and function of the thylakoid membranes. Materials and methods
Plant culture Sugarbeet plants (Beta vulgaris L. cv. F58554H1) were grown hydroponically with two different levels of phosphate in the culture medium in growth chambers at 25°C with an irradiance of 500 ~mol quanta photosynthetically active radiation (PAR) m -2 s-1 supplied over a 16-h photoperiod. Low-P and control plants were raised as described in Rao et al. [8]. Pi supply to low-P plants was increased by raising the P concentration in the culture medium [8]. Thylakoid isolation Thylakoids were obtained as described in Abadia et al. [9]. Thylakoid polypeptides and Chl-proteins Thylakoid polypeptides were separated in 10--20% lithium dodecyl sulfate (LIDS)polyacrylamide gels following the procedure of Nishio et al. [10]. The gels were stained with silver [11]. The separation of the pigment-protein complexes was performed according to the m e t h o d s described in Abadia et al. [9]. A trazine-binding sites The concentration of atrazine-binding sites was determined using the m e t h o d of Tischer and Strotmann [12] as modified by Vermaas et al. [13]. Cy toch romes The concentrations of cytochromes were determined spectrophotometrically using r o o m temperature difference spectra. Thylakoids were resuspended at 50 pg Chl/ml and
the spectra recorded using an Aminco DW-2C s p e c t r o p h o t o m e t e r in the split beam mode. C y t f and Cyt bss9 were determined from hydroquinone-reduced minus ferricyanideoxidized spectra while Cyt bs63 was determined from the dithionite-reduced minus ferricyanide-oxidized spectra. The final concentrations of hydroquinone and ferricyanide were 200 and 120 pM, respectively, and dithionite was added as a few solid grains. Cytochrome contents were calculated from the equations of Heber et al. [14].
Cytochrome f dark reduction Cyt f photooxidation and dark reduction were carried out as described in Ruhle and Wild [15]. For illumination, we used red light (Corning filter CS 2--64, 20--40 p m o l quanta m -2 s-1) or far red light (interference filter transmitting 710 nm with 10 nm bandwidth, 8 ~mol quanta m -2 s-l). The decrease in absorbance which occurs at 554 nm when leaf pieces are illuminated with red light is believed to be due to the p h o t o o x i d a t i o n of Cyt f [15]. When the light was turned off, Cyt f was reduced and the signal disappeared. The rates of Cyt f-dark reduction were similar, regardless of whether red light or 710 nm light were used. Photosyn thesis, photosyn the tic quan turn yield and electron transport The measurements of net photosynthesis and quantum yield of attached leaves were carried o u t as described in Terry [16]. Light-saturated rates of photosynthetic electron transport were measured polarographically at 25°C with a temperaturecontrolled, water-jacketed Rank Oxygen electrode connected to a strip chart recorder [17]. PS I-mediated electron flow, from ascorbate plus 2,6-dichlorophenol-indophenol (DCIP) to methyl viologen (MV), was determined by measuring oxygen uptake. The reaction mixture contained 30 mM Na4 P2 07 (pH 8.0), 5 mM MgC12, 10 mM NaC1, 0.2 mM MV, 2.5 mM NH4C1 as uncoupler, 1 mM Na-ascorbate, 0.1 mM DCIP, and chloroplast
51 membranes (20--30 ~g Chl ml-1). PS IImediated electron flow from water to DMBQ was determined by measuring oxygen evolution. The reaction mixture contained 50 mM HEPES (pH 7.6), 5 mM MgCI:, 10 mM NaC1, 1 mM EDTA, 0.25 mM 2,5-dimethyl benzoquinone (DMBQ), 2.5 mM NH4C1 as uncoupler, and chloroplast membranes (20-30 ug Chl ml-1).
and leaf discs were taken and immediately d r o p p e d into liquid N2. The leaf discs were ground to a p o w d e r in a mortar containing liquid N2 and 4 ml of water was added. An aliquot containing 5 pg chlorophyll (Chl) was placed in a precooled cuvette for fluorescence measurement. The illumination and collection of the fluorescence were carried o u t with a trifurcated fibre-optics system.
