Planta
Planta (1990)181:310 315
9 Springer-Verlag1990
The impact of decreased activity of starch-branching enzyme on photosynthetic starch synthesis in leaves of wrinkled-seeded peas Alison M. Smith 1, H. Ekkehard Neuhaus 2, and Mark Stitt 2 1 John Innes Institute and AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UH, UK 2 Lehrstuhl ftir Pflanzenphysiologie, Universit/it Bayreuth, D-8580 Bayreuth, Federal Republic of Germany
Abstract. The effect of a reduction of the activity of starch-branching enzyme (1,4-~-D-glucan, 1,4-~-D-glucan-6-glycosyl transferase; EC 2.4.1.18) on photosynthetic starch synthesis and photosynthate partitioning has been studied in leaves of pea (Pisum sativum L.). Leaves of wrinkled-seeded peas, recessive at the rugosus locus (rr), contained lower activity of branching enzyme than leaves of near-isogenic round-seeded peas, dominant at the rugosus locus ( R R ) . Western blots showed that one isoform of the enzyme is absent from rr leaves, corresponding to the isoform that is absent from rr embryos. R R and rr leaves had identical rates of starch synthesis and photosynthesis at low irradiances. At high irradiances the rate of starch synthesis was decreased by up to 40% in rr relative to R R leaves. There was no corresponding increase of sucrose synthesis in rr leaves; instead, the rate of photosynthesis was decreased. This inhibition of photosynthesis was more marked at low than at high temperatures and was accompanied by increased oscillatory behaviour, rr leaves contained higher levels of A D P glucose and glycerate 3-phosphate than R R leaves in low and high light. The contribution of these results to our understanding of the distribution of control in the pathways of starch and sucrose synthesis is discussed.
homozygous recessive at this locus (rr) are wrinkled when mature, whereas pea seeds that are heterozygous (Rr) or homozygous dominant ( R R ) at this locus are round when mature (Mendel 1865; Hedley et al. 1986). Mature wrinkled (rr) seeds contain less starch, with a lower proportion of amylopectin to amylose (Greenwood and T h o m s o n 1962; Kooistra 1962), but more sucrose and lipid (Kooistra 1962; Coxon and Davies 1982) than mature round seeds. It has recently been demonstrated that a gene which encodes an isoform (isoform I) of starch-branching enzyme is located at the r locus (Bhattacharyya et al. 1990). The phenotype of the rr seed is the result of a mutation in this gene which prevents its expression and thus considerably reduces the total activity of starch-branching enzyme in the developing embryo (Smith 1988; Bhattacharyya etal. 1990). The aim of the following experiments was to investigate whether the mutation at the r locus also decreases the activity of starch-branching enzyme in leaves, and, if so, to investigate how a reduced activity of this enzyme affects the rate and regulation of starch formation during photosynthesis.
Material and methods Key words: Mutant (P&um) Photosynthate partitioning - Photosynthesis (metabolic control) - Pisum (starch synthesis) - Starch-branching enzyme
Introduction The r-locus has profound effects on the m o r p h o l o g y and storage-product composition of pea seeds. Peas that are Abbreviations: ADPGlc = adeninediphosphoglucose; Chl = chlorophyll; Frul,6bisP=fructose-l,6-bisphosphate; Fru2,6bisP=fructose-2,6-bisphosphate; kDa=kilodalton; PAR =photosynthetically active radiation; Q A = the primary acceptor for photosystem II
