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Genetic variation of photosynthetic electron transport in barley: identification of plastocyanin as a potential limiting factor
plan cience ELSEVIER SCIENCE IRELAND
Plant Science 98 (1994) 177-187
Genetic variation of photosynthetic electron transport in barley: identification of plastocyanin as a potential limiting factort Kent O. Burkey United States Department of Agriculture, Agricultural Research Service, Departments of Crop Science and Botany, North Carolina State University, Raleigh, NC 27695-7631, USA (Received 8 September 1993; revision received 20 December 1993; accepted 21 December 1993)
Abstract
Cultivated (Hordeum vulgate) and wild (Hordeum spontaneum) genotypes of barley were compared for differences in photosynthetic electron transport activity and chloroplast membrane composition. Plants were grown at 21°C in a controlled environment chamber (500 #mol photons m -2 s-I). Thylakoid membranes were isolated from vegetative and flag leaves and analyzed for uncoupled electron transport activity. Significant genetic variability in chloroplast electron transport was found with fourfold differences in activity observed in the genotypes tested. Quantitative measurements were made for each component of the chloroplast electron transport chain, including the development of an immunochemical assay for plastocyanin. The genetic differences in electron transport activity were related to the concentration of plastocyanin in the thylakoid membrane. The fivefold differences in plastocyanin content observed within the genotypes tested corresponded to a plastocyanin pool size of 0.8-3.8 mol of plastocyanin per mol of photosystem I reaction center. Therefore, plastocyanin was a significant limiting factor in barley genotypes expressing low photosynthetic electron transport activity.
Key words: Barley; Chloroplast; Electron transport; Genetic variation; Photosynthesis; Plastocyanin
Correspondance address: Plant Science Research, 3127 Ligon Street, Raleigh, NC 27607, USA. tCooperative investigations of the United States, Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 276957643. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture, or the North Carolina Agricultural Research Service and does not imply its approval to the exclusion of other products that may also be suitable.
Abbreviations: BCIP/NPT, 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt/p-nitro blue tetrazolium chloride; BSA, bovine serum albumin; ehl, chlorophyll; DBMIB, 2,5-dibromo3-methyl-6-isopropyl-p-benzoquinone; DCIP, 2,6-dichlorophenolindophenol;DCMU, diuron; P700, photosystem I reaction center; PSI, photosystem I; PSII, photosystem I1; SDS, sodium dodecylsulfate;TBS, 20 mM Tris-HCl (pH 7.5), 500 mM NaCI; TTBS, 20 mM Tris-HCI (pH 7.5), 500 mM NaCI, 0.05% (w/v) polyoxyethylenesorbitanmonolaurate (Tween-20).
0168-9452/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0168-9452(94)03798-E
178 1. Introduction Genetics provides a number of approaches for the study of higher plant photosynthesis. Mutagenesis can been used to probe the function and organization of individual photosynthetic proteins and a number of valuable mutants have been described [1,2]. Differences in photosynthesis between normal genotypes provide an approach to identify rate-limiting steps in a fully functional photosynthetic apparatus. Screening of germplasm has revealed variation in photosynthesis [3,41, and breeding for enhanced photosynthesis has been correlated with improved yield [5]. To date, little is known about the biochemical basis for genetic differences in photosynthesis. Chloroplast electron transport is one aspect of photosynthesis for which genetic variation exists [6,7]. The objectives of this study were to identify genetic variation in barley photosynthetic electron transport and identify biochemical factors under genetic control that limit electron transport activity. 2. Materials and methods
2.1. Plant material and growth conditions Genotypes of cultivated (H. vulgate) and wild (H. spontaneum) barley were examined in this study (Table 1). H. spontaneum genotypes were obtained from the USDA-ARS National Small Grains Collection. Plants were grown in 1-1 pots of soil at 20-21°C with a 16 h per day photoperiod in a growth chamber under fluorescent (Philips F72T12/CW/ VHO) and incandescent (Philips 60A-52A/99/EW) lamps that provided an irradiance of 500 #mol photons m -2 s -l. Plant density was carefully controlled to minimize self-shading which is known to affect chloroplast electron transport capacity [8] and would prevent a clear interpretation of genetic differences. A density of eight to ten plants per pot was used for the analysis of vegetative leaves in 2week old plants. For flag leaf studies, a single plant per pot was grown in a greenhouse and then moved to the growth chamber at the time of flag leaf emergence. Three-week old flag leaves were analyzed following expansion and development under the growth chamber conditions described
K.O. Burkey/Plant Sci. 97 (1994) 177-187 Table 1 Barley genotypesevaluated in this study Genotype
Region
Leaf chl content (#gcm -2)
Hordeum vulgare cv. WB 158-1 cv. GA 78-394 cv. Boone cv. Blenheim
USA USA USA Europe
57 ± 55 + 68 ± 64 ±
Hordeum spontaneum PI 282597 PI 220523 PI 249983 PI 282625 PI 296797
Israel Afghanistan lran Israel Israel
53 ± 6 56 + 5 49 ± 3 n.d. n.d.
2 2 4 4
n.d., Not determined. Leaf chl content was determined independently for the primary, second, and third leavesof 2-week old barley plants. The data for the three leaf positions werecombined for presentation here. Each number represents the average ±S.E. of nine independent measurements, three from each leaf position.
above. Individual pots or groups of pots were selected at random and treated as independent replications.
2.2. Thylakoid membrane isolation Leaf tissue was harvested during the first 2 h of the photoperiod. Thylakoid membranes were isolated from a 5-cm apical leaf segment as previously described [9]. The grind buffer contained 0.4 M sorbitol, 10 mM NaC1, 5 mM MgC12, 0.2% (w/v) BSA, and 50 mM T r i c i n e - N a O H (pH 7.8). The membranes were washed once in cold grind buffer, resuspended in grind buffer, stored on ice during electron transport measurements, and then frozen in liquid nitrogen before long-term storage at -75°C. 2.3. Chlorophyll determ&ation Chl content of fresh leaf disks and thylakoid membrane preparations was determined by extraction of pigments with dimethylformamide followed by spectrophotometric analysis [10]. 2.4. Photosynthetic electron transport assays Uncoupled whole chain electron transport was
K.O. Burkey / Plant Sci. 97 (1994) 177-187
assayed as either DCIP reduction or as methyl viologen mediated oxygen uptake with water serving as the electron donor in both cases [11]. In certain assays, DCIP reduction was measured in the presence of DBMIB to block electron flow from the plastoquinone pool to the cytochrome b6/f complex. Details have been published elsewhere [81.
2.5. Analysis of thylakoid membrane components The concentration of PSII reaction centers was determined by measuring the specific binding of [14C]atrazine to the high affinity site on thylakoid membranes [12]. C y t o c h r o m e f c o n t e n t was determined from reduced (hydroquinone) minus oxidized (potassium ferricyanide) difference spectra using an extinction coefficient of 18 mM -~ cm -l [13]. The concentration of PSI reaction centers was determined from the reversible light-induced P700 absorbance change at 697 nm using an extinction coefficient of 64 mM -l cm -l [14]. Details have been published elsewhere [8]. Plastoquinone pool size was estimated from measurements of the area over the chl fluorescence induction curve in the absence and presence of the electron transport inhibitor DCMU [15]. Chl fluorescence induction was measured at room temperature with a Hansatech PEA Plant Efficiency Analyzer (Hansatech Instruments Ltd.) using a prototype cell holder. Thylakoid membranes were suspended in grind buffer at a concentration of 60 /~g chl ml -t with DCMU added to a final concentration of 10 /~M for assays conducted in the presence of the inhibitor. One milliliter of the suspension was placed in a capped vial compatible with the cell holder and the sample dark-adapted for 10 min prior to illumination with actinic light. The area over the induction curve was calculated automatically by the instrument software.
2.6. Western blotting Polyacrylamide gel electrophoresis was conducted in 15% (w/v) gels using the procedure of Laemmli [16] with the lithium salt of dodecylsulfate. Acetone precipitated thylakoid proteins (see below) were dissolved in electrophoresis sample buffer, and denatured at 50°C for 15 min. Gels were loaded on the basis of chl equivalents in
179
the sample prior to acetone precipitation. Electrophoresis was conducted overnight at 8°C. Polypeptides were electroblotted from the gels onto nitrocellulose filters (BioRad Laboratories, Richmond, CA) using the buffer system of Towbin et al. [17] without methanol. Transfer was conducted for 4 h in a cooled tank system using a current of 1 A. Nitrocellulose filters were blocked by incubation overnight at 4°C in TBS buffer containing 1% (w/v) BSA and 0.02% (w/v) NAN3. Nitrocellulose filters were incubated for 2 h at room temperature in 40 ml of primary antibody solution consisting of TBS, 2% (w/v) BSA and either a 500-fold dilution of plastocyanin antiserum or a 5-fold dilution of affinity-purified antibody stock solution (see below). Following four washes with TTBS buffer, filters were incubated for 1 h at room temperature in secondary antibody solution consisting of TTBS, 1% (w/v) BSA and a 3000-fold dilution of goat anti-rabbit IgG alkaline phosphatase conjugate (BioRad Laboratories, Richmond, CA). Following three washes with TTBS and one wash with TBS, color development was conducted with the BCIP/NBT substrate system [18].