Chlorophyll fluorescence
Chlorophyll
Fluorescence induction curves at room temperature were obtained by illuminating leaf pieces with 10 W m -2 of blue light (Coming CS 4--96) and collecting the fluorescence through a 685 nm interference filter. Signal recovery and processing were implem e n t e d by a Hewlett-Packard 3437A digital voltmeter interfaced with an on-line HP86B computer. The kinetic analysis of the data was p e r f o r m e d by the computer and the results plotted on a HP7475A plotter. Leaf pieces were removed from leaves of plants taken from the growth chamber at the end of the dark period and illuminated with blue light at room temperature. On excitation of the sample, fluorescence at 685 nm rapidly increased to an initial level, initial chlorophyll fluorescence yield (F0), which represents the fluorescence level when the first quinonetype acceptor of PS II (Q) is maximally oxidized. There was then a slower fluorescence rise until a maximal level, maximum chlorophyU fluorescence yield (Fro), was attained (Q maximally reduced). The difference in the fluorescence yield at Fm and Fo is termed the fluorescence of variable yield, Fv [ 18]. The distribution of excitation energy between PS II and P S I was determined according to the m e t h o d s outlined in Percival and Baker [18] by measuring the fluorescence at 680 and 730 nm (10-nm bandwidth) for leaf pieces illuminated with 480-nm light (10-nm bandwidth). Chlorophyll fluorescence at 77 ° K was measured using the 'dry powder' technique of Weis [ 1 9 ] . Leaves were either dark- or light-adapted in growth chambers for 2 h
Chl a, Chl b, Total Chl (a + b) and Chl a/Chl b ratios were determined according to Arnon [20]. Results and discussion When sugar beet plants were subjected to low-P treatment for a period of 2 weeks, acid-soluble and total leaf P declined by 77--78% {Table I). By far the most important change physiologically in these low-P treated plants was the reduction in leaf growth: total leaf area in low-P plants was decreased by 80% compared to the controls {Table I). Total plant dry matter was diminished to a comparable extent (77% decrease with low-P, Table I). The photosynthetic rate was reduced to a much smaller extent (32% decrease compared to controls, Table I), the decrease due to low-P being greatest at light saturation and diminishing with lower light intensities (data n o t shown). It is clear from these results that the major effect of low-P treatment on the accumulation of p h o t o s y n t h a t e was to reduce the rate of expansion of the photosynthetic surface rather than to decrease photosynthesis/area. Other data show that low-P effects on certain aspects of photosynthesis, particularly the 'dark reactions', can be quite pronounced. F o r example, low-P diminishes the levels of Calvin cycle intermediates substantially [ 5,6,21] with ribulose-l,5-bisphosphate being decreased by as much as 90% [21]. Furthermore, low-P treatment can markedly change the activities of various photosynthetic
52 Table I. Effect of low-P treatment on certain growth, gas exchange and photochemical characteristics of 5-week-old sugar beet plants. Characteristics
Treatment Con~ol
Acid soluble leafP (% dry wt.) 0.41 Total leaf P (% dry wt.) 0.57 Leaf area/plant (cm 2) 2269 Total dry matter (g plant -1 ) 23.5 Photosynthesis/area (~ mol CO 2 m-: s -1) 30.3 Chl (nmol cm-:) in: chlorophyll-protein-1 8.6 LHC II 21.3 CPa 6.9 Cytochromes (pmol cm -2) Cyt f 71 Cyt bss 9 120 Cyt bs~~ 282 Atrazine binding sites (pmol cm -2 ) 273 Chl a/Chl b (mol/mol) 3.7 Chl (nmol cm -2 ) 47.5 Specific leaf dry wt. (rag cm -2) 3.65 Soluble leaf protein (rag cm -2) 0.74 Electron transport (umol O: cm-2 h-l) PS I 40.2 PS II 15.7 Photosynthetic quantum yield (~) 0.117 Cyt f dark reduction (A 554--A551 × 103/rain) 12.6 Room temperature fluorescence: Fv/Fm 0.63
Low-P 0.09 0.13 450 5.3 20.6 11.1 26.8 7.0 80 120 340 294 3.5 58.8 4.76 0.83 41.3 13.8 0.109 6.7 0.50
FI/Fn a
1 min 9 rain 77 ° K fluorescence:
0.69 1.0
0.78 0.91
0.87 1.0
0.89 0.92
FI/FI]b Dark Light
aThe values of FI/Fn found in the control after 9 rain of illumination were arbitrarily taken as 1.0. bThe values of FI/FI! for the control in the light were arbitrarily taken as 1.0.
e n z y m e s o f the s t r o m a a n d c y t o s o l [ 2 1 , 2 2 ] . The photochemical apparatus however, a p p a r e n t l y u n d e r g o e s m u c h less c h a n g e in r e s p o n s e t o low-P as is s h o w n below.
Effects of low-P on thylakoid membrane composition L o n g , d e n a t u r i n g gels ( L I D S p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ) s h o w e d t h a t t h e r e w e r e n o m a j o r c h a n g e s in t h e relative a m o u n t s o f the numerous p o l y p e p t i d e s resolved (Fig. 1), i.e. low-P t r e a t m e n t did n o t signific a n t l y c h a n g e the p o l y p e p t i d e c o m p o s i t i o n of the t h y l a k o i d m e m b r a n e . L o w - P treatm e n t did a p p e a r t o i n f l u e n c e the a m o u n t s of s o m e m e m b r a n e c o m p o n e n t s relative t o the a m o u n t o f PS II c o m p o n e n t s in the m e m b r a n e . A n a l y s i s o f the p i g m e n t - p r o t e i n s s h o w e d t h a t low-P t r e a t m e n t increased the a m o u n t s o f Chl a s s o c i a t e d w i t h light harvesting c h l o r o p h y l l a/b p r o t e i n ( L H C ) I I a n d CP1 relative to the a m o u n t o f C P a / C h l ( t h e c o m p l e x t h a t has b e e n s u p p o s e d t o c o n t a i n the PS II r e a c t i o n c e n t e r ) . CPa a n d L H C II increased b y a b o u t 2 6 - - 2 8 % ( o n an a r e a basis) w i t h low-P t r e a t m e n t while t h e a m o u n t o f CPa Chl was u n c h a n g e d ( T a b l e I). F u r t h e r m o r e , low-P t r e a t m e n t i n c r e a s e d the a m o u n t s of C y t f a n d C y t bs63 b y 13 a n d 21%, respectively, while the a m o u n t o f C y t bss9 ( o f t e n a s s o c i a t e d w i t h PS II) a n d the n u m b e r of a t r a z i n e b i n d i n g sites p e r area (believed to be a m e a s u r e o f the n u m b e r o f PS II r e a c t i o n centers} were u n c h a n g e d ( T a b l e I). Chl a / C h l b r a t i o s were ~slightly d e c r e a s e d b y low-P, p r o b a b l y b e c a u s e o f the increase in light h a r v e s t i n g Chl a / C h l b c o m p l e x e s . T o t a l C h l / a r e a was i n c r e a s e d w i t h low-P a l o n g w i t h an increase in the t o t a l a m o u n t of leaf tissue p e r area (see specific leaf w e i g h t a n d soluble leaf p r o t e i n , T a b l e I).