All experiments were carried out using near-isogenic round (RR) and wrinkled (rr) -seeded lines of Pisum sativum L. derived from JI 430 (John Innes germplasm collection) as described by Hedley et al. (1986). Plants were grown in a greenhouse in daylight supplemented with 250 ~tmol quanta photosynthetically active radiation (PAR)-m-2-s 1, and fully expanded leaves were taken from fourto six-week-old plants. Polyaerylamide-sodium dodecyl sulfate gels and Western blots were prepared, run and developed precisely according to Smith (1988) and Bhattacharyya et al. (1990). The antibody to starchbranching enzyme was that described by Bhattacharyya et al. (1990). Extracts for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were prepared by homogenising 0.15 g leaf or developing embryo (approx. 350 mg fresh weight) in 1.5 ml ice-cold medium containing 100 mM 3-(N-morpholino)propanesulfonic acid (Mops, pH 7.4), 5 mM dithiothreitol, 1 mM
A.M. Smith et al. : Impact of decreased starch-branching enzyme on leaf starch synthesis
311
ethylenediaminetetraacetic acid (EDTA), 100 rag" ml- 1 polyvinylpolypyrrolidone and centrifuging the homogenate (10 rain, 10000.g, 4~ C). The supernatant was diluted with extraction medium to a protein concentration of 1-2 mg.ml-a, mixed with an equal volume of double-strength gel sample buffer (Laemmli 1970) and boiled for 2 min prior to loading onto gels. The supernatant was also assayed for branching-enzyme activity by the phosphorylase-stimulation method (Smith 1988). The assay for wild-type leaves contained 500 i11 of 200 mM 2-(N-morpholino)ethanesulfonic acid (Mes pH 6.6), 50 mM [U-l~C]glucose1-phosphate (150 MBq.mol 1), 0.3 units phosphorylase a and 15 txl extract. For mutant leaves, the assay contained 50 mM Nacitrate (pH 7.0) instead of Mes, and 50 ~1 of extract was used. Photosynthesis was measured by Oz evolution in a leaf-disc Oz-electrode (Hansatech, Kings Lynn, Norfolk, UK) in saturating COz as described by Stitt (1986), and chlorophyll fluorescence was measured using a PAM chlorophyll-fluorescence measuring system (Heinz Walz, Effeltrich, FRG) as in Stitt and GroBe (1988a). Photochemical chlorophyll fluorescence quenching was estimated as in Schreiber et al. (1986), using the Fo value obtained immediately after darkening the leaves at a given irradiance. Starch and sucrose synthesis were measured by 14C incorporation over 20 min as in Kruckeberg et al. (1989). Leaf material was frozen in liquid N2, extracted and assayed for fructose-2,6-bisphosphate (Fru2,6bisP) and other metabolites as described by Stitt and GroBe (1988a), using a Sigma dual-wavelength photometer (Biochemie, Mtinchen, FRG). Adeninediphosphoglucose (ADPGlc) was measured using high-performance liquid chromatography (HPLC) by a method modified from Edwards et al. (1988). The CHCl3/methanol extract was fractionated on a Partisil SAX10 column (Kontron, Mfinchen) and the absorption monitored at 258 nm. The column was washed for 5 min with 25 mM KH2PO 4 (pH 2.8), then for 16 rain with a gradient from 25 to 51 mM KHzPO 4 (pH 2.8), then for 10 min with a gradient from 51 to 200 mM KH2PO4 (pH 2.8), at a flow rate of 1.2 ml. min-1. The ADPGlc was identified and quantified by comparison with added, authentic ADPGlc. The retention time was 18 min [compared to 29 min for uridine diphosphoglucose (UDPGIc)]. The level of detection was about 0.2 nmol'mg -~ of chlorophyll (Chl). The reliability of the extraction and assay was checked for each metabolite by carrying out recovery experiments, in which amounts of each metabolite were included in the killing mixture. The amount added was the same as that expected in the leaf material on the basis of previous measurements. Recoveries (as a percentage of that added, mean__SE, n=3) were ADPGlc, 87__5;
Fru2,6bisP, 79_+6; glucose-6-phosphate, 96_+ 1 ; fructose-6-phosphate, 94 _+3 ; glucose-1 -phosphate, 102 _ 3 ; UDPGlc, 97 _+3 ; triose phosphate, 103 _+8 ; fructose-i ,6-bisphosphate (Fru1,6bisP), 96 + 9 glycerate-3-phosphate, 102 _+5.
