Interaction of cytochrome b5 with surfactant vesicles.

Lysates of protoplasts from the endosperm of developing grains of wheat (Triticum aestivum) were fractionated on density gradients of Nycodenz to give amyloplasts. Enzyme distribution on the gradients suggested that: (i) starch synthase and ADP-glucose pyrophosphorylase are confined to the amyloplasts; (ii) pyrophosphate: fructose-6-phosphate 1-phosphotransferase and UDP-glucose pyrophosphorylase are confined to the cytosol; (iii) a significant proportion (23-45%) of each glycolytic enzyme, from phosphoglucomutase to pyruvate kinase inclusive, is in the amyloplast. Starch synthase, ADP-glucose pyrophosphorylase and each of the glycolytic enzymes showed appreciable latency when assayed in unfractionated lysates of protoplasts. No activity of fructose-1,6-bisphosphatase was found in amyloplasts or in homogenates of endosperm. Antibody to plastidic fructose-1,6-bisphosphatase did not react positively, in an immunoblot analysis, with any protein in extracts of wheat endosperm. It is argued that wheat endosperm lacks significant plastidic fructose-1,6-bisphosphatase and that carbon for starch synthesis does not enter the amyloplast as a C-3 compound but probably as hexose phosphate.     

Enzymic properties of amyloplasts form suspension cultures of soybean

The aim of this work was to determine which enzymes of carbohydrate metabolism are present in amyloplasts. Protoplasts from 4- to 5-day-old suspension cultures of soybean, Glycine max, were lysed and fractionated on a sucrose gradient. This gave an amyloplast fraction that contained stromal enzymes and was not seriously contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. Studies of this fraction provide evidence that, in soybean cells, starch synthase and ADPglucose pyrophosphorylase are confined to amyloplasts; invertase, sucrose synthetase and UDPglucose pyrophosphorylase are absent from the amyloplast and probably confined to the cytosol; the following enzymes, though predominantly cytosolic, are present in the amyloplasts in activities high enough to mediate the rate of starch synthesis observed in vivo: glyceraldehyde-phosphate dehydrogenase (NAD), triosephosphate isomerase, fructose-1, 6-bisphosphate aldolase, fructose-bisphosphatase, glucosephosphate isomerase and phosphoglucomutase. The pathway from sucrose to starch in non-photosynthetic cells is discussed; particularly the possibility that sucrose is converted to triose phosphate for entry into the amyloplast.     

Enzyme activities associated with maize kernel amyloplasts

Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day-old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development.     

Starch synthesis by isolated amyloplasts from wheat endosperm

The aim of this work was to discover which compound(s) cross the amyloplast envelope to supply the carbon for starch synthesis in grains of Triticum aestivum L. Amyloplasts were isolated, on a continuous gradient of Nycodenz, from lysates of protoplasts of endosperm of developing grains, and then incubated in solutions of ¹⁴C-labelled: glucose, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glycerol 3-phosphate. Only glucose 1-phosphate gave appreciable labelling of starch that was dependent upon the integrity of the amyloplasts. Incorporation into starch was linear with respect to time for 2 h. At the end of the incubations, 98% of the ¹⁴C in the soluble fraction of the incubation mixture was recovered as [¹⁴C]glucose 1-phosphate. Thus it is unlikely that the added [¹⁴C glucose 1-phosphate was extensively metabolized prior to uptake by the amyloplasts. It is argued that the behaviour of the isolated amyloplasts, and previously published data on the labelling of starch by [¹³C]glucose, are consistent with the view that in wheat grains it is a C-6, not a C-3, compound that enters the amyloplast to provide the carbon for starch synthesis.Abbreviations PPase alkaline inorganic pyrophosphatase - UDPglucose uridine 5-diphosphoglucose     

Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non-photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.     

Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.)

The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.)

The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.     