Effects of low-P on thylakoid function T h y l a k o i d m e m b r a n e f u n c t i o n was a f f e c t e d to o n l y a small e x t e n t b y low-P t r e a t m e n t . T h e r e was n o e f f e c t of low-P on P S I e l e c t r o n t r a n s p o r t a n d v e r y little e f f e c t o n PS II e l e c t r o n t r a n s p o r t (12% decrease w i t h low-P, T a b l e I). Q u a n t u m yield was u n a f f e c t e d by low-P t r e a t m e n t . In c o n t r a s t t o o u r results, B r o o k s [ 5] o b t a i n e d a significant r e d u c t i o n in q u a n t u m yield w i t h low-P t r e a t m e n t ; this m a y have b e e n d u e to the f a c t t h a t we used
53 Low -P
,.
F-- Control
i
MW Markers (KD) CPI
9 2 . 5 ---)
6 6 . 2 ----> aCF
/~CF PSTrreoction center opo PS1T internol ontenno opo
4 5 . 0 ---)
8CF Heine
stain
PS IT bonds Green bond "CP 2 9 " _ _ PSIT Green bond LHCP ~.PS]]. bonds LHCPopo Iopo LH CP opo
2 9 . 0 ---)
LHClopo LHCI opo
--
}PS~Tbonds
14.4 - ~
Fig. 1. Effect of low-P t r e a t m e n t on t h y l a k o i d p o l y p e p t i d e composition. Silver stained L i D S / T X - 1 0 0 / p o l y a c r y l amide gels were e m p l o y e d . The origin of the gels was at the top; gels were subjected to electrophoresis overnight at a c o n s t a n t p o w e r of 4 W. The p o l y p e p t i d e bands were identified by i m m u n o d e c o r a t i o n of western blots (Abadia et al,, unpublished results) or 3,3',5,5'-tetramethylbenzidene (TMBZ) stain [ 1 0 ] . Molecular weight markers are s h o w n at the e x t r e m e left.
54 a different m e t h o d to induce P-deficiency, i.e. we supplied small amounts of P to plants during their growth while Brooks added no P after withholding P. Low-P treatment appeared to affect the rates in vivo of Cyt f dark-reduction after p h o t o o x i d a t i o n with 710-nm light. Low-P t r e a t m e n t decreased the rates of Cyt f dark reduction (Table I). When these plants were resupplied with phosphate, the rates increased significantly within 5 h (data n o t shown). The rapidity of the recovery would seem to preclude major changes in membrane composition with respect to the C y t b--f complex under low-P treatment. Low-P t r e a t m e n t had some small effects on chlorophyll fluorescence. Fv/Fm ratios measured on leaf pieces at room temperature decreased by 21% in response to low Ptreatment (Table I). Brooks [5] reported a similar effect of P-deficiency on fluorescence. The decrease in Fv/F m with low-P may have been due to a reduction in PS II antenna size (although there are other possible explanations, e.g. an increased oxidation state of plastoquinone). R o o m temperature Chl fluorescence emission spectra has been used to determine energy distribution between the Chlcontaining components of the p h o t o s y n t h e t i c membranes. The energy distribution between P S I and PS II is determined by comparing the fluorescence emissions at 730 and 680 nm, respectively. In control plants, the ratio of P S I fluorescence (FI) to PS II fluorescence (FII) increased by 45% during illumination with blue light (Table I). In low-P plants 1 min after illumination, FI/F]I was 13% larger than in control plants; however, by 9 min, Fi/Fii-values were about the same in the two treatments. The increases in FI/Fii-values on illumination (i.e. changes in room temperature fluorescence from 1 to 9 min after illumination) could be interpreted in terms of a movement of antenna from PS II to P S I resulting from the phosphorylation of antenna Chl-proteins [23,24]. The phosphorylation and dephosphorylation
of the antenna polypeptides occurs by the action of a light activated membrane-bound kinase and a membrane-bound phosphatase, respectively [25]. In low-P plants, the increase was much smaller than in control plants suggesting that P deficiency impaired the movement of antenna from PS II to PS I in some way. An alternative approach to determining the distribution of excitation energy between PS II and P S I is to use 77°K fluorescence. It is assumed that, after rapid cooling to 77°K, the conformational state of the photochemical pigment apparatus is fixed and that only primary photochemical reactions occur; secondary biochemical influences on the photosystems are frozen [26,27]. The room temperature fluorescence data indicated that low-P treatment may have increased the antenna size of PSI vs. PS II; this is because the FI/FII values 1 min after the onset of illumination were greater in the low-P treat~ m e n t than in the control. However, the 77°K fluorescence data for dark adapted leaves indicated little or no increase in FI/FII with low-P t r e a t m e n t (Table I). As with room temperature fluorescence, there was an increase in FI/FII values at 77°K on illumination, a p h e n o m e n o n which has been found by other workers [27]. Also in keeping with the room temperature fluorescence data, the increase on illumination was less in the low-P compared to control plants. In conclusion, our results show that low-P treatment had very little effect on thylakoid membrane composition and function. There was some evidence that low-P increased PSI and light harvesting Chl-protein complexes, and Cyt f and bs63, relative to the PS II complex (CPa), atrazine binding sites and Cyt bssg, and that low-P may have affected the redistribution of excitation energy between PS II and P S I which occurs on illumination. Acknowledgments We thank R. Huston, C. Carlson and J.