Fig. 1. Analysis by SDS-PAGE and Western blotting of crude extracts of wild-type and mutant embryos and leaves of pea. Lanes d, SDS-7.5% polyacrylamide gels of approx. 10 gg soluble protein from crude extracts of: a, wild-type embryo; b, wild-type leaf; c, mutant embryo; d, mutant leaf. Lanes e-l, Western blots of SDS7.5% polyacrylamide gels of approx. 20 gg soluble protein from crude extracts of: e, i, wild-type embryo; f, j, wild-type leaf; g, k, mutant embryo; h, /, mutant leaf. Lanes e-h, developed with
1/200 dilution of crude rabbit serum containing antibodies to the 114-kDa protein of isoform I of starch-branching enzyme; lanes i1, developed with 1/200 dilution of crude pre-immune serum from the same rabbit. Approximate molecular weights (kDa) of proteins specifically recognised by the antibodies are shown. 114+ 108 kDa=isoform I, 100 kDa=isoform II, 80 kDa=putative leaf isoform
Results
Branching-enzyme activity. B r a n c h i n g e n z y m e was ass a y e d b y the p h o s p h o r y l a s e - s t i m u l a t i o n m e t h o d ( S m i t h 1988). A s s a y c o n d i t i o n s were o p t i m i s e d to give m a x i m a l activities in extracts o f each i n d i v i d u a l g e n o t y p e . F o r e a c h g e n o t y p e , the a s s a y was s h o w n to be free f r o m i n t e r f e r e n c e b y s t a r c h a n d s t a r c h - d e g r a d i n g e n z y m e s in the e x t r a c t ( S m i t h 1988). M i x i n g e x p e r i m e n t s in w h i c h w i l d - t y p e a n d m u t a n t leaves, o r l e a f a n d d e v e l o p i n g emb r y o , were c o - e x t r a c t e d , i n d i c a t e d t h a t n o loss o f a c t i v i t y d u e to d e g r a d a t i o n o r i n h i b i t i o n o f the e n z y m e o c c u r r e d d u r i n g e x t r a c t i o n o f either g e n o t y p e . E s t i m a t e d a c t i v i t y o f s t a r c h - b r a n c h i n g e n z y m e in w i l d - t y p e (RR) leaves was 2 3 . 5 _ 1 . 4 1 a m o l glucose i n c o r p o r a t e d . m i n - 1 . ( g fresh w e i g h t ) - 1 , w h e r e a s in m u t a n t (rr) leaves it was o n l y 3.5 _+0.5 ( m e a n _+ SE, n = 5). Western blots of crude extracts of wild-type and mut a n t leaves, d e v e l o p e d w i t h a n a n t i b o d y to i s o f o r m I ( m o l e c u l a r weights 114 a n d 108 k i l o d a l t o n s , k D a ) o f starch-branching enzyme from developing embryos ( S m i t h 1988; B h a t t a c h a r y y a et al. 1990), r e v e a l e d t h a t this i s o f o r m was p r e s e n t in b o t h e m b r y o s a n d leaves o f w i l d - t y p e p l a n t s ( t r a c k s e a n d f, Fig. 1), b u t was absent f r o m b o t h e m b r y o s a n d leaves o f m u t a n t p l a n t s ( t r a c k s g a n d h, Fig. 1). T h e a n t i b o d y also c r o s s - r e a c t e d specifically with a p r o t e i n o f a p p a r e n t m o l e c u l a r w e i g h t a b o u t 80 k D a , t h a t was p r e s e n t in b o t h w i l d - t y p e a n d m u t a n t leaves b u t a p p e a r e d to be a b s e n t f r o m e m b r y o s . This m a y r e p r e s e n t a n o t h e r i s o f o r m o f s t a r c h - b r a n c h i n g e n z y m e which is r e s t r i c t e d to leaves.
