Multiple forms of maize endosperm adp-glucose pyrophosphorylase and their control by shrunken-2 and brittle-2

Heat-labile and heat stable forms of ADP-glucose pyrophosphorylase were identified in the maize endosperm. The heat-labile form is destroyed by normal electrophoretic conditions. The heat-stable form corresponds to pyrophosphorylase B. In wild type, 96% of the total activity is heat labile. Both forms are reduced in 11 brittle-2 (bt2) and 12 shrunken-2 (sh2) mutants. The heat-labile form is reduced to a greater extent than is the heat-stable form in each of the 23 mutants. Deletion of sh2 abolishes both forms. The original ratio of the two forms is restored after sh2 function is expressed via transposition of Dissociation from sh2. The possible roles of these genes in the control of ADP-glucose pyrophosphorylase are discussed.     

Increased hexokinase levels in endosperm of mutant maize

Hexokinase activity was measured in endosperms of shrunken-2 (sh₂) and starchy maize. Initial increases in hexokinase were observed for developing endosperms of both genotypes, and the enzyme declined in both as the seeds matured. A higher level of hexokinase was observed in developing sh₂ than in starchy endosperm. This difference persisted throughout maturation and occurred also in germinating seeds. Soluble hexokinase activity per endosperm continued to increase in sh₂ for about 8 days (22–30 days after pollination) after the enzyme in starchy endosperm had attained maximum activity and begun to decline. Hexokinase was predominantly soluble in both genotypes so the differences observed are not due to altered distribution of enzyme between particulate and soluble fractions.     

Effect of sucrose accumulation on zein synthesis in maize starch-deficient mutants

Starch-deficient maize (Zea mays) mutants, brittle-2 (bt2), brittle-1 (bt), and shrunken-2 (sh2), which accumulated large quantities of sucrose, had less than normal amounts of zein (the major storage protein) in the endosperm. Reduction of zein synthesis in the starch-deficient mutants was negatively correlated with the accumulation of sucrose and low osmotic potential in the developing endosperms. When radioactive amino acids were injected into the shank below ears that segregated for the starch-deficient mutant and normal kernels at 28 days post-pollination, mutant kernels absorbed only ca 22–36% of the labelled amino acids found in their normal controls. Thus, a low osmotic potential in the mutant endosperm may favour water movement but reduce solute movement. The inability of amino acids to move into the mutant endosperms, therefore, in part explains the reduction of zein accumulation in starch-deficient mutant endosperms.     

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.     

Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants

ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds and is likely the most important determinant of seed strength. The Escherichia coli mutant glgC gene (glgC16), which encodes a highly active and allosterically insensitive AGPase, was introduced into maize (Zea mays L.) under the control of an endosperm-specific promoter. Developing seeds from transgenic maize plants showed up to 2–4-fold higher levels of AGPase activity in the presence of 5 mM inorganic phosphate (Pi). Transgenic plants with higher cytoplasmic AGPase activity under Pi-inhibitory conditions showed increases (13–25%) in seed weight over the untransformed control. In addition, in all transgenic maize plants, the seeds were fully filled, and the seed number of transgenic plants had no significant difference compared with that of untransformed control. These results indicate that increasing cytoplasmic AGPase activity has a marked effect on sink activity and, in turn, seed weight in transgenic maize plants.     

Gene expression studies on developing kernels of maize sucrose synthase (SuSy) mutants show evidence for a third SuSy gene

Previous studies have identified two tissue- and cell-specific, yet functionally redundant, sucrose synthase (SuSy) genes, Sh1 and Sus1, which encode biochemically similar isozymes, SH1 and SUS1 (previously referred to as SS1 and SS2, respectively). Here we report evidence for a third SuSy gene in maize, Sus3, which is more similar to dicot than to monocot SuSys. RNA and/or protein blot analyses on developing kernels and other tissues show evidence of expression of Sus3, although at the lowest steady-state levels of the three SuSy gene products and without a unique pattern of tissue specificity. Immunoblots of sh1sus1-1 embryos that are either lacking or deficient for the embryo-specific SUS1 protein have shown a protein band which we attribute to the Sus3 gene, and may contribute to the residual enzyme activity seen in embryos of the double mutant. We also studied developing seeds of the double mutant sh1sus1-1, which is missing 99.5% of SuSy enzyme activity, for evidence of co-regulation of several genes of sugar metabolism. We found a significant reduction in the steady-state levels of Miniature-1 encoded cell wall invertase2, and Sucrose transporter (Sut) mRNAs in the double mutant, relative to the lineage-related sh1Sus1 and sh1Sus1 kernels. Down-regulation of the Mn1 gene was also reflected in significant reductions in cell wall invertase activity. Co-regulatory changes were not seen in the expression of Sucrose phosphate synthase, UDP-glucose pyrophosphorylase, and ADP-glucose pyrophosphorylase.     

