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First published online April 30, 2004; 10.1104/pp.103.036236 Plant Physiology 135:121-128 (2004) © 2004 American Society of Plant Biologists Light Induces Phosphorylation of Glucan Water Dikinase, Which Precedes Starch Degradation in Turions of the Duckweed Spirodela polyrhiza1,2Institute of General Botany and Plant Physiology (R.R., M.H., K.-J.A.) and Institute of Nutritional Sciences (B.M.), University of Jena, 07743 Jena, Germany
Degradation of storage starch in turions, survival organs of Spirodela polyrhiza, is induced by light. Starch granules isolated from irradiated (24 h red light) or dark-stored turions were used as an in vitro test system to study initial events of starch degradation. The starch-associated pool of glucan water dikinase (GWD) was investigated by two-dimensional gel electrophoresis and by western blotting using antibodies raised against GWD. Application of this technique allowed us to detect spots of GWD, which are light induced and absent on immunoblots prepared from dark-adapted plants. These spots, showing increased signal intensity following incubation of the starch granules with ATP, became labeled by randomized [   -33P]ATP but not by [-33P]ATP and were removed by acid phosphatase treatment. This strongly suggests that they represent a phosphorylated form(s) of GWD. The same light signal that induces starch degradation was thus demonstrated for the first time to induce autophosphorylation of starch-associated GWD. The in vitro assay system has been used to study further effects of the light signal that induces autophosphorylation of GWD and starch degradation. In comparison with starch granules from dark-adapted plants, those from irradiated plants showed increase in (1) binding capacity of GWD by ATP treatment decreased after phosphatase treatment; (2) incorporation of the -phosphate group of ATP into starch granules; and (3) rate of degradation of isolated granules by starch-associated proteins, further enhanced by phosphorylation of starch. The presented results provide evidence that autophosphorylation of GWD precedes the initiation of starch degradation under physiological conditions.
Degradation of starch in cereal endosperm has been thoroughly investigated over a long period (see Ritchie et al., 2000  ). However, the mechanisms discovered in these systems are not applicable to the metabolic mechanisms of starch in other plant cells, e.g. transitory starch in chloroplasts of leaves or storage starch in amyloplasts of tubers, roots, or turions (see Smith et al., 2003). The main reason for this difference is the a-cellular character of endosperm at the time of starch degradation. The network of regulatory processes is certainly different in living cells. In the last few years, a hitherto unexpected aspect of starch degradation has been revealed (see Blennow et al., 2002). A protein was discovered associated with starch granule surfaces (Lorberth et al., 1998; Ritte et al., 2000b), which showed reversible binding depending on the metabolic status of the cell (Ritte et al., 2000b; Reimann et al., 2002). By analysis of antisense plants and mutants, it became clear that this protein is necessary for phosphorylation of starch and that starch degradation is impaired in its absence (Lorberth et al., 1998; Yu et al., 2001). Later, the biochemical function of this protein, now named  -glucan water dikinase (GWD; EC 2.7.9.4), was revealed (Ritte et al., 2002; Mikkelsen et al., 2004). This protein becomes autophosphorylated by the -phosphate group of ATP and transfers this phosphate group further to starch. An in vitro assay of GWD has recently been described (Ritte et al., 2003). Site-directed mutagenesis of a conserved His residue resulted in an inactive enzyme and loss of autophosphorylation (Mikkelsen et al., 2004). The Glc moieties were phosphorylated at C6 and C3 positions; possible structural consequences have been described (Blennow et al., 2003; Engelsen et al., 2003).
In turions, survival organs of the duckweed Spirodela polyrhiza, the main storage compound is starch (Henssen, 1954 In this paper, the newly discovered enzyme GWD has been investigated in the turion system. An in vitro assay has been developed, which uses isolated starch granules and investigates their starch-associated proteins, phosphorylation of starch, and degradation of starch. The granules were isolated from irradiated (starch degradation-induced) or dark-adapted (no starch degradation in vivo) turions. This assay allows us to characterize the effect of the inducing light signal on different steps of starch degradation. We tested a working hypothesis that a light signal, which induces starch degradation, also induces autophosphorylation of GWD, resulting in the phosphorylation of starch. By this mechanism, early events of starch degradation are initiated.