Activities of key enzymes for starch synthesis in relation to growth of superior and inferior grains on winter wheat (Triticum aestivum L.) spike

Weight of individual grains is a major yield component in wheat. The non-uniform distribution of single grain weight on a wheat spike is assumed to be closely associated with starch synthesis in grains. The present study was undertaken to determine if the enzymes involved in starch synthesis cause the differences in single grain weight between superior and inferior grains on a wheat spike. Using two high-yield winter wheat (Triticum aestivum L.) varieties differing in grain weight and three nitrogen rates for one variety, the contents of amylose and amylopectin, and activities of enzymes involved in starch synthesis in both superior and inferior grains were investigated during the entire period of grain filling. Superior grains showed generally higher starch accumulation rates and activities of enzymes including SS (sucrose synthase), UDPGPPase (UDP-glucose pyrophosphorylase), ADPGPPase (ADP-glucose pyrophosphorylase), SSS (soluble starch synthase) and GBSS (starch granule bound starch synthase) and subsequently produced much higher single grain weight than inferior grains. Nitrogen increased enzyme activities and starch accumulation rates, and thus improved individual grain weight, especially for inferior grains. The SS, ADPGPPase and SSS were significantly correlated to amylopectin accumulation, while SS, ADPGPPase, SSS and GBSS were significantly correlated to amylose accumulation. This infers that SS, ADPGPPase and starch synthase play key roles in regulating starch accumulation and grain weight in superior and inferior grains on a wheat spike.     

In Vitro Biosynthesis of Phosphorylated Starch in Intact Potato Amyloplasts

Intact amyloplasts from potato (Solanum tuberosum L.) were used to study starch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best preparations were selected by searching for amyloplasts containing small starch grains. The isolated, small amyloplasts were 80% intact and were free from cytosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-¹⁴C]glucose-6-phosphate (Glc-6-P) was an efficient precursor for starch synthesis in a manner highly dependent on amyloplast integrity. Starch biosynthesis from [1-¹⁴C]Glc-1-P in small, intact amyloplasts was 5-fold lower and largely independent of amyloplast intactness. When [³³P]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch was produced. Isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated α-glucans. Acid hydrolysis of the phosphorylated α-glucans and high-performance anion-exchange chromatography analyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independent of intactness and most likely catalyzed by starch phosphorylase bound to naked starch grains.     

Effect of temperature on starch synthesis in potato tuber tissue and in amyloplasts

A sharp temperature optimum is observed at 21.5°C when the incorporation of [¹⁴C]sucrose into starch is measured with discs cut from developing tubers of potato (Solanum tuberosum L. cv Desirée). By contrast, increasing temperatures over the range 9 to 31°C only enhance release of ¹⁴C to respiratory CO₂ and incorporation of ¹⁴C into the ethanolsoluble fraction. By comparison, starch synthesis in discs from developing corms of cocoyam (Colocasia esculenta L. Schott) is increased by raising the temperature from 15 to 35°C. The significance of a relatively low temperature optimum for starch synthesis in potato is discussed in relation to the yield limitations imposed by continuously high soil temperatures. Amyloplasts isolated from protoplasts prepared from developing potato tubers contain activities of alkaline pyrophosphatase, NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, and phosphoglucomutase in addition to ADP-glucose-pyrophosphorylase, starch phosphorylase and starch synthase. Cell-free amyloplasts released by thinly slicing developing potato tubers synthesize starch from [¹⁴C]triose-phosphate generated from [¹⁴C]fructose-1,6-bisphosphate in the reaction medium. This starch synthesis is inhibited by addition of 10 millimolar inorganic phosphate and requires amyloplast integrity, suggesting the operation of a triose-phosphate/inorganic phosphate exchange carrier at the amyloplast membrane. The temperature optimum at 21.5°C observed with tissue discs is not observed with amyloplasts.     

Dry weight accumulation and starch-synthesis enzymes in grain of cultured ears of three winter wheat varieties

Detached ears of three winter wheat ( Triticum aestivum L.) varieties were cultured in solution for 12 days with sucrose levels varying from 36.5 to 292 m M. The dry weight and starch content of grains increased asymptotically with the sucrose level in the solution. At 4 days of culture, glucose phosphate isomerase (EC 5.3.1.9) activity grain⁻¹ was lower with 36.5 m M than with higher sucrose levels in the medium; at 8 days, adenosinc diphosphoglucose pyrophosphorylase (EC 2.7.7.27) and (soluble plus bound) starch synthase (EC 2.4.1.21) activities grain⁻¹ were higher with 146 and 292 m M sucrose than with 36.5 and 73 m M sucrose. The multiple regression of starch content over these enzyme activities showed that starch synthase was relatively more important as an independent variable. The dry weight and starch content of grains were higher in the variety Maris Huntsman than in Splendeur and Hobbit. The water content of grains was lower in Splendeur than in the other two varieties. At 4 days the glucose phosphate isomerase, adenosine diphosphoglucose pyrophosphorylase and starch synthase activities grain⁻¹ were smaller in Splendeur than in Hobbit and Maris Huntsman and al 8 days they were higher in Maris Huntsman than in Hobbit and Splendeur. The varietal differences in starch content of grains were related to the activities of glucose phosphate isomerase and especially of starch synthase.     