55 Krall for excellent technical assistance. We a r e g r a t e f u l t o D r s . A . M e l i s a n d R. G l i c k f o r measurements of room temperature fluorescence and for discussion of the manuscript.
References 1 D.A. Walker, in: Physiological Aspects of Crop Productivity, "Der Bund" AG, Bern/Switzerland, 1980, p. 195. 2 J. Preiss, Annu. Rev. Plant Physiol., 33 (1982) 431. 3 A.R. Portis, Plant Physiol., 71 (1983) 936. 4 M.N. Sivak and D.A. Walker, New Phytol., 102 (1986) 499. 5 A. Brooks, Aust. J. Plant Physiol., 13 (1986) 221. 6 K.-J. Dietz and C. Foyer, Planta, 167 (1986) 376. 7 C. F o y e r and C. Spencer, Planta, 167 (1986) 369. 8 I.M. Rao, J. Abadia and N. Terry, Plant Sci., 44 (1986) 133. 9 J. Abadia, J.N. Nishio and N. Terry, Photosyn. Res., 7 (1986) 237. 10 J.N. Nishio, S.E. Taylor and N. Terry, Plant Physiol., 77 (1985) 705. 11 D.A. Young, B.P. Voris, E.V. Maytin and R.A. Colbert, Meth. Enzymol., 91 (1983) 190. 12 W. Tischer and H. Strotmann, Biochim. Biophys. Acta, 460 (1977) 113.
13 W.F.J. Vermaas, C.J. Arntzen, L-Q. Gu, C-A. Yu, Biochim. Biophys. Acta, 723 (1983) 266. 14 U. Heber, N.K. Boardman and J.M. Anderson, Biochim. Biophys. Acta, 423 (1976) 275. 15 W. Ruhle and A. Wild, Planta, 146 (1979) 377. 16 N. Terry, Plant Physiol., 65 (1980) 114. 17 J.N. Nishio, J. Abadia and N. Terry, Plant Physiol., 78 (1985) 296. 18 M.P. Percival and N.R. Baker, Plant, Cell Environ., 8 (1985) 41. 19 E. Weis, Photosyn. Res., 6 (1985) 73. 20 D.I. Arnon, Plant Physiol., 24 (1949) 1. 21 I.M. Rao, J. Abadia and N. Terry, in: J. Biggins (Ed.), Proc. 7th International Congress on Photosynthesis, Martinus Nijhoff/Dr. Junk Publishers, The Hague, 1986, in press. 22 I.M. Rao, J. Abadia and N. Terry, in: J. Biggins (Ed.), Proc. 7th International Congress on Photosynthesis, Martinus Nijhoff/Dr. Junk Publishers, The Hague, 1986, in press. 23 J. Barber, Photobiochem. Photobiophys., 5 (1983) 181. 24 D.C. F o r k and K. Satoh, Annu. Rev. Plant Physiol., 37 (1986) 335. 25 J. Bennett, Biochem. J., 212 (1983) 1. 26 E. Weis, in: C. Sybesma (Ed.), Adv. Photosyn. Res. Vol. 3, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague, 1984, p. 291. 27 G.H. Krause and E. Weis, Photosyn. Res., 5 (1984) 139.
49
Elsevier Scientific Publishers Ireland Ltd.
CHANGES IN LEAF PHOSPHATE STATUS HAVE ONLY SMALL EFFECTS PHOTOCHEMICAL APPARATUS OF SUGAR BEET LEAVES
ON THE
JAVIER ABADIA*, I. MADHUSUDANA RAO and NORMAN TERRY** D e p a r t m e n t o f Plant and Soil Biology, University o f California, Berkeley, CA 94 720 (U.S.A.)