Photosynthesis. M u t a n t a n d w i l d - t y p e leaves h a d identical rates o f p h o t o s y n t h e s i s at low light, b u t p h o t o s y n t h e -
312
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A,M. Smith et al. : Impact of decreased starch-branching enzyme on leaf starch synthesis
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Fig. 2. Typical light-response curves of photosynthesis at 30~ C, 22 ~ C, 15~ C and 10~ C. The net rate of 02 evolution is shown for the leaf discs from wild-type ( o - - o ) and mutant ( o - - o ) pea plants. Points are measurements made on samples of four leaf discs, all on a single day. The experiment was repeated on three separate days, with essentially the same results each time
30 (~
Fig. 3A, B. Effect or temperature on QA reduction at irradiances of 576 (A) and 302 Hmol quanta P A R . m - 2 s- 1 (B). Reduction of QA was estimated for leaves of wild-type ( o - - o ) and mutant ( o - - o ) peas as described by Schreiber et al. (1986)
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sis saturated at lower irradiances in m u t a n t than in wildtype leaves (Fig. 2). This effect w a s particularly m a r k e d at lower temperatures. For e x a m p l e , at an irradiance o f 300 g m o l quanta P A R - m - 2. s - 1, the rate o f p h o t o synthesis o f m u t a n t leaves relative to that o f wild-type leaves was reduced by 10, 18, 30 and 32% at 30, 20, 15 and 10 ~ C, respectively (Fig. 2). T h e extent o f reduction o f QA (the acceptor for phot o s y s t e m II) c o n f i r m s that the m u t a n t had a selective decrease in the rate o f p h o t o s y n t h e s i s at l o w temperatures and high light (Fig. 3). Thus, QA reduction was essentially identical in wild-type and m u t a n t leaves at 302 lamol quanta P A R - m - 2 - s -1 at 2 0 ~ and 3 0 ~ (Fig. 3B). H o w e v e r , QA w a s m o r e reduced in m u t a n t than w i l d - t y p e leaves at 576 lamol quanta P A R , and at 302 lamol quanta P A R . m - Z - s -~ at 15 ~ C and 10 ~ C. W h e n leaf discs which had been illuminated in saturating light were darkened for 3 0 - 6 0 s then re-illuminated, the recovery o f p h o t o s y n t h e s i s was different for m u t a n t and wild-type leaves (Fig. 4). O x y g e n e v o l u t i o n by wild-type leaf discs recovered rapidly to its former level, whereas O2 e v o l u t i o n by m u t a n t leaf discs oscillated considerably o v e r several m i n u t e s before a steady rate w a s recovered. F l u o r e s c e n c e e m i s s i o n by the leaf disc oscillated over the s a m e period (Fig. 4).
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Fig. 4 A, B. Traces of the recovery of Oz evolution and fluorescence after a 30-s dark period in leaves of wild-type (RR) (A) and mutant (rr) (B) peas. Leaves were illuminated (576 gmol quanta PAR" m-Z.s 1) at 15~ in saturating CO2 for 12 rain, darkened for 30 s, and then reilluminated at the original fluence rate. The upper trace (1) is the rate of 02 evolution, and the lower trace (2) shows the fluorescence emission. Fo, fluorescence of dark-adapted sample with measuring bean of negligible actinic intensity
Starch and sucrose synthesis. Leaf discs f r o m m u t a n t peas had considerably lower rates o f starch synthesis than wild-type leaf discs at saturating irradiances (Fig. 5). At sub-saturating intensities, the rate o f starch
A.M. Smith et al. : Impact of decreased starch-branching enzyme on leaf starch synthesis
leaves at low and high irradiances. Although the difference in glycerate 3-phosphate levels was small, it was observed consistently in three separate experiments. Since the higher level of glycerate-3-phosphate was not accompanied by a higher level of triose phosphate, the glycerate-3-phosphate: triose phosphate ratio was higher in mutant than in wild-type leaves. There were no consistent differences in levels of Fru2,6bisP and Frul,6bisP (Table i), or of the hexose phosphates (not shown) between mutant and wild-type leaves.