Starch-synthesizing Enzymes in the Endosperm and Pollen of Maize

Two mutations, amylose-extender and waxy, which affect the proportion of amylose and amylopectin of starch synthesized in the endosperm of maize (Zea mays L.) seeds, are also expressed in the pollen. However, most mutations that affect starch synthesis in the maize endosperm are not expressed in the pollen. In an attempt to understand the nonconcordance between the endosperm and pollen, extracts of mature pollen grains were assayed for a number of the enzymes possibly implicated in starch synthesis in the endosperm. Sucrose synthetase (sucrose-UDP glucosyl transferase, EC 2.4.1.13) activity was not detectable in either mature or immature pollen grains of nonmutant maize, but both bound and soluble invertase (EC 3.2.1.26) exhibited much greater specific activity (per milligram protein) in pollen extracts than in 22-day-old endosperm extracts. Phosphorylase (EC 2.4.1.1) activity was also higher in pollen than in endosperm extracts. ADP-Glucose pyrophosphorylase (EC 2.7.7.27) activity was much lower in pollen than endosperm extracts, but mutations that drastically reduced ADP-glucose pyrophosphorylase activity in the endosperm (brittle-2 and shrunken-2) did not markedly affect enzymic activity in the pollen. Specific activities of other enzymes implicated in starch synthesis were similar in endosperm and pollen extracts.     

Biosynthesis of bacterial glycogen. Kinetic studies of a glucose-1-phosphate adenylyltransferase (EC 2.7.7.27) from a glycogen-deficient mutant of Escherichia coli B.

An Escherichia coli B mutant, SG14, accumulates glycogen at 28% the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to alpha- and beta-amylosis, chain length determination, and I2-complex absorption spectra. The SG14 mutant contains normal glycogen synthase and branching enzyme activity but has an ADP-glucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and requires a 12-fold higher concentration of fructose-P2 or a 26 fold higher concentration of pyridoxal-P than the parent type enzyme for 50% of maximal allosteric activation. TPNH, an effective activator of the E. coli B enzyme, does not activate the SG14 ADP-glucose pyrophosphorylase. Other studies show that for the SG14 enzyme the concentrations of ATP and Mg2+ in the synthesis direction and the concentrations of ADP-glucose and PPi in the pyrophosphorolysis direction required to give 50% of maximal activity are 3- to 6-fold higher than those observed for the parent E. coli B ADP-glucose pyrophosphorylase. The Km for alpha-glucose-1-P at saturating to half-saturating concentrations of the activator, fructose-P2, are about the same for both enzymes. However, in the presence of no activator, the concentration of glucose-1-P required for half-maximal activity is about 1.8-fold higher for the SG14 enzyme. Thus SG14 ADP-glucose pyrophosphorylase has lower affinity for its substrates than does the parent enzyme. Previously the SG14 enzyme had been shown to be less sensitive to inhibition by 5'-AMP than the E. coli B enzyme. This ensensitivity to inhibition renders the SG14 enzyme less responsive to energy charge than the E. coli B ADP-glucose pyrophosphorylase. On the basis of the above results and taking into account the reported concentrations of fructose-P2, of pyridoxal-P, and of the adenine nucleotide pool and its energy charge in E. coli strains, it is concluded that furctose-P2 is the important physiological allosteric activator of E. coli ADP-glucose pyrophosphorylase. Furthermore, the 1.7-fold increased rate of accumulation of glycogen observed when E. coli B or SG14 shifts from exponential phase to stationary phase of growth in nitrogen-limiting media can be accounted for by the 2.4-fold increase of the levels of the glycogen biosynthetic enzymes, glycogen synthase, and ADP-glucose pyrophosphorylase. Thus both allosteric regulation of the ADP-glucose pyrophosphorylase as well as the genetic regulation of the biosynthesis of the glycogen biosynthetic enzymes are involved in the regulation of glycogen accumulation in E. coli B.     

