Peter L. Keeling

From Glycogen to Amylopectin: A Model for the Biogenesis of the Plant Starch Granule

A major feature of the model we propose is that it gives us access to the third dimension of granule growth. The crystal lamella is a planar arrangement allowing for the three dimensional piling of glucan double helices (Figure 1Figure 1). The amorphous lamella on the other hand will not be planar but space-filling as can be predicted by the synthesis of phytoglycogen. At this stage the processing of phytoglycogen can lead to a variety of three dimensional structures that will allow for three dimensional extension of the amylopectin molecule. It is easy to understand how this is needed to accomodate regular concentric growth of the starch granule. Oostergetel and van Bruggen (1993) have very recently examined sections of potato starch granules by electron optical tomography and by cryo–electron diffraction. Their data imply a superhelical arrangement of both amorphous and crystalline lamellae. Moreover distinct superhelices are interlocked through their respective amorphous and crystalline lamellae to yield a tetragonal symmetry (Figure 3Figure 3). In this three dimensional arrangement, the double helical glucans are pointing in the axis of the superhelix towards the surface of the granule. This will of course allow for synthesis and growth of the crystals at the surface. This structure raises several questions with respect to biosynthesis, namely what determines the superhelical growth and how can this unidirectional growth account for concentric growth of the starch granule. We believe these questions can be presently addressed by our model. If we assume that the branching enzymes are setting the invariant amylopectin cluster size through their minimal catalytic requirements (see above), then once the first turn of the superhelix is synthesized the following turns will be dictated through this requirement. Concentric growth of the granule will call for synthesis of novel superhelices. These can be readily synthesized by allowing the amorphous lamella to fill vacant spaces between the growing superhelices. When sufficient space is available a novel superhelix will be made to grow by induced fit with the neighboring tetragonal organization. Debranching enzymes remain required at the surface to prevent glycogen synthesis and allow the trimming of the amorphous lamellae. The induced fit hypothesis for starch growth only requires the understanding of amylopectin cluster synthesis as proposed in our two dimensional model. Understanding how the first turn of the superhelices are generated will require further insight as to the priming events occurring at the granule core.Figure 3A Superhelical Model for the Three Dimensional Organization of Starch(A) The superhelical three dimensional organization of a section of the starch granule (based onOostergetel and van Bruggen 1993xOostergetel, G.T. and van Bruggen, E.F.J. Carbohydr. Polym. 1993; 21: 7–12Crossref | Scopus (138)See all ReferencesOostergetel and van Bruggen 1993). The top of the figure corresponds to the granules surface. The shaded areas correspond to the amorphous lamellae of the amylopectin molecules.(B) An enlargement of a single turn of the superhelix to display the double helices of the crystal lamellae. The shaded section would have overall structures similar to those shown for the amorphous lamellae in Figure 1Figure 1. Each superhelix is interlocked to neighboring superhelices to generate a tetragonal organization. We propose that vacant spaces are filled with amorphous material until sufficient room is available to yield a novel superhelix.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The Major Form of ADP-Glucose Pyrophosphorylase in Maize Endosperm Is Extra-Plastidial

Preparations enriched in plastids were used to investigate the location of ADP-glucose pyrophosphorylase (AGPase) in the developing endosperm of maize (Zea mays L.). These preparations contained more than 25% of the total activity of the plastid marker enzymes alkaline pyrophosphatase and soluble starch synthase, less than 2% of the cytosolic marker enzymes alcohol dehydrogenase and pyrophosphate, fructose 6-phosphate 1-phosphotransferase, and approximately 3% of the AGPase activity. Comparison with the marker enzyme distribution suggests that more than 95% of the activity of AGPase in maize endosperm is extra-plastidial. Two proteins were recognized by antibodies to the small subunit of AGPase from maize endosperm Brittle-2 (Bt2). The larger of the two proteins was the major small subunit in homogenates of maize endosperm, and the smaller, less abundant of the two proteins was enriched in preparations containing plastids. These results suggest that there are distinct plastidial and cytosolic forms of AGPase, which are composed of different subunits. Consistent with this was the finding that the bt2 mutation specifically eliminated the extra-plastidial AGPase activity and the larger of the two proteins recognized by the antibody to the Bt2 subunit.