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Planta (1990)181:310 315
9 Springer-Verlag1990
The impact of decreased activity of starch-branching enzyme on photosynthetic starch synthesis in leaves of wrinkled-seeded peas Alison M. Smith 1, H. Ekkehard Neuhaus 2, and Mark Stitt 2 1 John Innes Institute and AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UH, UK 2 Lehrstuhl ftir Pflanzenphysiologie, Universit/it Bayreuth, D-8580 Bayreuth, Federal Republic of Germany
Abstract. The effect of a reduction of the activity of starch-branching enzyme (1,4-~-D-glucan, 1,4-~-D-glucan-6-glycosyl transferase; EC 2.4.1.18) on photosynthetic starch synthesis and photosynthate partitioning has been studied in leaves of pea (Pisum sativum L.). Leaves of wrinkled-seeded peas, recessive at the rugosus locus (rr), contained lower activity of branching enzyme than leaves of near-isogenic round-seeded peas, dominant at the rugosus locus ( R R ) . Western blots showed that one isoform of the enzyme is absent from rr leaves, corresponding to the isoform that is absent from rr embryos. R R and rr leaves had identical rates of starch synthesis and photosynthesis at low irradiances. At high irradiances the rate of starch synthesis was decreased by up to 40% in rr relative to R R leaves. There was no corresponding increase of sucrose synthesis in rr leaves; instead, the rate of photosynthesis was decreased. This inhibition of photosynthesis was more marked at low than at high temperatures and was accompanied by increased oscillatory behaviour, rr leaves contained higher levels of A D P glucose and glycerate 3-phosphate than R R leaves in low and high light. The contribution of these results to our understanding of the distribution of control in the pathways of starch and sucrose synthesis is discussed.
homozygous recessive at this locus (rr) are wrinkled when mature, whereas pea seeds that are heterozygous (Rr) or homozygous dominant ( R R ) at this locus are round when mature (Mendel 1865; Hedley et al. 1986). Mature wrinkled (rr) seeds contain less starch, with a lower proportion of amylopectin to amylose (Greenwood and T h o m s o n 1962; Kooistra 1962), but more sucrose and lipid (Kooistra 1962; Coxon and Davies 1982) than mature round seeds. It has recently been demonstrated that a gene which encodes an isoform (isoform I) of starch-branching enzyme is located at the r locus (Bhattacharyya et al. 1990). The phenotype of the rr seed is the result of a mutation in this gene which prevents its expression and thus considerably reduces the total activity of starch-branching enzyme in the developing embryo (Smith 1988; Bhattacharyya etal. 1990). The aim of the following experiments was to investigate whether the mutation at the r locus also decreases the activity of starch-branching enzyme in leaves, and, if so, to investigate how a reduced activity of this enzyme affects the rate and regulation of starch formation during photosynthesis.
Material and methods Key words: Mutant (P&um) Photosynthate partitioning - Photosynthesis (metabolic control) - Pisum (starch synthesis) - Starch-branching enzyme
Introduction The r-locus has profound effects on the m o r p h o l o g y and storage-product composition of pea seeds. Peas that are Abbreviations: ADPGlc = adeninediphosphoglucose; Chl = chlorophyll; Frul,6bisP=fructose-l,6-bisphosphate; Fru2,6bisP=fructose-2,6-bisphosphate; kDa=kilodalton; PAR =photosynthetically active radiation; Q A = the primary acceptor for photosystem II