(Received September 29th, 1986) (Revision received January 3rd, 1987 ) (Accepted January 5th, 1987 )
The effects of orthophosphate (Pi) on the structure and function of thylakoid membranes was investigated by monitoring changes in thylakoid polypeptides, pigment-proteins, cytochromes (Cyt), atrazine-binding sites, leaf fluorescence characteristics, photosynthetic quantum yield and electron transport (including Cyt f dark reduction) in leaves of sugar beet (Beta vulgaris L. cv. F58-554H1 ) plants cultured hydroponically with different levels of Pi supply. Low-P treatment (i.e. low external Pi supply) decreased soluble leaf phosphorus (P) by 78% and total plant dry matter by 77%, and decreased light-saturated photosynthetic CO2 fixation by 32%. The results show that low-P treatment had only minor effects on thylakoid membrane composition and function. There was some evidence that low-P increased the amounts of photosystem (PS) I and light harvesting chlorophyll-protein complexes and of the Cyt f and bs63, relative to the amounts of PS II reaction center complex, the number of atrazine binding sites and Cyt bs59. Low-P treatment had very little effect, if any, on photosynthetic quantum yield and only small effects on chlorophyll fluorescence. Key words: thylakoid membrane; chlorophyll fluorescence; leaf phosphate; sugar beet
Introduction *Recipient of a postdoctoral fellowship from the Spanish Superior Council of Scientific Re,search. Present address: Estacion Experimental de Aula Dei, Apartado 202, Zaragoza, Spain. **To whom correspondence should be sent. Abbreviations: Chl, chlorophyll; CF, coupling factor; Cyt, cytochrome; DCIP, 2,6-dichlorophenol-indophenol; DMBQ, 2,5-dimethyl benzoquinone; FI, emission peak related to photosystem I; FII , lowwavelength emission peak related to photosystem II; F0, initial chlorophyll fluorescence yield; Fro, maximum chlorophyll fluorescence yield; F v = F m -- Fo, variable chlorophyll fluorescence yield; LHCP, light harvesting chlorophyll a/b protein; LIDS, lithium dodecyl sulfate; MV, methyl viologen; PAR, photosynthetically active radiation; Pi, orthophosphate; PS, photosystem; Q, first quinone-type acceptor of photosystem II; TMBZ, 3,3',5,5'-tetramethylbenzidene.
In r e c e n t years there has been m u c h interest in the idea t h a t the level o f o r t h o p h o s p h a t e (Pi) in p l a n t tissues m a y have a role in the r e g u l a t i o n of various aspects of p h o t o synthesis [ 1 - - 7 ] . O n e a p p r o a c h to the s t u d y of the role of Pi in p h o t o s y n t h e s i s in vivo is to v a r y Pi levels in leaves n u t r i t i o n a l l y a n d t h e n m o n i t o r the c h a n g e s which o c c u r in various aspects o f p h o t o s y n t h e s i s [ 5 - - 8 ] . In a s t u d y of this kind, B r o o k s [5] observed that P nutrition affected thylakoid membranes; she f o u n d t h a t low-P t r e a t m e n t diminished p h o t o s y n t h e t i c quantum yield a n d caused changes in 7 7 ° K fluorescence. R a o et al. [8] s h o w e d t h a t low-P t r e a t m e n t
0618-9452/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
50 affected the light scattering and Chl fluorescence characteristics of leaves and that these effects were reversible when the Pi supply was increased. The objective of the present work was to further explore the effects of P nutrition on the structure and function of the thylakoid membranes. Materials and methods
Plant culture Sugarbeet plants (Beta vulgaris L. cv. F58554H1) were grown hydroponically with two different levels of phosphate in the culture medium in growth chambers at 25°C with an irradiance of 500 ~mol quanta photosynthetically active radiation (PAR) m -2 s-1 supplied over a 16-h photoperiod. Low-P and control plants were raised as described in Rao et al. [8]. Pi supply to low-P plants was increased by raising the P concentration in the culture medium [8]. Thylakoid isolation Thylakoids were obtained as described in Abadia et al. [9]. Thylakoid polypeptides and Chl-proteins Thylakoid polypeptides were separated in 10--20% lithium dodecyl sulfate (LIDS)polyacrylamide gels following the procedure of Nishio et al. [10]. The gels were stained with silver [11]. The separation of the pigment-protein complexes was performed according to the m e t h o d s described in Abadia et al. [9]. A trazine-binding sites The concentration of atrazine-binding sites was determined using the m e t h o d of Tischer and Strotmann [12] as modified by Vermaas et al. [13]. Cy toch romes The concentrations of cytochromes were determined spectrophotometrically using r o o m temperature difference spectra. Thylakoids were resuspended at 50 pg Chl/ml and
the spectra recorded using an Aminco DW-2C s p e c t r o p h o t o m e t e r in the split beam mode. C y t f and Cyt bss9 were determined from hydroquinone-reduced minus ferricyanideoxidized spectra while Cyt bs63 was determined from the dithionite-reduced minus ferricyanide-oxidized spectra. The final concentrations of hydroquinone and ferricyanide were 200 and 120 pM, respectively, and dithionite was added as a few solid grains. Cytochrome contents were calculated from the equations of Heber et al. [14].