2. 7. Purification of plastocyanin antibodies Affinity purification of plastocyanin antibodies was conducted as follows using the barley plastocyanin rabbit antiserum described previously [19]. Thirty nanomoles of purified barley plastocyanin were denatured at 50°C for 15 min in 50 mM Tris-HCl (pH 7.5) containing 0.2% (w/v) SDS at an SDS/protein ratio of 3. The denatured plastocyanin was diluted to 0.005% (w/v) SDS with 50 mM Tris-HC1 (pH 7.5) and incubated with a 3.0 x 5.0 cm nitrocellulose filter hydrated in the Tris buffer without detergent. Binding of the denatured plastocyanin to the filter was conducted overnight at room temperature with NaN3 added to 0.02% (w/v) to prevent microbial growth. The filter was then blocked overnight at 4°C in TBS containing 1% (w/v) BSA and 0.02% (w/v) NaN 3. The affinity filter was incubated in 5 ml of plastocyanin antiserum for 2 h on ice and then washed five times with 5 ml of TTBS containing 0.1% (w/v) BSA to remove non-specifically bound serum proteins. Plastocyanin antibodies were released from
180
the filter by a 30-s incubation with 3.5 ml of elution buffer [5 mM glycine (pH 2.3), 500 mM NaC1, 0.05% (w/v) Tween-20, 0.1% (w/v) BSA and 0.01% (w/v) NAN3]. The antibody preparation was immediately neutralized with 0.35 ml 500 mM Tris-HCl (pH 7.5). Three additional elutions were performed and combined with the first. The neutralized antibody preparation was diluted with four volumes of TBS containing 2% (w/v) BSA to produce an antibody stock solution that was stored at - 2 0 ° C .
2.8. Quantitation of plastocyanin Unknowns were prepared by extracting 30 ~g chl aliquots of thylakoid membranes with four volumes of ice-cold acetone and retaining the precipitate. Because plastocyanin was released from the thylakoid membranes following a freeze/ thaw cycle (data not shown), quantitative determination required that thawed membranes be used without additional washing steps. The acetoneprecipitated proteins, including BSA from the isolation buffer, were collected by centrifugation at 12 000 x g for 3 min. Pellets were dissolved in 50 mM Tris-HCl (pH 7.5) containing 1% (w/v) SDS at an SDS/chl ratio of 20 based on the chl equivalents in the sample prior to acetone precipitation. The sample was denatured at 50°C for 15 min. Samples were diluted to 0.1% (w/v) SDS with Tris buffer, heated as above, and then diluted with Tris buffer to a final SDS concentration of 0.01% (w/v) before application to nitrocellulose dot blot filters. Standards were prepared by denaturation of a known quantity of purified barley plastocyanin in 50 mM Tri-HC1 (pH 7.5) containing 0.2% (w/v) SDS at an SDS/protein ratio of 3. The denatured protein was heated at 50°C for 15 min and then diluted with 50 mM Tris-HCl (pH 7.5) to a final concentration of 1000 pmol of plastocyanin per ml. Individual aliquots of this standard stock solution were stored at -20°C. For each dot blot experiment, an aliquot of the 1000 pmoi/ml stock solution was heated at 50°C for 15 min and a dilution series of plastocyanin standards (200-2 pmol/ml) was prepared in 50 mM Tris-HCl (pH 7.5). Dot blots were prepared using a HYBRI. DOT
K.O. Burkey/Plant Sci. 97 (1994) 177-187
Manifold (Bethesda Research Laboratories) and nitrocellulose filters hydrated with distilled water. For each filter, plastocyanin standards (0.1-10 pmol) were replicated three times to obtain a standard curve for comparison with unknowns. Unknowns were loaded on the basis of the chl content of the thylakoid sample prior to acetone precipitation. For each unknown, aliquots equivalent to 0.125, 0.25, 0.5 and 1.0 ~g of chl were included to demonstrate a linear response and obtain dot blot signals that were in the range of the standard curve. Dot blots were blocked, incubated with affinity-purified primary antibody and secondary antibody, and subjected to color development as described under Western Blotting. Developed blots were scanned with a laser densitometer (Pharmacia LKB). Blots were hydrated with deionized water prior to densitometry to reduce the background absorbance. 3. Results and discussion
3.1. Immunochemical assay for plastocyanin Several critical factors were discovered during development of the immunochemical assay for barley plastocyanin. First, acetone precipitation of thylakoid proteins was required to remove pigments and iipids that bind to nitrocellulose and interfere with antibody-antigen interactions on the membrane surface (data not shown). Previous work showed that plastocyanin is quantitatively recovered by acetone precipitation [19]. Second, the SDS concentration of solubilized protein pellets and denatured plastocyanin standards had to be diluted to 0.01% (w/v) prior to application on nitrocellulose filters to facilitate complete binding of the protein (data not shown). Binding was significantly reduced at 0.1% (w/v) SDS and completely eliminated at 1% (w/v) SDS. Finally, affinity purified barley plastocyanin antibodies were required to eliminate background problems. The barley plastocyanin antiserum used for this study was specific for plastocyanin [19], but was of low titer so that non-specific binding was apparent on western blots (Fig. 1, upper arrow in lane 1) and produced a significant background signal on dot blots (data not shown). Affinity purification of plastocyanin specific antibodies eliminated both
K.O. Burkey/Plant Sci. 97 (1994) 177-187
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Fig. 1. Comparison of barley plastocyanin antiserum with afffinity-purified barley plastocyanin antibodies. Acetone precipitated thylakoid membrane proteins from H. vulgate cultivar Boonewere separated on polyacrylamidegels, transferred to a nitrocellulosefilter, and the western blot developed as described in the Materials and Methods. Each lane was loaded with 10 tzg chl based on the chl content of the sample prior to acetone precipitation. Migration of the samples was from the cathode (-) at the top of the figure towards the anode (+) at the bottom of the figure. Lane 1 shows the result for plastocyanin rabbit antiserum with the non-specificbinding indicated by the upper arrow. Lane 2 shows the result for the affinitypurified antibody preparation where no non-specific reaction was observed.
the b a c k g r o u n d p r o b l e m observed on western blots (Fig. l, lane 2) a n d non-specific signals on dot blots (data not shown). The a m o u n t of colored alkaline phosphatase product formed on the nitrocellulose dot blot filter
Fig. 2. Quantitation of barley plastocyanin dot blots. The amount of each standard was based on the oxidized copper absorbance of the native plastocyanin stock solution prior to denaturation and dilution as described under Materials and Methods. After color development,dots blots were analyzed by densitometry and peak areas were integrated to generate the standard curve presented here.
was quantitated by densitometry. Purified barley plastocyanin standards in the 0. l - l 0 pmol range were used to generate a standard curve for each dot blot (Fig. 2). The signal increased in the 0 . 1 - 5 . 0 pmol range a n d saturated at higher amounts. The dot blot assay for plastocyanin was 10-fold more sensitive a n d permitted a more rapid analysis of u n k n o w n s compared with the western blot assay reported previously [19]. However, similar results were obtained in a side by side comparison with the two methods. The plastocyanin content of three i n d e p e n d e n t thylakoid preparations from H. vulgare cultivar Boone was determined to be 5.0 ± 0.8 a n d 6.6 ± 1.2 mmol (tool chl) -l by the dot blot a n d western blot methods, respectively.