Influence of Light on GWD Turions of S. polyrhiza were irradiated with red light (R) for 24 h or 48 h, or were dark-adapted as a control. Starch granules were isolated, and starch-associated proteins were extracted and investigated by two-dimensional SDS gel electrophoresis (2D-GE), followed by western analysis using antiserum raised against GWD (Fig. 1). The analysis of the dark control (Fig. 1; D/–ATP) showed a cluster of immunosignals at a molecular mass of 154 kD and a pI of approximately 5.5. To investigate a possible influence of autophosphorylation on starch-associated GWD, starch granules were incubated with ATP and further investigated by 2D-GE/western analysis. In the dark-control sample incubated with ATP (Fig. 1; D/+ATP), the antibodies detected a range of new spots slightly shifted toward the acidic region. When samples from irradiated turions (1 d R) were analyzed, two new signals appeared in the acidic region with a pI of approximately 4.0 (see arrow in Fig. 1; 1dR/–ATP), and one weaker signal at approximately 5.0. These new signals correspond to the same molecular weight as that of the polypeptides detected in the dark control. After 2 d of irradiation (Fig. 1; 2dR/–ATP), the original cluster of signals observed in the dark control disappeared completely so that only the two spots in the acidic region were detected. When irradiated samples were treated with ATP (1 d R), no new spots were found. Instead, the signal intensities of the R-produced spots were significantly enhanced (Fig. 1; 1dR/+ATP). The comparison of ATP-treated starch granules isolated from dark-adapted and R-irradiated turions suggests that the ATP treatment enhances the effect of R irradiation in vivo on GWD but cannot replace it.
To confirm that the light-induced spots in the acidic region represent phosphorylated forms of GWD, starch granules were incubated with radioactive ATP. The granules were isolated from irradiated turions (1 d R), and either common -ATP or randomized -ATP was used for incubation. Subsequently, the starch-associated proteins were extracted, separated by 2D-GE, and transferred to a polyvinylidene difluoride membrane. To ensure comparison of the signals, the same membranes were used for immunodetection of GWD using antibodies and for phosphor imaging (Fig. 2). The intensity of the immunosignals in the acidic range (Fig. 2A) clearly showed successful phosphorylation of GWD by the ATP treatment (compare with Fig. 1). No radioactive signal of any polypeptide in the investigated range of molecular weights was detected when -ATP was used (Fig. 2B). However, using randomized -ATP, the spots detected by immunostaining in the acidic region were also labeled by radioactivity (Fig. 2C). This shows that phosphate in the -position of ATP specifically autophosphorylated the protein recognized by GWD antibodies. Additionally, starch granules isolated from irradiated turions (1 d R) were treated with acid phosphatase. As a result of this enzymatic treatment, no polypeptides in the acidic region were recognized by the GWD antibodies, demonstrating independently that these GWD spots represent phosphorylated forms of the protein (Fig. 2D).
The soluble pool of GWD, not associated with the starch granule surface, was also investigated (Fig. 3). This pool represents more than 90% of the total amount of GWD (compare with Reimann et al., 2002 ). Regardless of the light conditions applied (1 d R or darkness), the immunoblots were almost identical to each other and comparable to the dark control of starch-associated GWD (Fig. 1; D/–ATP). No effect of ATP treatment (100 µM) was detected (Fig. 3; sol/–ATP, sol/+ATP). When soluble recombinant GWD from potato (Solanum tuberosum) was used for ATP treatment, two signals were detected in the acidic region that are very weak in comparison with the signals detected at the pI of approximately 5.5 (compare with Fig. 3; rec/–ATP, rec/+ATP). The position of these signals was the same as those of starch-associated GWD from irradiated samples treated with ATP (Fig. 1; 1dR/+ATP).