Cytochrome f dark reduction Cyt f photooxidation and dark reduction were carried out as described in Ruhle and Wild [15]. For illumination, we used red light (Corning filter CS 2--64, 20--40 p m o l quanta m -2 s-1) or far red light (interference filter transmitting 710 nm with 10 nm bandwidth, 8 ~mol quanta m -2 s-l). The decrease in absorbance which occurs at 554 nm when leaf pieces are illuminated with red light is believed to be due to the p h o t o o x i d a t i o n of Cyt f [15]. When the light was turned off, Cyt f was reduced and the signal disappeared. The rates of Cyt f-dark reduction were similar, regardless of whether red light or 710 nm light were used. Photosyn thesis, photosyn the tic quan turn yield and electron transport The measurements of net photosynthesis and quantum yield of attached leaves were carried o u t as described in Terry [16]. Light-saturated rates of photosynthetic electron transport were measured polarographically at 25°C with a temperaturecontrolled, water-jacketed Rank Oxygen electrode connected to a strip chart recorder [17]. PS I-mediated electron flow, from ascorbate plus 2,6-dichlorophenol-indophenol (DCIP) to methyl viologen (MV), was determined by measuring oxygen uptake. The reaction mixture contained 30 mM Na4 P2 07 (pH 8.0), 5 mM MgC12, 10 mM NaC1, 0.2 mM MV, 2.5 mM NH4C1 as uncoupler, 1 mM Na-ascorbate, 0.1 mM DCIP, and chloroplast
51 membranes (20--30 ~g Chl ml-1). PS IImediated electron flow from water to DMBQ was determined by measuring oxygen evolution. The reaction mixture contained 50 mM HEPES (pH 7.6), 5 mM MgCI:, 10 mM NaC1, 1 mM EDTA, 0.25 mM 2,5-dimethyl benzoquinone (DMBQ), 2.5 mM NH4C1 as uncoupler, and chloroplast membranes (20-30 ug Chl ml-1).
and leaf discs were taken and immediately d r o p p e d into liquid N2. The leaf discs were ground to a p o w d e r in a mortar containing liquid N2 and 4 ml of water was added. An aliquot containing 5 pg chlorophyll (Chl) was placed in a precooled cuvette for fluorescence measurement. The illumination and collection of the fluorescence were carried o u t with a trifurcated fibre-optics system.
Chlorophyll fluorescence
Chlorophyll
Fluorescence induction curves at room temperature were obtained by illuminating leaf pieces with 10 W m -2 of blue light (Coming CS 4--96) and collecting the fluorescence through a 685 nm interference filter. Signal recovery and processing were implem e n t e d by a Hewlett-Packard 3437A digital voltmeter interfaced with an on-line HP86B computer. The kinetic analysis of the data was p e r f o r m e d by the computer and the results plotted on a HP7475A plotter. Leaf pieces were removed from leaves of plants taken from the growth chamber at the end of the dark period and illuminated with blue light at room temperature. On excitation of the sample, fluorescence at 685 nm rapidly increased to an initial level, initial chlorophyll fluorescence yield (F0), which represents the fluorescence level when the first quinonetype acceptor of PS II (Q) is maximally oxidized. There was then a slower fluorescence rise until a maximal level, maximum chlorophyU fluorescence yield (Fro), was attained (Q maximally reduced). The difference in the fluorescence yield at Fm and Fo is termed the fluorescence of variable yield, Fv [ 18]. The distribution of excitation energy between PS II and P S I was determined according to the m e t h o d s outlined in Percival and Baker [18] by measuring the fluorescence at 680 and 730 nm (10-nm bandwidth) for leaf pieces illuminated with 480-nm light (10-nm bandwidth). Chlorophyll fluorescence at 77 ° K was measured using the 'dry powder' technique of Weis [ 1 9 ] . Leaves were either dark- or light-adapted in growth chambers for 2 h
Chl a, Chl b, Total Chl (a + b) and Chl a/Chl b ratios were determined according to Arnon [20]. Results and discussion When sugar beet plants were subjected to low-P treatment for a period of 2 weeks, acid-soluble and total leaf P declined by 77--78% {Table I). By far the most important change physiologically in these low-P treated plants was the reduction in leaf growth: total leaf area in low-P plants was decreased by 80% compared to the controls {Table I). Total plant dry matter was diminished to a comparable extent (77% decrease with low-P, Table I). The photosynthetic rate was reduced to a much smaller extent (32% decrease compared to controls, Table I), the decrease due to low-P being greatest at light saturation and diminishing with lower light intensities (data n o t shown). It is clear from these results that the major effect of low-P treatment on the accumulation of p h o t o s y n t h a t e was to reduce the rate of expansion of the photosynthetic surface rather than to decrease photosynthesis/area. Other data show that low-P effects on certain aspects of photosynthesis, particularly the 'dark reactions', can be quite pronounced. F o r example, low-P diminishes the levels of Calvin cycle intermediates substantially [ 5,6,21] with ribulose-l,5-bisphosphate being decreased by as much as 90% [21]. Furthermore, low-P treatment can markedly change the activities of various photosynthetic