Elevated temperature reduces starch deposition in wheat endosperm by reducing the activity of soluble starch synthase

Temperatures of more than 25° C adversely affect the activity of soluble starch synthase (SSS), an amyloplastic enzyme, in endosperm of wheat (Triticum aestivum L. cv. Mardler). Enzyme rate was found to have a temperature optimum between 20 and 25°C. This effect was apparently reversible after a short period of exposure to elevated temperature. We also found that with a prolonged period of exposure to elevated temperature there was another temperature-related phenomenon which caused a loss of enzyme activity that appeared to be much slower to reverse. We have termed this effect of temperature on SSS activity “knockdown”. The knockdown in SSS activity also occurred in-vivo. However, elevated temperature did not affect the activities of several other enzymes in the pathway of starch synthesis (ADP-glucose pyrophosphorylase, UDP-glucose pyrophosphorylase, sucrose synthase, phosphoglucomutase, phosphoglucose isomerase, bound starch synthase or hexokinase). Because the knockdown effect appeared to be specific to the enzyme SSS, we quantified the effect of knockdown on flux of carbon into starch and used these data to calculate the flux-control coefficient for SSS. Using data at 10–20°C the flux-control coefficient was CStarch10–20C = 0.50, whereas at 20–30° C the flux-control coefficient was CStarch20–30C = 1.38, and between 30–40°C the flux-control coefficient was CStarch30–40C = 0.69. Using data at 10–30°C the flux-control coefficient was CStarch10–30C = 1.15, and at 10–40°C the flux-control coefficient was CStarch10–40C = 0.82. In conclusion, we suggest that SSS is a major site of regulation of starch synthesis in developing wheat grain. During periods of high temperature the control point in the pathway of starch synthesis is apparently not associated exclusively with ADP-glucose pyrophosphorylase. In field conditions, in which temperatures are fluctuating, there will likely be periods of control of starch synthesis being exerted predominantly by SSS. During periods at lower temperature, control of flux may be exerted by SSS, perhaps in combination with other flux-controlling enzymes in the pathway. Our data point-out a crucial new aspect of quantifying control strengths of enzymes in plants: the determination of enzyme control strengths should be done in carefully regulated temperature conditions. Thus, since temperature is a major determinant of real flux through a pathway and the individual enzymes can respond differently to changing temperature conditions, the control strengths of individual steps in a pathway may vary with changing environmental conditions. This is particularly pronounced in starch deposition, because of the temperature instability of SSS.

Proteins from Multiple Metabolic Pathways Associate with Starch Biosynthetic Enzymes in High Molecular Weight Complexes: A Model for Regulation of Carbon Allocation in Maize Amyloplasts

Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds.

Physical Association of Starch Biosynthetic Enzymes with Starch Granules of Maize Endosperm (Granule-Associated Forms of Starch Synthase I and Starch Branching Enzyme II)

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.

Biochemistry and Genetics of Starch Synthesis

Enormous progress has been made in understanding the genetics and biochemistry of starch synthesis in crop plants. Furthermore, starch remains at the very epicenter of the worlds food and feed chains and has even now become one of the worlds most important sources of biorenewable energy (biofuel). Yet, despite this remarkable progress and the obvious economic importance, very little has been achieved in terms of adding value to starch or increasing starch yield, particularly in cereal crops. Here, we review the genetics and biochemistry of starch synthesis in crop plants, particularly maize. With all this know-how in place and a chasm of opportunity ahead, the time is right to see science deliver progress into a new frontier. Thus, in our view the stage is set for a new era of changes in starch synthesis, delivering enhancements in functionality and yield.

Influence of Gene Dosage on Carbohydrate Synthesis and Enzymatic Activities in Endosperm of Starch-Deficient Mutants of Maize

In cereals, starch is synthesized in endosperm cells, which have a ploidy level of three. By studying the allelic dosage of mutants affecting starch formation in maize (Zea mays L.) kernels, we determined the effect of down-regulated enzyme activity on starch accumulation and the activity of associated enzymes of carbohydrate metabolism. We found a direct relationship between the amount of starch produced in the endosperm and the gene dosage of amylose extender-1, brittle-2, shrunken1, and sugary-1 mutant alleles. Changes in starch content were found to be caused by changes in the duration as well as the rate of starch synthesis, depending on the mutant. Branching enzyme, ADP-glucose pyrophosphorylase, and sucrose synthase activities were linearly reduced in endosperm containing increasing dosages of amylose extender-1, brittle-2, and shrunken-1 alleles, respectively. De-branching enzyme activity declined only in the presence of two or three copies of sugary-1. No enzyme-dosage relationship occurred with the dull1 mutant allele. All mutants except sugary-1 displayed large increases (approximately 2- to 5-fold) in activity among various enzymes unrelated to the structural gene. This occurred in homozygous recessive genotypes, as did elevated concentrations of soluble sugars, and differed in magnitude and distribution among enzymes according to the particular mutation.