3.2. Genetic variation in photosynthetic electron transport activity H. vulgare a n d H. spontaneum genetic lines were
K.O. Burkey / Plant Sci. 97 (1994) 177-187
182
evaluated for differences in photosynthetic electron transport activity. For screening genetic lines, uncoupled whole chain electron transport was measured as DCIP reduction because this rapid spectrophotometric method allowed the analysis of large numbers of samples. Because DCIP can accept electrons from both PSI and PSII [11], a number of controls were conducted to define the reaction for barley thylakoid membranes. Based on DBMIB inhibitor effects on chloroplast membranes, electrons from PSI reduced 70-80% of the DCIP in all genotypes tested with the remaining 20-30% reduced directly by PSII (data not shown). Addition of excess reduced DCIP to the assay had no effect on the observed rate of electron transport (data not shown), a demonstration that reduced DCIP generated in the assay was not a significant PSI electron donor under the conditions employed here. H. vulgare cultivars exhibited twofold variation in activity with a fourfold variation observed in the H. spontaneum plant introductions (Fig. 3). Clearly, genetic background has a major effect on photosynthetic electron transport capacity in barley. Genetic differences of a similar magnitude have been observed previously in tall fescue [6] and wheat [7]. One implication from the large differences in photosynthetic electron transport is that significant genetic variation may exist in the capacity to provide reductant for the assimilation of carbon, nitrogen and sulfur. Although the assimilations of various nutrients do not compete for reductant [20,21], overall nutrient assimilation could be reduced in genotypes with low electron transport activity.
3.3. Biochemical basis for genetic differences in photosynthetic electron transport activity One mechanism for genetic control of photosynthetic electron transport would be to regulate the concentration of the individual thylakoid membrane components that participate in chloroplast electron transport. Because seed supplies of the H. spontaneum plant introductions were limited, high and low activity genotypes of H. vulgare were analyzed in more detail for chloroplast membrane activity and composition. Boone was compared with Blenheim because the two cultivars had
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Leaf Position Fig. 3. Leaf profiles of photosynthetic electron transport. H. vulgare cultivars were WB 158-1 (O), GA 78-394 (El), Boone (/X) and Blenheim (<)). H. spontaneum plant introductions were 282597 (O), 220523 (1~, 249983 (A) and 296797 (0). Thylakoid membranes from 2-week old plants were assayed for maximum whole chain electron transport activity under uncoupled conditions. Each point represents the average + SD of three independent sets of plants.
similar leaf chl content (Table 1), yet exhibited twofold differences in electron transport when assayed as either DCIP reduction or oxygen uptake (Table 2). The differences in activity were apparent in both vegetative (primary) and reproductive (flag) leaves, providing evidence that the genetic effects are expressed during the early and late stages of plant development. Genetic effects on the concentration of the major thylakoid membrane-protein complexes did not explain the differences in electron transport
K.O. Burkey/Plant Sci. 97 (1994) 177-187
183
Table 2 Biochemical comparison of H. vulgare cultivars 'Boone' and 'Blenheim' expressing differences in photosynthetic electron transport Cultivar
Boone
Blenheim
Ratio
A. Primary leaf Electron transport activity mmol DCIP (mol chl) -t s -1 mmol O2-uptake (mol chl)-Is -j
125 4- 3 55 4- 3
65 4- 3 26 4- 1
1.92 2.12
PSII reaction centers mmol atrazine-bound (mol chl) -I
3.1 4- 0.2
3.3 4- 0.2
0.94
Plastoquinone pool size Areafl-DCMU/Arean+DCMU
34 4- 2
43 4- 4
0.79
Cytochrome b6f complexes mmol cytochrome f ( m o l chl) -t
2.1 4- 0.1
1.8 4- 0.1
1.17
14.6 4- 0.6
4.5 4- 0.9
3.24
PSI reaction centers mmol P700 (mol chl) -1
3.2 4- 0.1
2.4 ± 0.1
1.33
Plastocyanin pool size a
4.7 4- 0.8
1.9 4- 0.5
2.47
Electron transport activity mmol DCIP (mol chl)-ls -I
102 ± 1
57 + 2
1.79
PSII reaction centers mmol atrazine-bound (mol chl) -]
3.3 ± 0.1
3.4 ± 0.1
0.97
Cytochrome boC complexes mmol cytochrome f ( m o l chl) -l
2.1 ± 0.1
1.7 ± 0.1
1.24
Plastocyanin mmol (mol chl) -I
6.1 ± 1.4
2.3 ± 0.6
2.65
PSI reaction centers mmol P700 (mol chl) -t
2.5 4- 0.1
2.5 4- 0.1
1.00
Plastocyanin pool size a
2.4 4- 0.5
0.9 4- 0.3
2.67
Plastocyanin mmol (mol chl) -j
B. Flag leaf
aPlastocyanin/P700 (mol/mol). Two-week-old primary or 3-week-old flag leaves were analyzed. Each number represents the average ± S.E. of three independent thylakoid membrane preparations.
activity between Boone and Blenheim. The two cultivars contained similar levels of PSII reaction centers assayed as the stoichiometric binding of atrazine (Table 2). The number of PSI reaction centers and cytochrome b6/f complexes were slightly elevated in Boone relative to Blenheim, but the 20-30% differences were much smaller than the twofold differences observed in whole chain activity (Table 2). An alternative possibility was that genetic
variability in activity was related to the pool sizes of plastoquinone and plastocyanin, the mobile carriers that transfer electrons between the membrane-protein complexes. Plastoquinone pool size was estimated from the area over the chl fluorescence induction curves [15]. Genetic differences in electron transport were not related to plastoquinone pool size because the low activity Blenheim cultivar contained more plastoquinone than the high activity Boone cultivar (Table 2). In
K.O. Burkey / Plant Sci. 97 (1994) 177-187
184 contrast, cultivar Boone c o n t a i n e d two- to threefold higher levels o f p l a s t o c y a n i n than did cultivar Blenheim (Table 2). T h e results o f this quantitative survey showed that p l a s t o c y a n i n was the only electron t r a n s p o r t c o m p o n e n t with genetic differences similar to the observed differences in electron t r a n s p o r t activity. W i t h i n all o f the H. vulgare and H. spontaneum genotypes examined in this study, p l a s t o c y a n i n levels varied between 2.3 and 14.8 m m o l (mol chl) - l a n d generally corresponded with electron t r a n s p o r t activity (Tables 2 and 3). Previous studies with tall fescue indicated that genotypes with high electron t r a n s p o r t activity also c o n t a i n e d m o r e p l a s t o c y a n i n [6,22]. These correlative observations d o not prove that plastocyanin limits electron t r a n s p o r t in low activity genotypes, but certainly suggest that p l a s t o c y a n i n is an i m p o r t a n t factor for consideration. There were also indications that p l a s t o c y a n i n levels d e p e n d on leaf age or stage o f p l a n t growth. The two- to threefold difference in p l a s t o c y a n i n content was always a p p a r e n t when cultivars Boone and Blenheim were c o m p a r e d , but the absolute values for p l a s t o c y a n i n were d e p e n d e n t on the leaf tissue selected for study. Boone contained from 5 to 15 m m o l p l a s t o c y a n i n (mol chl) -1 c o m p a r e d with 2 - 5 m m o l p l a s t o c y a n i n
(mol chl) -t in Blenheim when t h y l a k o i d s were isolated from p r i m a r y leaf tissue (Table 2), a mixture o f y o u n g and old vegetative leaves (Table 3) or flag leaves (Table 2). Changes in plastocyanin during leaf development were also observed in a previous study where plastocyanin was found to accumulate in p r i m a r y barley leaves after leaf chl accumulation was complete [19].
3.4. Relationship between electron transport and plastocyanin pool size A n informative a p p r o a c h to evaluate plastocyanin limitations on electron t r a n s p o r t is to consider the plastocyanin p o o l size. The p o o l size is expressed as the plastocyanin/P700 ratio which compares the n u m b e r o f p l a s t o c y a n i n molecules with the n u m b e r o f PSI reaction centers available to accept electrons from this c o p p e r protein. Within the barley genotypes examined in this study, plastocyanin levels varied fivefold while P700 levels varied by a factor o f a p p r o x i m a t e l y 1.6 which c o r r e s p o n d e d to p l a s t o c y a n i n p o o l sizes o f 0.8-3.8 (Table 3). Clearly, genetic variability in plastocyanin pool size exists for barley. Also, the range o f plastocyanin pool sizes in barley is much larger than the values o f 2 - 3 reported for spinach [23,24].