Binding of GWD to Starch
The phosphate content of starch granules isolated from dark-adapted and irradiated turions was investigated by colorimetric measurements following acid digestion of starch samples. Irradiation of turions (1 d R) increased the phosphate content from 3.1 ± 0.2 µmol g–1 starch in dark-control samples to 3.8 ± 0.2 g–1 that is significant at the 5% level according to Student's t test. Approximately every 2,000th Glc moiety of starch is therefore phosphorylated in turions. This very low phosphate content in starch of turions is the reason why no phosphate was detectable by enzymatic determination of Glc-6-P as described by Nielsen et al. (1994) To confirm that phosphorylation of starch influences, the binding capacity of GWD in vitro and starch granules isolated from irradiated turions (1 d R) or from dark-adapted ones were incubated with ATP. These starch granules were deproteinized and then incubated with soluble proteins from irradiated turions (1 d R). Proteins bound to the granules during this incubation were extracted by SDS, and the amount of GWD was determined by one-dimensional SDS gel electrophoresis (1D-GE)/western analysis. In the starch granules isolated from irradiated turions, the amount of starch-associated GWD was enhanced by ATP treatment (Fig. 4). By contrast, the granules from dark-adapted turions did not show any effect of ATP treatment. It can be concluded that GWD has higher affinity to phosphorylated starch in an ATP-dependent manner in starch granules from irradiated turions.
As shown before (Fig. 1), starch-associated GWD is partly phosphorylated on the granules from irradiated turions, and the level of phosphorylation can be decreased by treatment with acid phosphatase (Fig. 2D). To investigate whether the phosphorylation status of GWD itself has an influence on the capacity of starch binding, the granules were isolated from irradiated turions (1 d R) and incubated in the presence of acid phosphatase (20 min, 25°C). Following centrifugation (5 min, 15,000g), the relative amount of GWD was determined in the supernatant (released from starch surface) and in the pellet (starch associated) by 1D-GE/western analysis (Fig. 5). In controls without phosphatase treatment, GWD was detected exclusively in the starch-associated form but not in the supernatant. During the dephosphorylation of GWD by phosphatase, a significant amount of GWD was detected in the supernatant, released from the starch surface. This result indicates that dephosphorylation of GWD in vitro decreased its capacity to bind starch.
In Vitro Phosphorylation of Starch
The in vitro assay was used to investigate whether preirradiation of turions has any influence on the phosphorylation of isolated starch granules. This type of experiment was carried out because it has been shown before that autophosphorylation of GWD precedes the phosphate transfer to glucan (Ritte et al., 2002
In Vitro Degradation of Starch Granules As stated in preliminary experiments, incubation of isolated starch granules in buffer at room temperature led to their degradation, and low molecular carbohydrates like Glc, maltose, and carbohydrates with polymerization degrees of 3 and 4 were abundant products (data not shown). Using the in vitro assay, we characterized degradation of native starch granules. The soluble carbohydrates formed by starch hydrolysis were further degraded by acid hydrolysis, and the resulting amount of Glc was measured. Starch degradation was higher in starch granules isolated from irradiated turions (1 d R), and it was strongly enhanced by the ATP treatment (Fig. 7). ATP did not affect the weaker degradation of the granules isolated from dark-adapted turions. In control experiments with deproteinized starch granules, no release of Glc was detected (data not shown).
Finally, starch granules were incubated with acid phosphatase to decrease the phosphorylation level of starch-associated GWD, and the influence of this treatment on starch degradation in vitro was tested. Starch granules were isolated from irradiated turions (1 d R) and incubated with acid phosphatase for 15 min at 25°C. Thereafter, starch granules were washed twice to remove acid phosphatase, and the rate of starch degradation was determined as described before. The phosphatase treatment had no influence on the rate of starch degradation (Fig. 7). Therefore, the phosphorylation status of GWD itself did not influence starch degradation in vitro.
In this study we discovered that R induces autophosphorylation of the recently described enzyme GWD (Lorberth et al., 1998 ; Yu et al., 2001; Ritte et al., 2002; Mikkelsen et al., 2004), a key enzyme in starch degradation. We conducted our experiments with turions of S. polyrhiza, which represent an excellent model system to study starch degradation since they are starch storage organs in these plants. Moreover, in contrast to degradation of transitory starch in chloroplasts of leafs (Lorberth et al., 1998), degradation of storage starch in amyloplasts of turions is induced by light (Doelger et al., 1997; Appenroth and Gabrys, 2001, 2003). Within the 24 h-period of irradiation, degradation of starch was induced by light, but significant breakdown of starch could be detected only in the following (light or dark) period because this biochemical process needs more time (Appenroth and Gabrys, 2001). Most interestingly, irradiation of turions induced autophosphorylation of GWD. This is evident by (1) two new forms of immunodetectable GWD shifted toward the acidic region of 2D maps. These two protein spots are induced by light. (2) The increase in staining intensity of the two acidic protein spots resulted from incubation of starch granules with ATP. The same two proteins, which were recognized by GWD antibodies, were labeled by incubation of isolated starch granules with [33P]ATP. This reaction uses specifically -ATP, whereas -ATP has no effect. (3) Phosphorylation of starch proceeds during incubation of isolated starch granules with -ATP, whereas -ATP has no effect. All these results are in agreement with the properties of GWD as revealed by biochemical investigations (Ritte et al., 2002, 2003; Mikkelsen et al., 2004). It can be concluded that the light signal starts a chain of events, which produces autophosphorylation of GWD and, finally, results in starch degradation. To the best of our knowledge, this is shown here for the first time.