All experiments were carried out using near-isogenic round (RR) and wrinkled (rr) -seeded lines of Pisum sativum L. derived from JI 430 (John Innes germplasm collection) as described by Hedley et al. (1986). Plants were grown in a greenhouse in daylight supplemented with 250 ~tmol quanta photosynthetically active radiation (PAR)-m-2-s 1, and fully expanded leaves were taken from fourto six-week-old plants. Polyaerylamide-sodium dodecyl sulfate gels and Western blots were prepared, run and developed precisely according to Smith (1988) and Bhattacharyya et al. (1990). The antibody to starchbranching enzyme was that described by Bhattacharyya et al. (1990). Extracts for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were prepared by homogenising 0.15 g leaf or developing embryo (approx. 350 mg fresh weight) in 1.5 ml ice-cold medium containing 100 mM 3-(N-morpholino)propanesulfonic acid (Mops, pH 7.4), 5 mM dithiothreitol, 1 mM
A.M. Smith et al. : Impact of decreased starch-branching enzyme on leaf starch synthesis
311
ethylenediaminetetraacetic acid (EDTA), 100 rag" ml- 1 polyvinylpolypyrrolidone and centrifuging the homogenate (10 rain, 10000.g, 4~ C). The supernatant was diluted with extraction medium to a protein concentration of 1-2 mg.ml-a, mixed with an equal volume of double-strength gel sample buffer (Laemmli 1970) and boiled for 2 min prior to loading onto gels. The supernatant was also assayed for branching-enzyme activity by the phosphorylase-stimulation method (Smith 1988). The assay for wild-type leaves contained 500 i11 of 200 mM 2-(N-morpholino)ethanesulfonic acid (Mes pH 6.6), 50 mM [U-l~C]glucose1-phosphate (150 MBq.mol 1), 0.3 units phosphorylase a and 15 txl extract. For mutant leaves, the assay contained 50 mM Nacitrate (pH 7.0) instead of Mes, and 50 ~1 of extract was used. Photosynthesis was measured by Oz evolution in a leaf-disc Oz-electrode (Hansatech, Kings Lynn, Norfolk, UK) in saturating COz as described by Stitt (1986), and chlorophyll fluorescence was measured using a PAM chlorophyll-fluorescence measuring system (Heinz Walz, Effeltrich, FRG) as in Stitt and GroBe (1988a). Photochemical chlorophyll fluorescence quenching was estimated as in Schreiber et al. (1986), using the Fo value obtained immediately after darkening the leaves at a given irradiance. Starch and sucrose synthesis were measured by 14C incorporation over 20 min as in Kruckeberg et al. (1989). Leaf material was frozen in liquid N2, extracted and assayed for fructose-2,6-bisphosphate (Fru2,6bisP) and other metabolites as described by Stitt and GroBe (1988a), using a Sigma dual-wavelength photometer (Biochemie, Mtinchen, FRG). Adeninediphosphoglucose (ADPGlc) was measured using high-performance liquid chromatography (HPLC) by a method modified from Edwards et al. (1988). The CHCl3/methanol extract was fractionated on a Partisil SAX10 column (Kontron, Mfinchen) and the absorption monitored at 258 nm. The column was washed for 5 min with 25 mM KH2PO 4 (pH 2.8), then for 16 rain with a gradient from 25 to 51 mM KHzPO 4 (pH 2.8), then for 10 min with a gradient from 51 to 200 mM KH2PO4 (pH 2.8), at a flow rate of 1.2 ml. min-1. The ADPGlc was identified and quantified by comparison with added, authentic ADPGlc. The retention time was 18 min [compared to 29 min for uridine diphosphoglucose (UDPGIc)]. The level of detection was about 0.2 nmol'mg -~ of chlorophyll (Chl). The reliability of the extraction and assay was checked for each metabolite by carrying out recovery experiments, in which amounts of each metabolite were included in the killing mixture. The amount added was the same as that expected in the leaf material on the basis of previous measurements. Recoveries (as a percentage of that added, mean__SE, n=3) were ADPGlc, 87__5;
Fru2,6bisP, 79_+6; glucose-6-phosphate, 96_+ 1 ; fructose-6-phosphate, 94 _+3 ; glucose-1 -phosphate, 102 _ 3 ; UDPGlc, 97 _+3 ; triose phosphate, 103 _+8 ; fructose-i ,6-bisphosphate (Fru1,6bisP), 96 + 9 glycerate-3-phosphate, 102 _+5.