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.     

Starchless mutants of Chlamydomonas reinhardtii lack the small subunit of a heterotetrameric ADP-glucose pyrophosphorylase

ADP-glucose synthesis through ADP-glucose pyrophosphorylase defines the major rate-controlling step of storage polysaccharide synthesis in both bacteria and plants. We have isolated mutant strains defective in the STA6 locus of the monocellular green alga Chlamydomonas reinhardtii that fail to accumulate starch and lack ADP-glucose pyrophosphorylase activity. We show that this locus encodes a 514-amino-acid polypeptide corresponding to a mature 50-kDa protein with homology to vascular plant ADP-glucose pyrophosphorylase small-subunit sequences. This gene segregates independently from the previously characterized STA1 locus that encodes the large 53-kDa subunit of the same heterotetramer enzyme. Because STA1 locus mutants have retained an AGPase but exhibit lower sensitivity to 3-phosphoglyceric acid activation, we suggest that the small and large subunits of the enzyme define, respectively, the catalytic and regulatory subunits of AGPase in unicellular green algae. We provide preliminary evidence that both the small-subunit mRNA abundance and enzyme activity, and therefore also starch metabolism, may be controlled by the circadian clock.     

Expression of a modified ADP-glucose pyrophosphorylase large subunit in wheat seeds stimulates photosynthesis and carbon metabolism

ADP-glucose pyrophosphorylase (AGP) is the rate-limiting step in seed starch biosynthesis. Expression of an altered maize AGP large subunit (Sh2r6hs) in wheat (Triticum aestivum L.) results in increased AGP activity in developing seed endosperm and seed yield. The yield phenotype involves increases in both seed number and total plant biomass. Here we describe stimulation of photosynthesis by the seed-specific Sh2r6hs transgene. Photosynthetic rates were increased in Sh2r6hs-expressing plants under high light but not low light growth conditions, peaking at roughly 7 days after flowering (DAF). In addition, there were significant increases in levels of fructose, glucose, and sucrose in flag leaves at both 7 and 14 DAF. In seeds, levels of carbon metabolites at 7 and 14 DAF were relatively unchanged but increases in glucose, ADP-glucose, and UDP-glucose were observed in seeds from Sh2r6hs positive plants at maturity. Increased photosynthetic rates relatively early in seed development appear to be key to the Sh2r6hs enhanced yield phenotype as no yield increase or photosynthetic rate changes were found when plants were grown in a suboptimal light environment. These findings demonstrate that stimulation of biochemical events in both source and sink tissues is associated with Sh2r6hs expression.     

Altered Maize Endosperm Adp-Glucose Pyrophosphorylases from Revertants of a SHRUNKEN-2-DISSOCIATION ALLELE

Four phenotypically wild-type seeds were obtained from separate Activator-induced events in the Dissociation-inhibited allele sh2-ml (shrunken-2, mutable-1). Endosperm adenosine diphosphoglucose pyrophosphorylase, the enzyme controlled by sh2, was extracted and partially purified from the four revertants and was compared to enzyme produced by the progenitor Sh2 allele and the sh2-m allele.

The revertants contained 50 to 140% of the activity conditioned by the progenitor allele. Each of the revertants appears to be unique as judged by differences in Km(glucose-1-PO₄), 3-phosphoglycerate(3-PGA) activation, and phosphate-inhibition. In one case the reversion event apparently increased the sensitivity of ADP-glucose pyrophosphorylate to 3-PGA activation.

     

A shrunken-2 transgene increases maize yield by acting in maternal tissues to increase the frequency of seed development

The maize (Zea mays) shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme, ADP-glucose pyrophosphorylase. Expression of a transgenic form of the enzyme with enhanced heat stability and reduced phosphate inhibition increased maize yield up to 64%. The extent of the yield increase is dependent on temperatures during the first 4 d post pollination, and yield is increased if average daily high temperatures exceed 33 °C. As found in wheat (Triticum aestivum) and rice (Oryza sativa), this transgene increases maize yield by increasing seed number. This result was surprising, since an entire series of historic observations at the whole-plant, enzyme, gene, and physiological levels pointed to Sh2 playing an important role only in the endosperm. Here, we present several lines of evidence that lead to the conclusion that the Sh2 transgene functions in maternal tissue to increase seed number and, in turn, yield. Furthermore, the transgene does not increase ovary number; rather, it increases the probability that a seed will develop. Surprisingly, the number of fully developed seeds is only ～50% of the number of ovaries in wild-type maize. This suggests that increasing the frequency of seed development is a feasible agricultural target, especially under conditions of elevated temperatures.     