Table 3 Genetic variation in plastocyanin pool size Genotype
Hordeum vulgare cv. WB 158-1 cv. GA 78-394 cv. Boone cv. Blenheim Hordeum spontaneurn PI 282597 PI 220523 PI 249983 PI 282625 PI 296797
Activity mmol DC1P (mol chl) -l s -1 134 ± 11 143 -4- 6 90 4. 8 43 -4- 4 99 71 56 44 25
± ± ± ± ±
5 7 5 1 2
Plastocyanin content PSI reaction centers mmol component (mol chl)-
Plastocyanin pool sizea
4.2 ± 2.9 ± 5.0 ± 2.5 ±
0.6 0.3 0.8 0.3
2.7 4. 0.1 2.6 4- 0.1 3.0 ± 0.1 3.2 ± 0.2
1.6 ± 1.1 ± 1.7 ± 0.8 ±
0.2 0.1 0.2 0.1
14.8 ± 12.3 ± 9.1 ± 5.5 ± 3.4 ±
1.7 1.0 1.2 0.5 0.1
3.9 ± 4.0 ± 4.1 ± 3.4 ± 3.5 ±
3.8 + 3.1 ± 2.2 ± 1.6 ± 1.0 ±
0.5 0.3 0.2 0.2 0.1
0.2 0.1 0.1 0.1 0.1
aplastocyanin/P700 (mol/mol). Thylakoid membranes were obtained from a mixture of primary, second and third leaves of 2-week old plants. Each number represents the average ± SE of three to five independent thylakoid preparations.
K.O. Burkey/Plant ScL 97 (1994) 177-187
When the H. vulgare cultivars and the H. spontaneum plant introductions were considered as separate groups, a clear trend was observed between electron transport activity and plastocyanin pool size (Table 3). However, the H. spontaneum plant introductions generally contained more plastocyanin and PSI, but exhibited lower rates of electron transport than did the H. vulgare cultivars. The results suggest that plastocyanin pool size is one factor that affects electron transport capacity, but is probably not the only factor that caused the genetic variation observed in this study. The hypothesis that plastocyanin limits photosynthetic electron transport must be compatible with the kinetics of the reactions involved. Typical kinetic experiments utilize short light flashes to induce reactions in dark-adapted thylakoids. Plastoquinol oxidation by cytochrome b6/f complexes was shown to be the slowest reaction in chloroplast electron transport [25]. Rates of cytochrome f oxidation and P700 ÷ reduction by plastocyanin were found to be at least an order of magnitude faster than plastoquinol oxidation [26]. Therefore, plastocyanin should not limit electron transport based on the reaction kinetics of each individual step between plastoquinol and PSI. However, plastocyanin inhibition studies suggest that plastocyanin has the potential to limit electron transport. Spinach thylakoids normally have a plastocyanin pool size in excess of the amount required to support rapid turnover of all the P700 [24]. When spinach thylakoids were treated with specific chemical-inhibitors to reduce plastocyanin levels, total P700 was not affected but the P700 ÷ reduction kinetics exhibited both a fast and a slow component [27]. The slow kinetic component represented a group of PSI reaction centers that could not utilize the remaining active plastocyanin to participate in rapid electron transport reactions. Both whole chain and PSI electron transport activities declined in response to the inhibitor treatment [27]. Clearly, lower plastocyanin levels had a negative effect on electron transport. By analogy, any factor that limits plastocyanin accumulation in the chloroplast would have the same negative effect as treatment of thylakoids with a plastocyanin inhibitor. Such a factor appeared to operate within the
185
barley genotypes examined in this study because electron transport activity declined as plastocyanin pool size decreased (Tables 2 and 3). Thus, the treatment of spinach thylakoids in vitro with plastocyanin inhibitors or a factor in barley that limits plastocyanin accumulation both effectively reduced plastocyanin levels to a point where linear electron transport was affected. The nature of this plastocyanin accumulation factor and the reason for the genetic variability in the expression of this factor are not known at present. The heterogeneous organization of the thylakoid membrane system into grana and stroma lamellae is also important in understanding how plastocyanin pool size could limit the rate of chloroplast electron transport. The two kinetic forms of PSI observed after partial plastocyanin inhibition defined two different domains in the thylakoid membrane [27]. The observed kinetic pattern could be explained by the preferential distribution of the remaining active plastocyanin into one domain and the inability of the plastocyanin to act as a mobile pool that rapidly exchanges electrons between the two domains [27]. The two PSI domains defined by kinetics could be the PSI found in the grana and stroma lamellae fractions separated by two-phase partitioning [28]. Twophase partitioning has also been used to define three domains for cytochrome f [29] that could explain the multiple kinetic forms reported for cytochrome f [26]. Thus, chloroplast thylakoids appear to contain more than one independent domain where cytochrome b6/f complexes, plastocyanin and PSI reaction centers interact. In this model, plastoquinone acts as the electron carrier to shuttle electrons long distances from PSII reaction centers to the cytochrome b6/f complexes while plastocyanin is localized within specific domains [27]. Each domain would require a minimum number of plastocyanin molecules to attain the maximum overall rate of electron transport. The low rates of electron transport observed in barley genotypes with a low plastocyanin pool size could represent a situation where the plastocyanin concentration in one or more of the domains declined below a critical level relative to the cytochrome b6/f complexes and PSI reaction centers.
186 The above discussion suggests that a m i n i m u m level of plastocyanin is required to express maxim u m rates of electron transport. This study normalized the data as the plastocyanin/P700 ratio in an attempt to identify a critical m i n i m u m value for plastocyanin content. The data showed a gradual decrease in electron transport activity as the plastocyanin/P700 ratio declined a n d no critical value was apparent. The determination of a m i n i m u m value for plastocyanin c o n t e n t may be difficult because there is evidence that the plastocyanin/P700 ratio changes with leaf age or stage of plant development (see discussion above). However, the data do suggest that m a x i m u m electron transport activity could be improved in genotypes with low plastocyanin c o n t e n t if the plastocyanin/P700 ratio was increased to a value of approximately three.
4. Acknowledgments
K.O. Burkey / Plant Sci. 97 (1994) 177-187
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The author thanks Sharyn R. Caudell, Sandra K. Dawson and Mitchell S. Hayes for technical assistance, and Barbara L. Leach for assistance in the production of the text a n d figures. Seeds for the H. vulgate cultivars were kindly provided by Dr. J. Paul M u r p h y of the Crop Science Department at N o r t h Carolina State University. Dr. Steven R. Spilatro of Marietta College provided helpful discussions on affinity purification of antibodies. The a u t h o r thanks Hansatech I n s t r u m e n t s Ltd. for the o p p o r t u n i t y to test the prototype of the single cell aqueous phase a d a p t o r for the P E A system.
5. References 1 D. Miles,The use of mutations to probe photosynthesisin higher plants, in: M. Edelman, R.B. Hallick and N.-H. Chua, (Eds.), Methods in Chloroplast Molecular Biology, Elsevier, Amsterdam, 1982, pp. 75-107. 2 D.J. Simpson and D. von Wettstein, Macromolecular physiology of plastids X1V. Viridis mutants in barley: genetic, fluoroscopic, and ultrastructural characterization. Carlsberg Res. Commun., 45 (1980) 283-314. 3 B.F. Carver, R.C. Johnson and A+L. Rayburn, Genetic analysis of photosynthetic variation in hexaploid and tetraploid wheat and their interspecific hybrids. Photosynth. Res., 20 (1989) 105-118. 4 R. Wells, L.L. Schulze, D.A. Ashley, H.R. Boerrna and
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19 20
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R.H. Brown, Cultivar differences in canopy apparent photosynthesis and their relationship to seed yield in soybean. Crop Sci., 22 (1982) 886-890. D.A. Ashley and H.R. Boerma, Canopy photosynthesis and its association with seed yield in advanced generations of a soybean cross. Crop Sci., 29 (1989) 1042-1045. R.W. Krueger and D. Miles, Photosynthesisin tall fescue llI. Rates of electron transport in a polyploid series of tall rescue plants. Plant Physiol., 68 (1981) 1110-1114. M.I. Zelenskii, G. Mogileva, I. Shitova and F. Fattakhova, Hill reaction of some species, varieties and cultivars of wheat. Photosynthetica, 12 (1978) 428-435. W.R.De la Torre and K.O. Burkey,Acclimationof barley to changes in light intensity: photosynthetic electron transport and components. Photosynth. Res., 24 (1990) 127-136. W.R.De la Torre and K.O. Burkey,Acclimationof barley to changes in light intensity: chlorophyll organization. Photosynth. Res., 24 (1990) 117-125. R. Moran, Formulae for determination of chlorophyllous pigments extracted with N,N-dimethylformamide.Plant Physiol., 68 (1982) 1376-1381. S. lzawa, Acceptors and donors for chloroplast electron transport. Methods Enzymol., 69 (1980) 413-434. W. Tischer and H. Strotmann, Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport. Biochim. Biophys. Acta, 460 (1977) 113-125. E. Hurt and G. Hauska, A cytochromeflb 6 complex of five polypeptides w i t h plastoquinol-plastocyaninoxidoreductase activity from spinach chloroplasts. Eur.J. Biochem., 117 (1981) 591-599. T. Himaya and B. Ke, Difference spectra and extinction coefficients of P-700. Biochim. Biophys. Acta, 267 (1972) 160-171. S.W. McCauley and A. Melis, Quantitation of plastoquinone photoreduction in spinach chloroplasts. Photosynth. Res., 8 (1986) 3-16. UK Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227 (1970) 680-685. H. Towbin, T. Staehelin and J. Gordon, Electrophoretic transfer of proteins from polyacrylamidegels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76 (1979) 4350-4354. M.S. Blake, K.H. Johnson, G.J. Russell-Jones and E.C. Gotschlich, A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal. Biochem., 136 (1984) 175-179. K.O. Burkey, Effect of growth irradiance on plastocyanin levels in barley. Photosynth. Res., 36 (1993) 103-110. J . M . Robinson, Carbon dioxide and nitrite photoassimilatory processes do not intercompete for reducing equivalents in spinach and soybean leaf chloroplasts. Plant Physiol., 80 (1986) 676-684. J.M. Robinson, Spinach leaf chloroplast CO2 and NO2photoassimilations do not compete for photogenerated
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23
24
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reductant: manipulation of reductant levels by quantum flux density titrations. Plant Physiol., 88 (1988) 1373-1380. R.W. Krueger, D.D. Randall and D. Miles, Photosynthesis in tall fescue V. Analysis of high PSI activity in a decaploid genotype. Plant Physiol., 76 (1984) 903-909. T. Graan and D. Ort, Quantitation of the rapid electron donors to PT00, the functional plastoquinone pool, and the ratio of the photosystems in spinach chloroplasts. J. Biol. Chem., 259 (1984) 14003-14010. W. Haehnel, R. Ratajczak and R. Robenek, Lateral distribution of plastocyanin in chloroplast thylakoids. J. Cell Biol., 108 (1989) 1397-1405. H.H. Stiehl and W.T. Witt, Quantitative treatment of the function of plastoquinone in photosynthesis. Z. Naturforsch, 24b (1969) 1588-1598.