52 Table I. Effect of low-P treatment on certain growth, gas exchange and photochemical characteristics of 5-week-old sugar beet plants. Characteristics
Treatment Con~ol
Acid soluble leafP (% dry wt.) 0.41 Total leaf P (% dry wt.) 0.57 Leaf area/plant (cm 2) 2269 Total dry matter (g plant -1 ) 23.5 Photosynthesis/area (~ mol CO 2 m-: s -1) 30.3 Chl (nmol cm-:) in: chlorophyll-protein-1 8.6 LHC II 21.3 CPa 6.9 Cytochromes (pmol cm -2) Cyt f 71 Cyt bss 9 120 Cyt bs~~ 282 Atrazine binding sites (pmol cm -2 ) 273 Chl a/Chl b (mol/mol) 3.7 Chl (nmol cm -2 ) 47.5 Specific leaf dry wt. (rag cm -2) 3.65 Soluble leaf protein (rag cm -2) 0.74 Electron transport (umol O: cm-2 h-l) PS I 40.2 PS II 15.7 Photosynthetic quantum yield (~) 0.117 Cyt f dark reduction (A 554--A551 × 103/rain) 12.6 Room temperature fluorescence: Fv/Fm 0.63
Low-P 0.09 0.13 450 5.3 20.6 11.1 26.8 7.0 80 120 340 294 3.5 58.8 4.76 0.83 41.3 13.8 0.109 6.7 0.50
FI/Fn a
1 min 9 rain 77 ° K fluorescence:
0.69 1.0
0.78 0.91
0.87 1.0
0.89 0.92
FI/FI]b Dark Light
aThe values of FI/Fn found in the control after 9 rain of illumination were arbitrarily taken as 1.0. bThe values of FI/FI! for the control in the light were arbitrarily taken as 1.0.
e n z y m e s o f the s t r o m a a n d c y t o s o l [ 2 1 , 2 2 ] . The photochemical apparatus however, a p p a r e n t l y u n d e r g o e s m u c h less c h a n g e in r e s p o n s e t o low-P as is s h o w n below.
Effects of low-P on thylakoid membrane composition L o n g , d e n a t u r i n g gels ( L I D S p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ) s h o w e d t h a t t h e r e w e r e n o m a j o r c h a n g e s in t h e relative a m o u n t s o f the numerous p o l y p e p t i d e s resolved (Fig. 1), i.e. low-P t r e a t m e n t did n o t signific a n t l y c h a n g e the p o l y p e p t i d e c o m p o s i t i o n of the t h y l a k o i d m e m b r a n e . L o w - P treatm e n t did a p p e a r t o i n f l u e n c e the a m o u n t s of s o m e m e m b r a n e c o m p o n e n t s relative t o the a m o u n t o f PS II c o m p o n e n t s in the m e m b r a n e . A n a l y s i s o f the p i g m e n t - p r o t e i n s s h o w e d t h a t low-P t r e a t m e n t increased the a m o u n t s o f Chl a s s o c i a t e d w i t h light harvesting c h l o r o p h y l l a/b p r o t e i n ( L H C ) I I a n d CP1 relative to the a m o u n t o f C P a / C h l ( t h e c o m p l e x t h a t has b e e n s u p p o s e d t o c o n t a i n the PS II r e a c t i o n c e n t e r ) . CPa a n d L H C II increased b y a b o u t 2 6 - - 2 8 % ( o n an a r e a basis) w i t h low-P t r e a t m e n t while t h e a m o u n t o f CPa Chl was u n c h a n g e d ( T a b l e I). F u r t h e r m o r e , low-P t r e a t m e n t i n c r e a s e d the a m o u n t s of C y t f a n d C y t bs63 b y 13 a n d 21%, respectively, while the a m o u n t o f C y t bss9 ( o f t e n a s s o c i a t e d w i t h PS II) a n d the n u m b e r of a t r a z i n e b i n d i n g sites p e r area (believed to be a m e a s u r e o f the n u m b e r o f PS II r e a c t i o n centers} were u n c h a n g e d ( T a b l e I). Chl a / C h l b r a t i o s were ~slightly d e c r e a s e d b y low-P, p r o b a b l y b e c a u s e o f the increase in light h a r v e s t i n g Chl a / C h l b c o m p l e x e s . T o t a l C h l / a r e a was i n c r e a s e d w i t h low-P a l o n g w i t h an increase in the t o t a l a m o u n t of leaf tissue p e r area (see specific leaf w e i g h t a n d soluble leaf p r o t e i n , T a b l e I).
Effects of low-P on thylakoid function T h y l a k o i d m e m b r a n e f u n c t i o n was a f f e c t e d to o n l y a small e x t e n t b y low-P t r e a t m e n t . T h e r e was n o e f f e c t of low-P on P S I e l e c t r o n t r a n s p o r t a n d v e r y little e f f e c t o n PS II e l e c t r o n t r a n s p o r t (12% decrease w i t h low-P, T a b l e I). Q u a n t u m yield was u n a f f e c t e d by low-P t r e a t m e n t . In c o n t r a s t t o o u r results, B r o o k s [ 5] o b t a i n e d a significant r e d u c t i o n in q u a n t u m yield w i t h low-P t r e a t m e n t ; this m a y have b e e n d u e to the f a c t t h a t we used
53 Low -P
,.