Starch synthesis and carbohydrate oxidation in amyloplasts from developing wheat endosperm

The rates of incorporation of various metabolites into starch by isolated amyloplasts from developing endosperm of spring wheat (Triticum aestivum L. cv. Axona) were examined. Of the metabolites tested that were likely to be present in the cytosol at concentrations sufficient to sustain starch synthesis, only glucose 1-phosphate (Glc1P) supported physiologically relevant rates of starch synthesis. Incorporation of Glc1P into starch was both dependent on the presence of ATP and intact organelles. The rate of incorporation of hexose into starch became saturated at a Glc1P concentration of less than 1 mol·m^-3 in the presence of 1 mol·m^-3 ATP. Starch synthesis from 5 mol · m^-3 ADP-glucose supplied to the organelles occurred at rates 15-fold higher than from similar concentrations of Glc1P, but it is argued that this is probably of little physiological relevance. The net incorporation of hexose units into starch from GlclP was inhibited 50% by 100 mmol.m^-3 carboxyatractyloside. Carbohydrate oxidation in the amyloplast was stimulated by the addition of 2-oxoglutarate and glutamine, and in such circumstances incorporation of¹⁴C-labelled metabolites into starch was reduced. Glucose 6-phosphate proved to be a better substrate for oxidative pathways than Glc1P. Our results suggest that Glc1P is the primary substrate for starch synthesis in developing wheat endosperm, and that ATP required for starch synthesis is imported via an adenylate translocator.     

Characterization of ADP-glucose transport across the cereal endosperm amyloplast envelope

Most of the carbon used for starch biosynthesis in cereal endosperms is derived from ADP-glucose (ADP-Glc) synthesized by extra-plastidial AGPase activity, and imported directly across the amyloplast envelope. The properties of the wheat endosperm amyloplast ADP-Glc transporter were analysed with respect to substrate kinetics and specificities using reconstituted amyloplast envelope proteins in a proteoliposome-based assay system, as well as with isolated intact organelles. Experiments with liposomes showed that ADP-Glc transport was dependent on counter-exchange with other adenylates. Rates of ADP-Glc transport were highest with ADP and AMP as counter-exchange substrates, and kinetic analysis revealed that the transport system has a similar affinity for ADP and AMP. Measurement of ADP and AMP efflux from intact amyloplasts showed that, under conditions of ADP-Glc-dependent starch biosynthesis, ADP is exported from the plastid at a rate equal to that of ADP-Glc utilization by starch synthases. Photo-affinity labelling of amyloplast membranes with the substrate analogue 8-azido-[alpha-32P]ADP-Glc showed that the polypeptide involved in substrate binding is an integral membrane protein of 38 kDa. This study shows that the ADP-Glc transporter in cereal endosperm amyloplasts imports ADP-Glc in exchange for ADP which is produced as a by-product of the starch synthase reaction inside the plastid.     

Immunocytochemical Localization of ADPglucose Pyrophosphorylase in Developing Potato Tuber Cells

The subcellular localization of ADPglucose pyrophosphorylase, a key regulatory enzyme in starch biosynthesis, was determined in developing potato tuber cells by immunocytochemical localization techniques at the light microscopy level. Specific labeling of ADPglucose pyrophosphorylase by either immunofluorescence or immunogold followed by silver enhancement was detected only in the amyloplasts and indicates that this enzyme is located exclusively in the amyloplasts in developing potato tuber cells. Labeling occurred on the starch grains and, in some instances, specific labeling patterns were evident which may be related to sites active in starch deposition.     

Is There an Alternative Pathway for Starch Synthesis?