187 26
W. Haehnel, A. Propper and H. Krause, Evidence for complexed plastocyanin as the immediate electron donor of P-700. Biochim. Biophys. Acta., 593 (1980) 384-399. 27 W. Haehnel, On the functional organization of electron transport from plastoquinone to photosystem I. Biochim. Biophys. Acta., 682 (1982) 245-257. 28 P. Svensson, E. Andreasson and P.A. Albertsson, Heterogeneity among photosystem I. Biochim. Biophys. Acta., 1060 (1991) 45-50. 29 P.A. Albertsson, E. Andreasson, P. Svensson and S.-G. Yu, Localization of cytochrome f in the thylakoid membrane: evidence for multiple domains. Biochim. Biophys. Acta., 1098 (1991) 90-94.
Plant Science 98 (1994) 177-187
Genetic variation of photosynthetic electron transport in barley: identification of plastocyanin as a potential limiting factort Kent O. Burkey United States Department of Agriculture, Agricultural Research Service, Departments of Crop Science and Botany, North Carolina State University, Raleigh, NC 27695-7631, USA (Received 8 September 1993; revision received 20 December 1993; accepted 21 December 1993)
Abstract
Cultivated (Hordeum vulgate) and wild (Hordeum spontaneum) genotypes of barley were compared for differences in photosynthetic electron transport activity and chloroplast membrane composition. Plants were grown at 21°C in a controlled environment chamber (500 #mol photons m -2 s-I). Thylakoid membranes were isolated from vegetative and flag leaves and analyzed for uncoupled electron transport activity. Significant genetic variability in chloroplast electron transport was found with fourfold differences in activity observed in the genotypes tested. Quantitative measurements were made for each component of the chloroplast electron transport chain, including the development of an immunochemical assay for plastocyanin. The genetic differences in electron transport activity were related to the concentration of plastocyanin in the thylakoid membrane. The fivefold differences in plastocyanin content observed within the genotypes tested corresponded to a plastocyanin pool size of 0.8-3.8 mol of plastocyanin per mol of photosystem I reaction center. Therefore, plastocyanin was a significant limiting factor in barley genotypes expressing low photosynthetic electron transport activity.
Key words: Barley; Chloroplast; Electron transport; Genetic variation; Photosynthesis; Plastocyanin
Correspondance address: Plant Science Research, 3127 Ligon Street, Raleigh, NC 27607, USA. tCooperative investigations of the United States, Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 276957643. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture, or the North Carolina Agricultural Research Service and does not imply its approval to the exclusion of other products that may also be suitable.
Abbreviations: BCIP/NPT, 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt/p-nitro blue tetrazolium chloride; BSA, bovine serum albumin; ehl, chlorophyll; DBMIB, 2,5-dibromo3-methyl-6-isopropyl-p-benzoquinone; DCIP, 2,6-dichlorophenolindophenol;DCMU, diuron; P700, photosystem I reaction center; PSI, photosystem I; PSII, photosystem I1; SDS, sodium dodecylsulfate;TBS, 20 mM Tris-HCl (pH 7.5), 500 mM NaCI; TTBS, 20 mM Tris-HCI (pH 7.5), 500 mM NaCI, 0.05% (w/v) polyoxyethylenesorbitanmonolaurate (Tween-20).
0168-9452/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0168-9452(94)03798-E
178 1. Introduction Genetics provides a number of approaches for the study of higher plant photosynthesis. Mutagenesis can been used to probe the function and organization of individual photosynthetic proteins and a number of valuable mutants have been described [1,2]. Differences in photosynthesis between normal genotypes provide an approach to identify rate-limiting steps in a fully functional photosynthetic apparatus. Screening of germplasm has revealed variation in photosynthesis [3,41, and breeding for enhanced photosynthesis has been correlated with improved yield [5]. To date, little is known about the biochemical basis for genetic differences in photosynthesis. Chloroplast electron transport is one aspect of photosynthesis for which genetic variation exists [6,7]. The objectives of this study were to identify genetic variation in barley photosynthetic electron transport and identify biochemical factors under genetic control that limit electron transport activity. 2. Materials and methods
2.1. Plant material and growth conditions Genotypes of cultivated (H. vulgate) and wild (H. spontaneum) barley were examined in this study (Table 1). H. spontaneum genotypes were obtained from the USDA-ARS National Small Grains Collection. Plants were grown in 1-1 pots of soil at 20-21°C with a 16 h per day photoperiod in a growth chamber under fluorescent (Philips F72T12/CW/ VHO) and incandescent (Philips 60A-52A/99/EW) lamps that provided an irradiance of 500 #mol photons m -2 s -l. Plant density was carefully controlled to minimize self-shading which is known to affect chloroplast electron transport capacity [8] and would prevent a clear interpretation of genetic differences. A density of eight to ten plants per pot was used for the analysis of vegetative leaves in 2week old plants. For flag leaf studies, a single plant per pot was grown in a greenhouse and then moved to the growth chamber at the time of flag leaf emergence. Three-week old flag leaves were analyzed following expansion and development under the growth chamber conditions described
K.O. Burkey/Plant Sci. 97 (1994) 177-187 Table 1 Barley genotypesevaluated in this study Genotype
Region
Leaf chl content (#gcm -2)
Hordeum vulgare cv. WB 158-1 cv. GA 78-394 cv. Boone cv. Blenheim
USA USA USA Europe
57 ± 55 + 68 ± 64 ±
Hordeum spontaneum PI 282597 PI 220523 PI 249983 PI 282625 PI 296797
Israel Afghanistan lran Israel Israel
53 ± 6 56 + 5 49 ± 3 n.d. n.d.
2 2 4 4
n.d., Not determined. Leaf chl content was determined independently for the primary, second, and third leavesof 2-week old barley plants. The data for the three leaf positions werecombined for presentation here. Each number represents the average ±S.E. of nine independent measurements, three from each leaf position.
above. Individual pots or groups of pots were selected at random and treated as independent replications.
2.2. Thylakoid membrane isolation Leaf tissue was harvested during the first 2 h of the photoperiod. Thylakoid membranes were isolated from a 5-cm apical leaf segment as previously described [9]. The grind buffer contained 0.4 M sorbitol, 10 mM NaC1, 5 mM MgC12, 0.2% (w/v) BSA, and 50 mM T r i c i n e - N a O H (pH 7.8). The membranes were washed once in cold grind buffer, resuspended in grind buffer, stored on ice during electron transport measurements, and then frozen in liquid nitrogen before long-term storage at -75°C. 2.3. Chlorophyll determ&ation Chl content of fresh leaf disks and thylakoid membrane preparations was determined by extraction of pigments with dimethylformamide followed by spectrophotometric analysis [10]. 2.4. Photosynthetic electron transport assays Uncoupled whole chain electron transport was
K.O. Burkey / Plant Sci. 97 (1994) 177-187
assayed as either DCIP reduction or as methyl viologen mediated oxygen uptake with water serving as the electron donor in both cases [11]. In certain assays, DCIP reduction was measured in the presence of DBMIB to block electron flow from the plastoquinone pool to the cytochrome b6/f complex. Details have been published elsewhere [81.