Fig. 1. Analysis by SDS-PAGE and Western blotting of crude extracts of wild-type and mutant embryos and leaves of pea. Lanes d, SDS-7.5% polyacrylamide gels of approx. 10 gg soluble protein from crude extracts of: a, wild-type embryo; b, wild-type leaf; c, mutant embryo; d, mutant leaf. Lanes e-l, Western blots of SDS7.5% polyacrylamide gels of approx. 20 gg soluble protein from crude extracts of: e, i, wild-type embryo; f, j, wild-type leaf; g, k, mutant embryo; h, /, mutant leaf. Lanes e-h, developed with
1/200 dilution of crude rabbit serum containing antibodies to the 114-kDa protein of isoform I of starch-branching enzyme; lanes i1, developed with 1/200 dilution of crude pre-immune serum from the same rabbit. Approximate molecular weights (kDa) of proteins specifically recognised by the antibodies are shown. 114+ 108 kDa=isoform I, 100 kDa=isoform II, 80 kDa=putative leaf isoform
Results
Branching-enzyme activity. B r a n c h i n g e n z y m e was ass a y e d b y the p h o s p h o r y l a s e - s t i m u l a t i o n m e t h o d ( S m i t h 1988). A s s a y c o n d i t i o n s were o p t i m i s e d to give m a x i m a l activities in extracts o f each i n d i v i d u a l g e n o t y p e . F o r e a c h g e n o t y p e , the a s s a y was s h o w n to be free f r o m i n t e r f e r e n c e b y s t a r c h a n d s t a r c h - d e g r a d i n g e n z y m e s in the e x t r a c t ( S m i t h 1988). M i x i n g e x p e r i m e n t s in w h i c h w i l d - t y p e a n d m u t a n t leaves, o r l e a f a n d d e v e l o p i n g emb r y o , were c o - e x t r a c t e d , i n d i c a t e d t h a t n o loss o f a c t i v i t y d u e to d e g r a d a t i o n o r i n h i b i t i o n o f the e n z y m e o c c u r r e d d u r i n g e x t r a c t i o n o f either g e n o t y p e . E s t i m a t e d a c t i v i t y o f s t a r c h - b r a n c h i n g e n z y m e in w i l d - t y p e (RR) leaves was 2 3 . 5 _ 1 . 4 1 a m o l glucose i n c o r p o r a t e d . m i n - 1 . ( g fresh w e i g h t ) - 1 , w h e r e a s in m u t a n t (rr) leaves it was o n l y 3.5 _+0.5 ( m e a n _+ SE, n = 5). Western blots of crude extracts of wild-type and mut a n t leaves, d e v e l o p e d w i t h a n a n t i b o d y to i s o f o r m I ( m o l e c u l a r weights 114 a n d 108 k i l o d a l t o n s , k D a ) o f starch-branching enzyme from developing embryos ( S m i t h 1988; B h a t t a c h a r y y a et al. 1990), r e v e a l e d t h a t this i s o f o r m was p r e s e n t in b o t h e m b r y o s a n d leaves o f w i l d - t y p e p l a n t s ( t r a c k s e a n d f, Fig. 1), b u t was absent f r o m b o t h e m b r y o s a n d leaves o f m u t a n t p l a n t s ( t r a c k s g a n d h, Fig. 1). T h e a n t i b o d y also c r o s s - r e a c t e d specifically with a p r o t e i n o f a p p a r e n t m o l e c u l a r w e i g h t a b o u t 80 k D a , t h a t was p r e s e n t in b o t h w i l d - t y p e a n d m u t a n t leaves b u t a p p e a r e d to be a b s e n t f r o m e m b r y o s . This m a y r e p r e s e n t a n o t h e r i s o f o r m o f s t a r c h - b r a n c h i n g e n z y m e which is r e s t r i c t e d to leaves.
Photosynthesis. M u t a n t a n d w i l d - t y p e leaves h a d identical rates o f p h o t o s y n t h e s i s at low light, b u t p h o t o s y n t h e -
312
T
A,M. Smith et al. : Impact of decreased starch-branching enzyme on leaf starch synthesis
300.
/ e
30~C
~
A
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~ 0.4.
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0
g
10
20
30
B
o~ 0.6. 7= O
200
400
u 400
2 0
600
600
E 200.
.,L
0.4. 10%
~5%
C~ 0.2.