Starch granules isolated from dark-adapted turions (which do not undergo starch degradation in vivo) show in vitro different properties than those from irradiated turions. The starch-associated GWD is not phosphorylated, as the two GWD isoforms present after
The shift into acidic region of the 2D-GE described above (approximately 1.5 units) that is caused by phosphorylation of GWD cannot be solely explained by phosphorylation itself. In the case of GWD sequences from potato and Arabidopsis (Lorberth et al., 1998
The results presented here show that effective autophosphorylation of GWD requires association of this protein with starch granule surfaces. This starch-associated fraction of GWD (Reimann et al., 2002
GWD binds reversibly to the starch surface in dependence on the physiological conditions (Ritte et al., 2000b
From starch excess mutants of potato (Lorberth et al., 1998
Taken together, the sequence of events leading to starch degradation may include the following steps (Fig. 8). (1) Absorption of light by phytochrome, which leads through some undiscovered intermediary steps to the activation (responsiveness toward phosphorylation) of starch-associated GWD. (2) Starch-associated GWD becomes autophosphorylated. (3) Starch becomes phosphorylated, which (4) enhances the binding capacity of enzymes as already known from
Cultivation of Plants, Light Sources, and Irradiation
All experiments were carried out with etiolated nondormant turions of the duckweed (Araceae) Spirodela polyrhiza L. Schleiden strain SJ (Appenroth et al., 1996
The isolation of starch granules and the extraction of soluble and starch-associated proteins have been described previously (Reimann et al., 2002
One-dimensional SDS gel electrophoresis, electrophoretic transfer, and immunostaining were carried out as described before (Reimann et al., 2002 After electrophoresis in the first dimension was finished, the IPG strips were removed from the tray and equilibrated for SDS-PAGE using two different equilibration solutions. The IPG strips were first incubated with solution 1 (50 mM Tris-HCl, pH 6.8; 6 M urea; 30% [v/v] glycerol; 2% [w/v] SDS; and 2% [w/v] dithioerythritol) for 12 min and afterward with solution 2 (Tris-HCl, pH 6.8; 6 M urea; 30% [v/v] glycerol; 2% [w/v] SDS; 2.5% [w/v] iodoacetamide; and 0.5% [w/v] bromphenol blue) for 5 min. The strips were then briefly washed with water, loaded on top of a prepared SDS-PAGE (8% acrylamide and 0.42% piperazine diacrylamide), and covered with 0.5% agarose. The SDS-PAGE was run at 8°C and 30 mA per gel using a Bio-Rad multi-cell apparatus (Hercules, CA).