2.5. Analysis of thylakoid membrane components The concentration of PSII reaction centers was determined by measuring the specific binding of [14C]atrazine to the high affinity site on thylakoid membranes [12]. C y t o c h r o m e f c o n t e n t was determined from reduced (hydroquinone) minus oxidized (potassium ferricyanide) difference spectra using an extinction coefficient of 18 mM -~ cm -l [13]. The concentration of PSI reaction centers was determined from the reversible light-induced P700 absorbance change at 697 nm using an extinction coefficient of 64 mM -l cm -l [14]. Details have been published elsewhere [8]. Plastoquinone pool size was estimated from measurements of the area over the chl fluorescence induction curve in the absence and presence of the electron transport inhibitor DCMU [15]. Chl fluorescence induction was measured at room temperature with a Hansatech PEA Plant Efficiency Analyzer (Hansatech Instruments Ltd.) using a prototype cell holder. Thylakoid membranes were suspended in grind buffer at a concentration of 60 /~g chl ml -t with DCMU added to a final concentration of 10 /~M for assays conducted in the presence of the inhibitor. One milliliter of the suspension was placed in a capped vial compatible with the cell holder and the sample dark-adapted for 10 min prior to illumination with actinic light. The area over the induction curve was calculated automatically by the instrument software.
2.6. Western blotting Polyacrylamide gel electrophoresis was conducted in 15% (w/v) gels using the procedure of Laemmli [16] with the lithium salt of dodecylsulfate. Acetone precipitated thylakoid proteins (see below) were dissolved in electrophoresis sample buffer, and denatured at 50°C for 15 min. Gels were loaded on the basis of chl equivalents in
179
the sample prior to acetone precipitation. Electrophoresis was conducted overnight at 8°C. Polypeptides were electroblotted from the gels onto nitrocellulose filters (BioRad Laboratories, Richmond, CA) using the buffer system of Towbin et al. [17] without methanol. Transfer was conducted for 4 h in a cooled tank system using a current of 1 A. Nitrocellulose filters were blocked by incubation overnight at 4°C in TBS buffer containing 1% (w/v) BSA and 0.02% (w/v) NAN3. Nitrocellulose filters were incubated for 2 h at room temperature in 40 ml of primary antibody solution consisting of TBS, 2% (w/v) BSA and either a 500-fold dilution of plastocyanin antiserum or a 5-fold dilution of affinity-purified antibody stock solution (see below). Following four washes with TTBS buffer, filters were incubated for 1 h at room temperature in secondary antibody solution consisting of TTBS, 1% (w/v) BSA and a 3000-fold dilution of goat anti-rabbit IgG alkaline phosphatase conjugate (BioRad Laboratories, Richmond, CA). Following three washes with TTBS and one wash with TBS, color development was conducted with the BCIP/NBT substrate system [18].
2. 7. Purification of plastocyanin antibodies Affinity purification of plastocyanin antibodies was conducted as follows using the barley plastocyanin rabbit antiserum described previously [19]. Thirty nanomoles of purified barley plastocyanin were denatured at 50°C for 15 min in 50 mM Tris-HCl (pH 7.5) containing 0.2% (w/v) SDS at an SDS/protein ratio of 3. The denatured plastocyanin was diluted to 0.005% (w/v) SDS with 50 mM Tris-HC1 (pH 7.5) and incubated with a 3.0 x 5.0 cm nitrocellulose filter hydrated in the Tris buffer without detergent. Binding of the denatured plastocyanin to the filter was conducted overnight at room temperature with NaN3 added to 0.02% (w/v) to prevent microbial growth. The filter was then blocked overnight at 4°C in TBS containing 1% (w/v) BSA and 0.02% (w/v) NaN 3. The affinity filter was incubated in 5 ml of plastocyanin antiserum for 2 h on ice and then washed five times with 5 ml of TTBS containing 0.1% (w/v) BSA to remove non-specifically bound serum proteins. Plastocyanin antibodies were released from
180
the filter by a 30-s incubation with 3.5 ml of elution buffer [5 mM glycine (pH 2.3), 500 mM NaC1, 0.05% (w/v) Tween-20, 0.1% (w/v) BSA and 0.01% (w/v) NAN3]. The antibody preparation was immediately neutralized with 0.35 ml 500 mM Tris-HCl (pH 7.5). Three additional elutions were performed and combined with the first. The neutralized antibody preparation was diluted with four volumes of TBS containing 2% (w/v) BSA to produce an antibody stock solution that was stored at - 2 0 ° C .
2.8. Quantitation of plastocyanin Unknowns were prepared by extracting 30 ~g chl aliquots of thylakoid membranes with four volumes of ice-cold acetone and retaining the precipitate. Because plastocyanin was released from the thylakoid membranes following a freeze/ thaw cycle (data not shown), quantitative determination required that thawed membranes be used without additional washing steps. The acetoneprecipitated proteins, including BSA from the isolation buffer, were collected by centrifugation at 12 000 x g for 3 min. Pellets were dissolved in 50 mM Tris-HCl (pH 7.5) containing 1% (w/v) SDS at an SDS/chl ratio of 20 based on the chl equivalents in the sample prior to acetone precipitation. The sample was denatured at 50°C for 15 min. Samples were diluted to 0.1% (w/v) SDS with Tris buffer, heated as above, and then diluted with Tris buffer to a final SDS concentration of 0.01% (w/v) before application to nitrocellulose dot blot filters. Standards were prepared by denaturation of a known quantity of purified barley plastocyanin in 50 mM Tri-HC1 (pH 7.5) containing 0.2% (w/v) SDS at an SDS/protein ratio of 3. The denatured protein was heated at 50°C for 15 min and then diluted with 50 mM Tris-HCl (pH 7.5) to a final concentration of 1000 pmol of plastocyanin per ml. Individual aliquots of this standard stock solution were stored at -20°C. For each dot blot experiment, an aliquot of the 1000 pmoi/ml stock solution was heated at 50°C for 15 min and a dilution series of plastocyanin standards (200-2 pmol/ml) was prepared in 50 mM Tris-HCl (pH 7.5). Dot blots were prepared using a HYBRI. DOT
K.O. Burkey/Plant Sci. 97 (1994) 177-187
Manifold (Bethesda Research Laboratories) and nitrocellulose filters hydrated with distilled water. For each filter, plastocyanin standards (0.1-10 pmol) were replicated three times to obtain a standard curve for comparison with unknowns. Unknowns were loaded on the basis of the chl content of the thylakoid sample prior to acetone precipitation. For each unknown, aliquots equivalent to 0.125, 0.25, 0.5 and 1.0 ~g of chl were included to demonstrate a linear response and obtain dot blot signals that were in the range of the standard curve. Dot blots were blocked, incubated with affinity-purified primary antibody and secondary antibody, and subjected to color development as described under Western Blotting. Developed blots were scanned with a laser densitometer (Pharmacia LKB). Blots were hydrated with deionized water prior to densitometry to reduce the background absorbance. 3. Results and discussion
3.1. Immunochemical assay for plastocyanin Several critical factors were discovered during development of the immunochemical assay for barley plastocyanin. First, acetone precipitation of thylakoid proteins was required to remove pigments and iipids that bind to nitrocellulose and interfere with antibody-antigen interactions on the membrane surface (data not shown). Previous work showed that plastocyanin is quantitatively recovered by acetone precipitation [19]. Second, the SDS concentration of solubilized protein pellets and denatured plastocyanin standards had to be diluted to 0.01% (w/v) prior to application on nitrocellulose filters to facilitate complete binding of the protein (data not shown). Binding was significantly reduced at 0.1% (w/v) SDS and completely eliminated at 1% (w/v) SDS. Finally, affinity purified barley plastocyanin antibodies were required to eliminate background problems. The barley plastocyanin antiserum used for this study was specific for plastocyanin [19], but was of low titer so that non-specific binding was apparent on western blots (Fig. 1, upper arrow in lane 1) and produced a significant background signal on dot blots (data not shown). Affinity purification of plastocyanin specific antibodies eliminated both
K.O. Burkey/Plant Sci. 97 (1994) 177-187
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Fig. 1. Comparison of barley plastocyanin antiserum with afffinity-purified barley plastocyanin antibodies. Acetone precipitated thylakoid membrane proteins from H. vulgate cultivar Boonewere separated on polyacrylamidegels, transferred to a nitrocellulosefilter, and the western blot developed as described in the Materials and Methods. Each lane was loaded with 10 tzg chl based on the chl content of the sample prior to acetone precipitation. Migration of the samples was from the cathode (-) at the top of the figure towards the anode (+) at the bottom of the figure. Lane 1 shows the result for plastocyanin rabbit antiserum with the non-specificbinding indicated by the upper arrow. Lane 2 shows the result for the affinitypurified antibody preparation where no non-specific reaction was observed.