O
E -i g
o
g
oj
o
tO0
0
-5 g
/ o
o" ,
n
0
10
20
Temperature
2()0
400
600
0
200
400
600
Irradiance (prnol q u a n t a P A R .rn-2-s-1 )
Fig. 2. Typical light-response curves of photosynthesis at 30~ C, 22 ~ C, 15~ C and 10~ C. The net rate of 02 evolution is shown for the leaf discs from wild-type ( o - - o ) and mutant ( o - - o ) pea plants. Points are measurements made on samples of four leaf discs, all on a single day. The experiment was repeated on three separate days, with essentially the same results each time
30 (~
Fig. 3A, B. Effect or temperature on QA reduction at irradiances of 576 (A) and 302 Hmol quanta P A R . m - 2 s- 1 (B). Reduction of QA was estimated for leaves of wild-type ( o - - o ) and mutant ( o - - o ) peas as described by Schreiber et al. (1986)
Wi[dfype
T
A
Hufont
Fm '~ B
200. I
o
0')
100-
E
sis saturated at lower irradiances in m u t a n t than in wildtype leaves (Fig. 2). This effect w a s particularly m a r k e d at lower temperatures. For e x a m p l e , at an irradiance o f 300 g m o l quanta P A R - m - 2. s - 1, the rate o f p h o t o synthesis o f m u t a n t leaves relative to that o f wild-type leaves was reduced by 10, 18, 30 and 32% at 30, 20, 15 and 10 ~ C, respectively (Fig. 2). T h e extent o f reduction o f QA (the acceptor for phot o s y s t e m II) c o n f i r m s that the m u t a n t had a selective decrease in the rate o f p h o t o s y n t h e s i s at l o w temperatures and high light (Fig. 3). Thus, QA reduction was essentially identical in wild-type and m u t a n t leaves at 302 lamol quanta P A R - m - 2 - s -1 at 2 0 ~ and 3 0 ~ (Fig. 3B). H o w e v e r , QA w a s m o r e reduced in m u t a n t than w i l d - t y p e leaves at 576 lamol quanta P A R , and at 302 lamol quanta P A R . m - Z - s -~ at 15 ~ C and 10 ~ C. W h e n leaf discs which had been illuminated in saturating light were darkened for 3 0 - 6 0 s then re-illuminated, the recovery o f p h o t o s y n t h e s i s was different for m u t a n t and wild-type leaves (Fig. 4). O x y g e n e v o l u t i o n by wild-type leaf discs recovered rapidly to its former level, whereas O2 e v o l u t i o n by m u t a n t leaf discs oscillated considerably o v e r several m i n u t e s before a steady rate w a s recovered. F l u o r e s c e n c e e m i s s i o n by the leaf disc oscillated over the s a m e period (Fig. 4).
'~ 0
0-
c O
E II)
(,0
O O r
-5
F0
O (1)
0
o
ii
1'0 t'~, 1'8
10 % 18 22 Time in light (min)
Fig. 4 A, B. Traces of the recovery of Oz evolution and fluorescence after a 30-s dark period in leaves of wild-type (RR) (A) and mutant (rr) (B) peas. Leaves were illuminated (576 gmol quanta PAR" m-Z.s 1) at 15~ in saturating CO2 for 12 rain, darkened for 30 s, and then reilluminated at the original fluence rate. The upper trace (1) is the rate of 02 evolution, and the lower trace (2) shows the fluorescence emission. Fo, fluorescence of dark-adapted sample with measuring bean of negligible actinic intensity
Starch and sucrose synthesis. Leaf discs f r o m m u t a n t peas had considerably lower rates o f starch synthesis than wild-type leaf discs at saturating irradiances (Fig. 5). At sub-saturating intensities, the rate o f starch
A.M. Smith et al. : Impact of decreased starch-branching enzyme on leaf starch synthesis
leaves at low and high irradiances. Although the difference in glycerate 3-phosphate levels was small, it was observed consistently in three separate experiments. Since the higher level of glycerate-3-phosphate was not accompanied by a higher level of triose phosphate, the glycerate-3-phosphate: triose phosphate ratio was higher in mutant than in wild-type leaves. There were no consistent differences in levels of Fru2,6bisP and Frul,6bisP (Table i), or of the hexose phosphates (not shown) between mutant and wild-type leaves.
I
9~ 30
STARCH
SUCROSE
t
tO
I...
r E
20
E
,9
O
"O