Insights into Glycogen Metabolism in Chemolithoautotrophic Bacteria from Distinctive Kinetic and Regulatory Properties of ADP-Glucose Pyrophosphorylase from Nitrosomonas europaea

Nitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO₂ via the Benson-Calvin cycle. Despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. Here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in N. europaea, ADP-glucose pyrophosphorylase and glycogen synthase. In other bacteria, ADP-glucose pyrophosphorylase catalyzes the regulatory step of the synthetic pathway and glycogen synthase elongates the polymer. In starch synthesis in plants, homologous enzymes play similar roles. We purified to homogeneity the recombinant ADP-glucose pyrophosphorylase from N. europaea and characterized its kinetic, regulatory, and oligomeric properties. The enzyme was allosterically activated by pyruvate, oxaloacetate, and phosphoenolpyruvate and inhibited by AMP. It had a broad thermal and pH stability and used different divalent metal ions as cofactors. Depending on the cofactor, the enzyme was able to accept different nucleotides and sugar phosphates as alternative substrates. However, characterization of the recombinant glycogen synthase showed that only ADP-Glc elongates the polysaccharide, indicating that ATP and glucose-1-phosphate are the physiological substrates of the ADP-glucose pyrophosphorylase. The distinctive properties with respect to selectivity for substrates and activators of the ADP-glucose pyrophosphorylase were in good agreement with the metabolic routes operating in N. europaea, indicating an evolutionary adaptation. These unique properties place the enzyme in a category of its own within the family, highlighting the unique regulation in these organisms.     

Starch Synthesis in Developing Potato Tubers

The activities of enzymes involved in starch metabolism were measured at intervals during tuberization and the early stages of tuber growth in Solanum tubersum grown in water culture under controlled environmental conditions. Starch synthase, ADPglucose pyrophosphorylase, UDPglucose pyrophosphorylase and phosphorylase activities all increased during tuber development, the most pronounced increases occurring in the activities of ADP-glucose pyrophosphorylase and phosphorylase. The activity ratio ADPglucose pyrophosphorylase/phosphorylase was lowest in slow growing tubers and hightest in fast growing tubers. In addition, high sugar concentrations in fast growing tubers and low sugar concentrations in slow growing tubers suggested that enzyme levels might be influenced by sugar concentration. The activities of starch synthase, phosphorylase and ADPglucose pyrophosphorylase were increased 2–2.5 fold by the presence of 100 mM K⁺. It is concluded that the major enzyme changes occur as a consequence of tuber initiation and that starch accumulation is controlled, at least in part, by the activities of ADPglucose pyrophosphorylase and phosphorylase.     

Comparison of Starch and ADP-Glucose Pyrophosphorylase Levels in Nonembryogenic Cells and Developing Embryos from Induced Carrot Cultures

Cultures of carrot (Daucus carota L.) in a medium without added 2,4-dichlorophenoxyacetic acid were separated into fractions of embryos at different stages of development (large globular and heart, torpedo, and germinating) and nonembryogenic cells. The average starch content per cell in these fractions was similar. However, due to the smaller sizes of the cells of the embryos relative to the nonembryogenic cells, starch content per weight of tissue was higher in the embryos. The ADP-glucose pyrophosphorylase activity per cell in the nonembryogenic cells was double that of the embryo cells. Furthermore, the ratio of ADP-glucose pyrophosphorylase to starch was over 2-fold higher in the nonembryogenic cells, indicating that starch content is not simply determined by ADP-glucose pyrophosphorylase levels. ADP-glucose pyrophosphorylase activity of all culture fractions was directly proportional to the level of a single 50 kilodalton polypeptide detected by immunoblot analysis, using antiserum raised to the purified spinach leaf enzyme. In the same immunoblot analysis, novel polypeptides of 63 and 100 kilodalton were detected in embryos but were absent from nonembryogenic cells. This is one of the few reported examples of specific proteins which differentially accumulate in embryos and nonembryogenic cells.