F-- Control
i
MW Markers (KD) CPI
9 2 . 5 ---)
6 6 . 2 ----> aCF
/~CF PSTrreoction center opo PS1T internol ontenno opo
4 5 . 0 ---)
8CF Heine
stain
PS IT bonds Green bond "CP 2 9 " _ _ PSIT Green bond LHCP ~.PS]]. bonds LHCPopo Iopo LH CP opo
2 9 . 0 ---)
LHClopo LHCI opo
--
}PS~Tbonds
14.4 - ~
Fig. 1. Effect of low-P t r e a t m e n t on t h y l a k o i d p o l y p e p t i d e composition. Silver stained L i D S / T X - 1 0 0 / p o l y a c r y l amide gels were e m p l o y e d . The origin of the gels was at the top; gels were subjected to electrophoresis overnight at a c o n s t a n t p o w e r of 4 W. The p o l y p e p t i d e bands were identified by i m m u n o d e c o r a t i o n of western blots (Abadia et al,, unpublished results) or 3,3',5,5'-tetramethylbenzidene (TMBZ) stain [ 1 0 ] . Molecular weight markers are s h o w n at the e x t r e m e left.
54 a different m e t h o d to induce P-deficiency, i.e. we supplied small amounts of P to plants during their growth while Brooks added no P after withholding P. Low-P treatment appeared to affect the rates in vivo of Cyt f dark-reduction after p h o t o o x i d a t i o n with 710-nm light. Low-P t r e a t m e n t decreased the rates of Cyt f dark reduction (Table I). When these plants were resupplied with phosphate, the rates increased significantly within 5 h (data n o t shown). The rapidity of the recovery would seem to preclude major changes in membrane composition with respect to the C y t b--f complex under low-P treatment. Low-P t r e a t m e n t had some small effects on chlorophyll fluorescence. Fv/Fm ratios measured on leaf pieces at room temperature decreased by 21% in response to low Ptreatment (Table I). Brooks [5] reported a similar effect of P-deficiency on fluorescence. The decrease in Fv/F m with low-P may have been due to a reduction in PS II antenna size (although there are other possible explanations, e.g. an increased oxidation state of plastoquinone). R o o m temperature Chl fluorescence emission spectra has been used to determine energy distribution between the Chlcontaining components of the p h o t o s y n t h e t i c membranes. The energy distribution between P S I and PS II is determined by comparing the fluorescence emissions at 730 and 680 nm, respectively. In control plants, the ratio of P S I fluorescence (FI) to PS II fluorescence (FII) increased by 45% during illumination with blue light (Table I). In low-P plants 1 min after illumination, FI/F]I was 13% larger than in control plants; however, by 9 min, Fi/Fii-values were about the same in the two treatments. The increases in FI/Fii-values on illumination (i.e. changes in room temperature fluorescence from 1 to 9 min after illumination) could be interpreted in terms of a movement of antenna from PS II to P S I resulting from the phosphorylation of antenna Chl-proteins [23,24]. The phosphorylation and dephosphorylation
of the antenna polypeptides occurs by the action of a light activated membrane-bound kinase and a membrane-bound phosphatase, respectively [25]. In low-P plants, the increase was much smaller than in control plants suggesting that P deficiency impaired the movement of antenna from PS II to PS I in some way. An alternative approach to determining the distribution of excitation energy between PS II and P S I is to use 77°K fluorescence. It is assumed that, after rapid cooling to 77°K, the conformational state of the photochemical pigment apparatus is fixed and that only primary photochemical reactions occur; secondary biochemical influences on the photosystems are frozen [26,27]. The room temperature fluorescence data indicated that low-P treatment may have increased the antenna size of PSI vs. PS II; this is because the FI/FII values 1 min after the onset of illumination were greater in the low-P treat~ m e n t than in the control. However, the 77°K fluorescence data for dark adapted leaves indicated little or no increase in FI/FII with low-P t r e a t m e n t (Table I). As with room temperature fluorescence, there was an increase in FI/FII values at 77°K on illumination, a p h e n o m e n o n which has been found by other workers [27]. Also in keeping with the room temperature fluorescence data, the increase on illumination was less in the low-P compared to control plants. In conclusion, our results show that low-P treatment had very little effect on thylakoid membrane composition and function. There was some evidence that low-P increased PSI and light harvesting Chl-protein complexes, and Cyt f and bs63, relative to the PS II complex (CPa), atrazine binding sites and Cyt bssg, and that low-P may have affected the redistribution of excitation energy between PS II and P S I which occurs on illumination. Acknowledgments We thank R. Huston, C. Carlson and J.
55 Krall for excellent technical assistance. We a r e g r a t e f u l t o D r s . A . M e l i s a n d R. G l i c k f o r measurements of room temperature fluorescence and for discussion of the manuscript.
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