the b a c k g r o u n d p r o b l e m observed on western blots (Fig. l, lane 2) a n d non-specific signals on dot blots (data not shown). The a m o u n t of colored alkaline phosphatase product formed on the nitrocellulose dot blot filter
Fig. 2. Quantitation of barley plastocyanin dot blots. The amount of each standard was based on the oxidized copper absorbance of the native plastocyanin stock solution prior to denaturation and dilution as described under Materials and Methods. After color development,dots blots were analyzed by densitometry and peak areas were integrated to generate the standard curve presented here.
was quantitated by densitometry. Purified barley plastocyanin standards in the 0. l - l 0 pmol range were used to generate a standard curve for each dot blot (Fig. 2). The signal increased in the 0 . 1 - 5 . 0 pmol range a n d saturated at higher amounts. The dot blot assay for plastocyanin was 10-fold more sensitive a n d permitted a more rapid analysis of u n k n o w n s compared with the western blot assay reported previously [19]. However, similar results were obtained in a side by side comparison with the two methods. The plastocyanin content of three i n d e p e n d e n t thylakoid preparations from H. vulgare cultivar Boone was determined to be 5.0 ± 0.8 a n d 6.6 ± 1.2 mmol (tool chl) -l by the dot blot a n d western blot methods, respectively.
3.2. Genetic variation in photosynthetic electron transport activity H. vulgare a n d H. spontaneum genetic lines were
K.O. Burkey / Plant Sci. 97 (1994) 177-187
182
evaluated for differences in photosynthetic electron transport activity. For screening genetic lines, uncoupled whole chain electron transport was measured as DCIP reduction because this rapid spectrophotometric method allowed the analysis of large numbers of samples. Because DCIP can accept electrons from both PSI and PSII [11], a number of controls were conducted to define the reaction for barley thylakoid membranes. Based on DBMIB inhibitor effects on chloroplast membranes, electrons from PSI reduced 70-80% of the DCIP in all genotypes tested with the remaining 20-30% reduced directly by PSII (data not shown). Addition of excess reduced DCIP to the assay had no effect on the observed rate of electron transport (data not shown), a demonstration that reduced DCIP generated in the assay was not a significant PSI electron donor under the conditions employed here. H. vulgare cultivars exhibited twofold variation in activity with a fourfold variation observed in the H. spontaneum plant introductions (Fig. 3). Clearly, genetic background has a major effect on photosynthetic electron transport capacity in barley. Genetic differences of a similar magnitude have been observed previously in tall fescue [6] and wheat [7]. One implication from the large differences in photosynthetic electron transport is that significant genetic variation may exist in the capacity to provide reductant for the assimilation of carbon, nitrogen and sulfur. Although the assimilations of various nutrients do not compete for reductant [20,21], overall nutrient assimilation could be reduced in genotypes with low electron transport activity.
3.3. Biochemical basis for genetic differences in photosynthetic electron transport activity One mechanism for genetic control of photosynthetic electron transport would be to regulate the concentration of the individual thylakoid membrane components that participate in chloroplast electron transport. Because seed supplies of the H. spontaneum plant introductions were limited, high and low activity genotypes of H. vulgare were analyzed in more detail for chloroplast membrane activity and composition. Boone was compared with Blenheim because the two cultivars had
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3
Leaf Position Fig. 3. Leaf profiles of photosynthetic electron transport. H. vulgare cultivars were WB 158-1 (O), GA 78-394 (El), Boone (/X) and Blenheim (<)). H. spontaneum plant introductions were 282597 (O), 220523 (1~, 249983 (A) and 296797 (0). Thylakoid membranes from 2-week old plants were assayed for maximum whole chain electron transport activity under uncoupled conditions. Each point represents the average + SD of three independent sets of plants.
similar leaf chl content (Table 1), yet exhibited twofold differences in electron transport when assayed as either DCIP reduction or oxygen uptake (Table 2). The differences in activity were apparent in both vegetative (primary) and reproductive (flag) leaves, providing evidence that the genetic effects are expressed during the early and late stages of plant development. Genetic effects on the concentration of the major thylakoid membrane-protein complexes did not explain the differences in electron transport
K.O. Burkey/Plant Sci. 97 (1994) 177-187
183
Table 2 Biochemical comparison of H. vulgare cultivars 'Boone' and 'Blenheim' expressing differences in photosynthetic electron transport Cultivar
Boone
Blenheim
Ratio
A. Primary leaf Electron transport activity mmol DCIP (mol chl) -t s -1 mmol O2-uptake (mol chl)-Is -j
125 4- 3 55 4- 3
65 4- 3 26 4- 1
1.92 2.12
PSII reaction centers mmol atrazine-bound (mol chl) -I
3.1 4- 0.2
3.3 4- 0.2
0.94
Plastoquinone pool size Areafl-DCMU/Arean+DCMU
34 4- 2
43 4- 4
0.79
Cytochrome b6f complexes mmol cytochrome f ( m o l chl) -t
2.1 4- 0.1
1.8 4- 0.1
1.17
14.6 4- 0.6
4.5 4- 0.9
3.24
PSI reaction centers mmol P700 (mol chl) -1
3.2 4- 0.1
2.4 ± 0.1
1.33
Plastocyanin pool size a
4.7 4- 0.8
1.9 4- 0.5
2.47
Electron transport activity mmol DCIP (mol chl)-ls -I
102 ± 1
57 + 2
1.79
PSII reaction centers mmol atrazine-bound (mol chl) -]
3.3 ± 0.1
3.4 ± 0.1
0.97
Cytochrome boC complexes mmol cytochrome f ( m o l chl) -l
2.1 ± 0.1
1.7 ± 0.1
1.24
Plastocyanin mmol (mol chl) -I
6.1 ± 1.4
2.3 ± 0.6
2.65
PSI reaction centers mmol P700 (mol chl) -t
2.5 4- 0.1
2.5 4- 0.1
1.00
Plastocyanin pool size a
2.4 4- 0.5
0.9 4- 0.3
2.67
Plastocyanin mmol (mol chl) -j
B. Flag leaf
aPlastocyanin/P700 (mol/mol). Two-week-old primary or 3-week-old flag leaves were analyzed. Each number represents the average ± S.E. of three independent thylakoid membrane preparations.
activity between Boone and Blenheim. The two cultivars contained similar levels of PSII reaction centers assayed as the stoichiometric binding of atrazine (Table 2). The number of PSI reaction centers and cytochrome b6/f complexes were slightly elevated in Boone relative to Blenheim, but the 20-30% differences were much smaller than the twofold differences observed in whole chain activity (Table 2). An alternative possibility was that genetic
variability in activity was related to the pool sizes of plastoquinone and plastocyanin, the mobile carriers that transfer electrons between the membrane-protein complexes. Plastoquinone pool size was estimated from the area over the chl fluorescence induction curves [15]. Genetic differences in electron transport were not related to plastoquinone pool size because the low activity Blenheim cultivar contained more plastoquinone than the high activity Boone cultivar (Table 2). In
K.O. Burkey / Plant Sci. 97 (1994) 177-187
184 contrast, cultivar Boone c o n t a i n e d two- to threefold higher levels o f p l a s t o c y a n i n than did cultivar Blenheim (Table 2). T h e results o f this quantitative survey showed that p l a s t o c y a n i n was the only electron t r a n s p o r t c o m p o n e n t with genetic differences similar to the observed differences in electron t r a n s p o r t activity. W i t h i n all o f the H. vulgare and H. spontaneum genotypes examined in this study, p l a s t o c y a n i n levels varied between 2.3 and 14.8 m m o l (mol chl) - l a n d generally corresponded with electron t r a n s p o r t activity (Tables 2 and 3). Previous studies with tall fescue indicated that genotypes with high electron t r a n s p o r t activity also c o n t a i n e d m o r e p l a s t o c y a n i n [6,22]. These correlative observations d o not prove that plastocyanin limits electron t r a n s p o r t in low activity genotypes, but certainly suggest that p l a s t o c y a n i n is an i m p o r t a n t factor for consideration. There were also indications that p l a s t o c y a n i n levels d e p e n d on leaf age or stage o f p l a n t growth. The two- to threefold difference in p l a s t o c y a n i n content was always a p p a r e n t when cultivars Boone and Blenheim were c o m p a r e d , but the absolute values for p l a s t o c y a n i n were d e p e n d e n t on the leaf tissue selected for study. Boone contained from 5 to 15 m m o l p l a s t o c y a n i n (mol chl) -1 c o m p a r e d with 2 - 5 m m o l p l a s t o c y a n i n
(mol chl) -t in Blenheim when t h y l a k o i d s were isolated from p r i m a r y leaf tissue (Table 2), a mixture o f y o u n g and old vegetative leaves (Table 3) or flag leaves (Table 2). Changes in plastocyanin during leaf development were also observed in a previous study where plastocyanin was found to accumulate in p r i m a r y barley leaves after leaf chl accumulation was complete [19].