MAIZE STARCH FINE STRUCTURES AFFECTED BY EAR DEVELOPMENTAL TEMPERATURE

Abstract Growing temperature is known to affect the grain yield and quality of maize. Two genetically unrelated normal dent maize inbreds, ICI63 and ICI92, with different heterotic backgrounds were grown in a greenhouse with the ears wrapped in temperature control devices set at 25 and 35 °C during the grain-filling period. Grain yield, kernel weight, and kernel density were less for ears at 35 °C than for those at 25 °C. The extent of the loss, however, varied with the variety: 13.1 and 37.9% kernel weight loss and 8.47 and 10.08% density loss for ICI63 and ICI92, respectively. The starch granular shape of ICI63 became more oval-shaped, but there was no shape change for ICI92. As developmental temperature increased, starch granule size decreased and gelatinization temperature increased. With increasing developmental temperature, the true amylose content of ICI63, determined by iodine affinity, decreased 2.39% and that for ICI92 decreased 2.20%; amylose molecular size of both varieties also decreased. Size exclusion chromatography and high-performance anion-exchange chromatography revealed an increased medium branch-chain fraction and decreased long and short branch-chain fractions for ICI63 amylopectin, whereas ICI92 amylopectin had increased long and medium branch-chain fractions and a decreased short branch-chain fraction, when the ear developed at 35 °C.

The effect of waxy mutations on the granule-bound starch synthases of barley and maize endosperms

The effects of waxy mutations on starch-granule-bound starch synthases (EC 2.4.1.18) in the developing endosperm of barley (Hordeum vulgare L.) and maize (Zea mays L.) have been investigated. Three granule-bound starch synthases in barley endosperm were identified by use of antibodies to known starch synthases, by reconstitution and assay of individual proteins from sodium dodecyl sulphate-polyacrylamide gels of granule-bound proteins, and by partial purification of proteins released by enzymic digestion of starch. These are proteins of 60, 77 and 90 kDa. Use of antibodies to known starch synthases and partial purification of proteins released by enzymic digestion of starch indicated that there may be at least four granule-bound starch synthases in maize endosperm: proteins of 59, 74, 77 and 83 kDa. Mutations at the waxy loci of both species affected only the 60- (barley) and 59-(maize) kDa isoforms. No evidence was found that other putative isoforms are altered in abundance or activity by the mutations. The contribution of our results to understanding of the starch synthase activity of intact starch granules and the mechanism of amylose synthesis is discussed.

Temperature effect on retrogradation rate and crystalline structure of amylose

Abstract Commercial potato amylose was used to study temperature effects on the retrogradation of amylose solutions (3.5mg/ml). The retrogradation rate decreased as incubation temperature increased (5 to 45 °C). The degree of retrogradation within 24 h decreased from 58.8 to 7.1% as incubation temperature increased from 5 to 45 °C. In the amylose solution, different-sized molecular subfractions retrograded at different rates. After incubating at 5 °C for 100 days, the majority of the amylose molecules retrograded and precipitated from the solution; at 45 °C, only amylose of the small-molecular subfraction (number average, DP n = 110; weight average, DP w = 150) retrograded and precipitated. Entanglement of molecules was observed in size exclusion chromatograms. The morphology of retrograded amylose observed by using a scanning electron microscope differed with the retrogradation temperature. The chain length of amylose crystalline segments, prepared by hydrolysis of retrograded amylose, showed a narrow distribution (polydispersity from 1.21 to 1.67). The chain lengths of resistant segments increased DP n from 39 to 52 and DP w from 47 to 72 for α-amylolysis and DP n from 34 to 40 and DP w from 48 to 67 for 16% sulfuric acid hydrolysis, when incubation temperature increased from 5 to 45 °C.