For the immunostaining, antibodies raised against GWD (EC 2.7.9.4) from potato (Solanum tuberosum; Lorberth et al., 1998
Starch granules (30 mg dry weight) were incubated in 0.5 mL of the following buffer: 50 mM HEPES-KOH (pH 7.5), 1 mM EDTA, 6 mM MgCl2, 5 µL of protease inhibitor (cocktail for plant cell and tissue extract; Sigma-Aldrich, Taufkirchen, Germany), and 0.1 mM ATP (Ritte et al., 2002
For the incubation of starch granules with radioactive ATP, two different labeled ATP preparations were used: [
Soluble proteins were extracted from turions (1g fresh weight) that had been irradiated with R for 24 h. The concentration of the total soluble protein was 0.1 mg in 0.5 mL. The plant extract was incubated with (100 µM) or without ATP and thereafter used for 2D-GE/western analysis. The same experiment was carried out with recombinant, partially purified GWD from potato (0.6 µg mL–1) as described before (Ritte et al., 2002
Starch granules (10 mg dry weight) were incubated in 1 mL of buffer containing 100 mM PIPES-HCl (pH 6.0) and 1 mM dithiothreitol in the presence of 10 units of acid phosphatase from potato (Roche Diagnostics, Mannheim, Germany). The suspension was incubated for 20 min at 26°C using a mixing rotor (15 rpm) and thereafter centrifuged for 5 min at 16,000g. The supernatant (which contained the starch-associated proteins released from starch grains during the phosphatase treatment) was precipitated with phenol-ether (Sauve et al., 1995
The pIs of GWD subjected to multiple potential phosphorylations were calculated using the algorithm from ExPASy's Compute pI/Mw program provided under http://scansite.mit.edu (see also Bjellqvist et al., 1993
Isolated starch granules (10 mg dry weight) were incubated with nonradioactive ATP as described above. The suspension was then centrifuged (5 min, 16,000g), and starch was deproteinized using toluene (Morrison and Karkalas, 1990
Starch granules (10 mg dry weight) were incubated in 0.5 mL of buffer containing 50 mM HEPES-KOH (pH 7.5) and 1 mM EDTA at 25°C under rotation. After the incubation, the granules were separated from the supernatant by centrifugation (5 min, 16,000g), and the sugar released during the incubation was measured (Waffenschmidt and Jaenicke, 1987 To investigate the influence of ATP on starch degradation, 0.1 mM ATP and 6 mM MgCl2 were added to the reaction mixture. The treatment with acid phosphatase was carried out in the same way as described above (see "Acid Phosphatase Treatment and Analysis of Starch-Associated GWD"). In all experiments, six independent samples were investigated. Data are given as average ± SE.
For the determination of the phosphate content of starch, 15 mg of isolated starch granules were deproteinized by toluene treatment (see "In Vitro Binding of Soluble GWD to Starch Granules"), lyophilized, and digested by pressurized microwave decomposition (equipment PMD2; Kürner, Rosenheim, Germany) with 1 mL of concentrated HNO3 (suprapur; Merck, Darmstadt, Germany) for 8 min. The resulting solutions were diluted with water to 5.0 mL, adjusted to pH 3.0 to 4.0 by 5 M NaOH, and the phosphate content was measured by the molybdate method (Worsfold et al., 1987
Without the support by Prof. Dr. M. Steup and Dr. G. Ritte, University of Potsdam, Germany, this work would not have been possible. We also thank Ms. Gabriele Lenk for skillful technical assistance and Prof. Halina Gabrys, Jagiellonian University, Krakow, Poland, for critical proofreading of the manuscript. Received November 20, 2003; returned for revision January 27, 2004; accepted January 28, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft, Bonn (grant no. Ap54/6 to K.-J.A.), and the Freistaat Thüringen (Nachwuchsgruppe Pflanzenphysiologie; grant to M.H.). 
2 Dedicated to Professor Dr. Aino Henssen, Marburg, Germany. Her pioneer work about starch metabolism in turions of Spirodela polyrhiza was published 50 years ago. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.036236. * Corresponding author; e-mail klaus.appenroth{at}uni-jena.de; fax 49 3641 949232.