3.4. Relationship between electron transport and plastocyanin pool size A n informative a p p r o a c h to evaluate plastocyanin limitations on electron t r a n s p o r t is to consider the plastocyanin p o o l size. The p o o l size is expressed as the plastocyanin/P700 ratio which compares the n u m b e r o f p l a s t o c y a n i n molecules with the n u m b e r o f PSI reaction centers available to accept electrons from this c o p p e r protein. Within the barley genotypes examined in this study, plastocyanin levels varied fivefold while P700 levels varied by a factor o f a p p r o x i m a t e l y 1.6 which c o r r e s p o n d e d to p l a s t o c y a n i n p o o l sizes o f 0.8-3.8 (Table 3). Clearly, genetic variability in plastocyanin pool size exists for barley. Also, the range o f plastocyanin pool sizes in barley is much larger than the values o f 2 - 3 reported for spinach [23,24].
Table 3 Genetic variation in plastocyanin pool size Genotype
Hordeum vulgare cv. WB 158-1 cv. GA 78-394 cv. Boone cv. Blenheim Hordeum spontaneurn PI 282597 PI 220523 PI 249983 PI 282625 PI 296797
Activity mmol DC1P (mol chl) -l s -1 134 ± 11 143 -4- 6 90 4. 8 43 -4- 4 99 71 56 44 25
± ± ± ± ±
5 7 5 1 2
Plastocyanin content PSI reaction centers mmol component (mol chl)-
Plastocyanin pool sizea
4.2 ± 2.9 ± 5.0 ± 2.5 ±
0.6 0.3 0.8 0.3
2.7 4. 0.1 2.6 4- 0.1 3.0 ± 0.1 3.2 ± 0.2
1.6 ± 1.1 ± 1.7 ± 0.8 ±
0.2 0.1 0.2 0.1
14.8 ± 12.3 ± 9.1 ± 5.5 ± 3.4 ±
1.7 1.0 1.2 0.5 0.1
3.9 ± 4.0 ± 4.1 ± 3.4 ± 3.5 ±
3.8 + 3.1 ± 2.2 ± 1.6 ± 1.0 ±
0.5 0.3 0.2 0.2 0.1
0.2 0.1 0.1 0.1 0.1
aplastocyanin/P700 (mol/mol). Thylakoid membranes were obtained from a mixture of primary, second and third leaves of 2-week old plants. Each number represents the average ± SE of three to five independent thylakoid preparations.
K.O. Burkey/Plant ScL 97 (1994) 177-187
When the H. vulgare cultivars and the H. spontaneum plant introductions were considered as separate groups, a clear trend was observed between electron transport activity and plastocyanin pool size (Table 3). However, the H. spontaneum plant introductions generally contained more plastocyanin and PSI, but exhibited lower rates of electron transport than did the H. vulgare cultivars. The results suggest that plastocyanin pool size is one factor that affects electron transport capacity, but is probably not the only factor that caused the genetic variation observed in this study. The hypothesis that plastocyanin limits photosynthetic electron transport must be compatible with the kinetics of the reactions involved. Typical kinetic experiments utilize short light flashes to induce reactions in dark-adapted thylakoids. Plastoquinol oxidation by cytochrome b6/f complexes was shown to be the slowest reaction in chloroplast electron transport [25]. Rates of cytochrome f oxidation and P700 ÷ reduction by plastocyanin were found to be at least an order of magnitude faster than plastoquinol oxidation [26]. Therefore, plastocyanin should not limit electron transport based on the reaction kinetics of each individual step between plastoquinol and PSI. However, plastocyanin inhibition studies suggest that plastocyanin has the potential to limit electron transport. Spinach thylakoids normally have a plastocyanin pool size in excess of the amount required to support rapid turnover of all the P700 [24]. When spinach thylakoids were treated with specific chemical-inhibitors to reduce plastocyanin levels, total P700 was not affected but the P700 ÷ reduction kinetics exhibited both a fast and a slow component [27]. The slow kinetic component represented a group of PSI reaction centers that could not utilize the remaining active plastocyanin to participate in rapid electron transport reactions. Both whole chain and PSI electron transport activities declined in response to the inhibitor treatment [27]. Clearly, lower plastocyanin levels had a negative effect on electron transport. By analogy, any factor that limits plastocyanin accumulation in the chloroplast would have the same negative effect as treatment of thylakoids with a plastocyanin inhibitor. Such a factor appeared to operate within the
185
barley genotypes examined in this study because electron transport activity declined as plastocyanin pool size decreased (Tables 2 and 3). Thus, the treatment of spinach thylakoids in vitro with plastocyanin inhibitors or a factor in barley that limits plastocyanin accumulation both effectively reduced plastocyanin levels to a point where linear electron transport was affected. The nature of this plastocyanin accumulation factor and the reason for the genetic variability in the expression of this factor are not known at present. The heterogeneous organization of the thylakoid membrane system into grana and stroma lamellae is also important in understanding how plastocyanin pool size could limit the rate of chloroplast electron transport. The two kinetic forms of PSI observed after partial plastocyanin inhibition defined two different domains in the thylakoid membrane [27]. The observed kinetic pattern could be explained by the preferential distribution of the remaining active plastocyanin into one domain and the inability of the plastocyanin to act as a mobile pool that rapidly exchanges electrons between the two domains [27]. The two PSI domains defined by kinetics could be the PSI found in the grana and stroma lamellae fractions separated by two-phase partitioning [28]. Twophase partitioning has also been used to define three domains for cytochrome f [29] that could explain the multiple kinetic forms reported for cytochrome f [26]. Thus, chloroplast thylakoids appear to contain more than one independent domain where cytochrome b6/f complexes, plastocyanin and PSI reaction centers interact. In this model, plastoquinone acts as the electron carrier to shuttle electrons long distances from PSII reaction centers to the cytochrome b6/f complexes while plastocyanin is localized within specific domains [27]. Each domain would require a minimum number of plastocyanin molecules to attain the maximum overall rate of electron transport. The low rates of electron transport observed in barley genotypes with a low plastocyanin pool size could represent a situation where the plastocyanin concentration in one or more of the domains declined below a critical level relative to the cytochrome b6/f complexes and PSI reaction centers.
186 The above discussion suggests that a m i n i m u m level of plastocyanin is required to express maxim u m rates of electron transport. This study normalized the data as the plastocyanin/P700 ratio in an attempt to identify a critical m i n i m u m value for plastocyanin content. The data showed a gradual decrease in electron transport activity as the plastocyanin/P700 ratio declined a n d no critical value was apparent. The determination of a m i n i m u m value for plastocyanin c o n t e n t may be difficult because there is evidence that the plastocyanin/P700 ratio changes with leaf age or stage of plant development (see discussion above). However, the data do suggest that m a x i m u m electron transport activity could be improved in genotypes with low plastocyanin c o n t e n t if the plastocyanin/P700 ratio was increased to a value of approximately three.
4. Acknowledgments
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The author thanks Sharyn R. Caudell, Sandra K. Dawson and Mitchell S. Hayes for technical assistance, and Barbara L. Leach for assistance in the production of the text a n d figures. Seeds for the H. vulgate cultivars were kindly provided by Dr. J. Paul M u r p h y of the Crop Science Department at N o r t h Carolina State University. Dr. Steven R. Spilatro of Marietta College provided helpful discussions on affinity purification of antibodies. The a u t h o r thanks Hansatech I n s t r u m e n t s Ltd. for the o p p o r t u n i t y to test the prototype of the single cell aqueous phase a d a p t o r for the P E A system.
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