In leaf tissue, carbon enters starch via the gluconeogenesis pathway where d-glycerate 3-phosphate formed from CO₂ fixation is converted into hexose monophosphates within the chloroplast stroma. In starch-containing sink organs, evidence has been obtained indicating that the flow of carbon into starch follows a different pathway whereby hexose monophosphates formed from sucrose are transported into the amyloplast, a plastid specialized in starch accumulation. In both chloroplasts and amyloplasts, the formation of ADPglucose, the substrate for starch synthase, is controlled by the activity of ADPglucose pyrophosphorylase, a key regulatory enzyme of starch synthesis localized in the plastid. Recently, an alternative pathway of starch synthesis has been proposed in which ADPglucose is synthesized from sucrose and transported directly into the plastid compartment, where it is used for starch synthesis. On the basis of the biochemical phenotypes exhibited by various plant mutants with defined genetic lesions, it is concluded that ADPglucose pyrophosphorylase is essential for starch synthesis, whereas the alternative pathway has only a minor role in this process.     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

Localization of Branching Enzyme in Potato Tuber Cells with the Use of Immunoelectron Microscopy

Potato branching enzyme, a key enzyme in the biosynthesis of starch, was localized in amyloplasts in starch-storage cells of potato (Solanum tuberosum L.) with the use of immunogold electron microscopy. Branching enzyme was found in the amyloplast stroma, concentrated at the interface of the stroma and the surface of the starch granule. ADP-glucose pyrophosphorylase, a key regulatory enzyme in starch synthesis, was localized for comparison to exclude possible artifacts. ADP-glucose pyrophosphorylase, in contrast with branching enzyme, proved to be evenly distributed throughout the stroma. Branching enzyme also appears to be present in a membrane-bounded inclusion body in the stroma, whereas ADP-glucose pyrophosphorylase is not. The presence of branching enzyme predominantly at the surface of the starch granule indicates that branching takes place at that surface and not throughout the amyloplast stroma.     

Metabolic pathways of the wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) endosperm amyloplast revealed by proteomics

Background  

By definition, amyloplasts are plastids specialized for starch production. However, a proteomic study of amyloplasts isolated from wheat (Triticum aestivum Butte 86) endosperm at 10 days after anthesis (DPA) detected enzymes from many other metabolic and biosynthetic pathways. To better understand the role of amyloplasts in food production, the data from that study were evaluated in detail and an amyloplast metabolic map was outlined.     

Gravitropism and starch statoliths in an Arabidopsis mutant

The mutant TC 7 of Arabidopsis thaliana (L.) Heynh. has been reported to be starch-free and still exhibit root gravitropism (T. Caspar and B. G. Pickard 1989, Planta 177, 185–197). This is not consistent with the hypothesis that plastid starch has a statolith function in gravity perception. In the present study, initial light microscopy using the same mutant showed apparently starch-free statocytes. However, ultrastructural examination detected residues of amyloplast starch grains in addition to the starch-depleted amyloplasts. Applying a point-counting morphometric method, the starch grains in the individual amyloplasts in the mutant were generally found to occupy more than 20% and in a few cases up to 60% of the amyloplast area. In the wild type (WT) the starch occupied on average 98 % of the amyloplast area and appeared as densely packed grains. The amyloplasts occupied 13.9% of the area of the statocyte in the mutant and 23.3% of the statocyte area in the WT. Sedimentation of starch-depleted amyloplasts in the mutant was not detected after 40 min of inversion while in the WT the amyloplasts sedimented at a speed of 6 m · h^-1. The gravitropic reactivity and the curvature pattern were also examined in the WT and the mutant. The time-courses of root curvature in the WT and the mutant showed that when cultivated under standard conditions for 60 h in darkness, the curvatures were 83° and 44°, respectively, after 25 h of continuous stimulation in the horizontal position. The WT roots curved significantly more rapidly and with a more normal gravitropic pattern than those of the mutant. These results are discussed in relation to the results previously obtained with the mutant and with respect to the starch-statolith hypothesis.Abbreviation WT wild type This work was supported by grants from Norwegian Research Council for Science and the Humanities (NAVF) which we gratefully acknowledge. We would also like to thank Dr. Timothy Caspar, Michigan State University, East Lansing, USA, for providing us with the seeds of TC 75.