Appenroth K-J, Augsten H (1990) Photophysiology of turion germination in Spirodela polyrhiza (L.) SCHLEIDEN. V. Demonstration of a calcium-requiring phase during phytochrome-mediated germination. Photochem Photobiol 52: 61–66 Appenroth K-J, Augsten H, Liebermann B, Feist H (1982) Effects of light quality on amino acid composition of proteins in Wolffia arrhiza (L.) WIMM. using a specially modified BRADFORD method. Biochem Physiol Pflanzen 177: 251–258 Appenroth K-J, Gabrys H (2001) Light-induced starch degradation in non-dormant turions of Spirodela polyrhiza. Photochem Photobiol 73: 77–82[Medline] Appenroth K-J, Gabrys H (2003) Ion antagonism between calcium and magnesium in phytochrome-mediated degradation of storage starch in Spirodela polyrhiza. Plant Sci 165: 1261–1265[CrossRef] Appenroth K-J, Teller S, Horn M (1996) Photophysiology of turion formation and germination in Spirodela polyrhiza. Biol Plant 38: 95–106 Bjellqvist B, Hughes GJ, Pasquali Ch, Paquet N, Ravier F, Sanchez J-C, Frutiger S, Hochstrasser DF (1993) The focusing positions of polypetides in immobilized pH gradients can be predicted from their amino-acid-sequences. Electrophoresis 14: 1023–1031[CrossRef][ISI][Medline] Blennow A, Engelsen SB, Nielsen TH, Baunsgaard L, Mikkelsen R (2002) Starch phosphorylation: a new front line in starch research. Trends Plant Sci 7: 445–450[CrossRef][ISI][Medline] Blennow A, Hansen M, Schulz A, Jørgensen K, Donald AM, Sanderson J (2003) The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy. J Struct Biol 143: 229–241[Medline] Doelger K, Tirlapur UK, Appenroth K-J (1997) Phytochrome-regulated starch degradation in germinating turions of Spirodela polyrhiza. Photochem Photobiol 66: 124–127 Engelsen SB, Madsen AO, Blennow A, Mottawia SM, Moller BL, Larsen S (2003) The phosphorylation site in double helical amylopectin as investigated by a combined approach using chemical synthesis, crystallography and molecular modelling. FEBS Lett 541: 137–144[Medline] Henssen A (1954) Die Dauerorgane von Spirodela polyrrhiza (L.) Schleid. in physiologischer Betrachtung. Flora 141: 523–566 Hippler M, Klein J, Fink A, Allinger T, Hoerth P (2001) Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii. Plant J 28: 595–606[CrossRef][ISI][Medline] Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline] Ley S, Doelger K, Appenroth K-J (1997) Carbohydrate metabolism as a possible modulator of dormancy in turions of Spirodela polyrhiza (L.) Schleiden. Plant Sci 129: 1–7[CrossRef] Lorberth R, Ritte G, Willmitzer L, Kossmann J (1998) Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening. Nat Biotechnol 16: 473–477[CrossRef][ISI][Medline]
Mikkelsen R, Baunsgaard L, Blennow A (2004) Functional characterization of Morrison WR, Karkalas J (1990) Starch. In PJ Lea, ed, Methods in Plant Biochemistry, Vol 2. Academic Press, London, pp 323–352 Nielsen TH, Wischmann B, Enevoldsen K, Moller BL (1994) Starch phosphorylation in potato-tubers proceeds concurrently with de-novo biosynthesis of starch. Plant Physiol 105: 111–117[Abstract]
Reimann R, Ritte G, Steup M, Appenroth K-J (2002) Association of Ritchie S, Swanson SJ, Gilroy S (2000) Physiology of the aleurone layer and starchy endosperm during grain development and early seedling growth: new insights from cell and molecular biology. Seed Sci Res 10: 193–212 Ritte G, Eckermann N, Haebel S, Lorberth R, Steup M (2000a) Compartmentation of the starch-related R1 protein in higher plants. Starch Staerke 52: 179–185[CrossRef]
Ritte G, Lloyd JR, Eckermann N, Rottmann A, Kossmann J, Steup M (2002) The starch-related R1 protein is an Ritte G, Lorberth R, Steup M (2000b) Reversible binding of the starch-related R1 protein to the surface of transitory starch granules. Plant J 21: 387–391[CrossRef][ISI][Medline] Ritte G, Steup M, Kossmann J, Lloyd JR (2003) Determination of the starch-phosphorylating enzyme activity in plant extracts. Planta 216: 798–801[Medline] Sauve DM, Ho DT, Roberge M (1995) Concentration of dilute protein for gel electrophoresis. Anal Biochem 226: 382–383[CrossRef][Medline]
Smith AM, Zeeman SC, Thorneycroft D, Smith AM (2003) Starch mobilization in leaves. J Exp Bot 54: 577–583 Waffenschmidt S, Jaenicke L (1987) Assay of reducing sugars in the nanomole range with 2,2'-bicinchoninate. Anal Biochem 165: 337–340[CrossRef][Medline] Worsfold PJ, Clinch JR, Casey H (1987) Spectrophotometric field-monitor for water-quality parameters: the determination of phosphate. Anal Chim Acta 197: 43–50[CrossRef]
Yu T-S, Kofler H, Hausler RE, Hille D, Flugge UI, Zeeman SC, Smith AM, Kossmann J, Lloyd J, Ritte G, et al. (2001) The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter. Plant Cell 13: 1907–1918 This article has been cited by other articles:
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