Subcellular localization of ADPglucose pyrophosphorylase in developing wheat endosperm and analysis of the properties of a plastidial isoform

The intracellular location of ADPglucose pyrophosphorylase (AGPase) in wheat during endosperm development was investigated by analysis of the recovery of marker enzymes from amyloplast preparations. Amyloplast preparations contained 20-28% of the total endosperm activity of two plastidial marker enzymes and less than 0.8% of the total endosperm activity of two cytosolic marker enzymes. Amylo plasts prepared at various stages of development, from 8-30 d post anthesis, contained between 2% and 10% of the total AGPase activity; this implies that between 7% and 40% of the AGPase in wheat endosperm is plastidial during this period of development. Two proteins were recognized by antibodies to both the large and small subunits of wheat AGPase. The larger of the two AGPases was the major form of the enzyme in whole cell extracts, and the smaller, less abundant, form of AGPase was enriched in plastid preparations. The results are consistent with data from other graminaceous endosperms, suggesting that there are distinct plastidial and cytosolic isoforms of AGPase composed of different subunits. The plastidial isoform of AGPase from wheat endosperm is relatively insensitive to the allosteric regulators 3-phosphoglycerate and inorganic orthophos phate compared with plastidial AGPase from other species. Amyloplast AGPase showed no sensitivity to physiological concentrations of inorganic orthophosphate. 15 mM 3-phosphoglycerate caused no stimulation of the pyrophosphorolytic reaction, and only 2-fold stimulation of the ADPglucose synthesizing reaction.     

Two Paralogous Genes Encoding Small Subunits of ADP-glucose Pyrophosphorylase in Maize, <Emphasis Type="Italic">Bt2</Emphasis> and <Emphasis Type="Italic">L2</Emphasis>, Replace the Single Alternatively Spliced Gene Found in Other Cereal Species

Two types of gene encoding small subunits (SSU) of ADP-glucose pyrophosphorylase, a starch-biosynthetic enzyme, have been found in cereals and other grasses. One of these genes encodes two SSU proteins. These are targeted to different subcellular compartments and expressed in different organs of the plant: the endosperm cytosol and the leaf plastids. The SSU gene encoding two proteins evolved from an ancestral gene encoding a single protein by the acquisition of an alternative first exon. Prior to the work reported here, this type of SSU gene had been found in all grasses examined except maize. In maize, two separate genes, Bt2 and L2, were known to have the same roles as the alternatively spliced gene found in other grasses. The evolutionary origin of these maize genes and their relationship to the SSU genes in other grasses were unclear. Here we show that Bt2 and L2 are paralogous genes that arose as a result of the tetraploidization of the maize genome. Both genes derive from an ancestral alternatively spliced SSU gene orthologous to that found in other grasses. Following duplication, the Bt2 and L2 genes diverged in function. Each took a different one of the two functions of the ancestral gene. Now Bt2 encodes the endosperm cytosolic SSU but does not contribute significantly to leaf AGPase activity. Similarly, L2 has lost the use of one of its two alternative first exons. It can no longer contribute to the endosperm cytosolic SSU but is probably responsible for the bulk of the leaf AGPase SSU. Reviewing Editor: Dr. Patrick Keeling Sequences have been deposited in GenBank under accession numbers AY727927, DQ118037, and DQ118038.     

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.     

A low-starch barley mutant,risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase,reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Ris? 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Ris? 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.     

Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize

Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.     

Isolation of a heat-stable maize endosperm ADP-glucose pyrophosphorylase variant

Heat stress reduces maize yield and several lines of evidence suggest that the heat lability of maize endosperm ADP-glucose pyrophosphorylase (AGPase) contributes to this yield loss. AGPase catalyzes a rate-limiting step in starch synthesis. Herein, we present a novel maize endosperm AGPase small subunit variant, termed BT2-TI that harbors a single amino acid change of residue 462 from threonine to isoleucine. The mutant was isolated by random mutagenesis and heterologous expression in a bacterial system. BT2-TI exhibits enhanced heat stability compared to wildtype maize endosperm AGPase.The TI mutation was placed into another heat-stable small subunit variant, MP. MP is composed of sequences from the maize endosperm and the potato tuber small subunit. The MP-TI small subunit variant exhibited greater heat stability than did MP. Characterization of heat stability as well as kinetic and allosteric properties suggests that MP-TI may lead to increased starch yield when expressed in monocot endosperms.     

Heat stability of maize endosperm ADP-glucose pyrophosphorylase is enhanced by insertion of a cysteine in the N terminus of the small subunit

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis. However, plant AGPases differ in several parameters, including spatial and temporal expression, allosteric regulation, and heat stability. AGPases of cereal endosperms are heat labile, while those in other tissues, such as the potato (Solanum tuberosum) tuber, are heat stable. Sequence comparisons of heat-stable and heat-labile AGPases identified an N-terminal motif unique to heat-stable enzymes. Insertion of this motif into recombinant maize (Zea mays) endosperm AGPase increased the half-life at 58 degrees C more than 70-fold. Km values for physiological substrates were unaffected, although Kcat was doubled. A cysteine within the inserted motif gives rise to small subunit homodimers not found in the wild-type maize enzyme. Placement of this N-terminal motif into a mosaic small subunit containing the N terminus from maize endosperm and the C terminus from potato tuber AGPase increases heat stability more than 300-fold.     

Subtle structural differences crucial for function in similarly engineered ADP-glucose pyrophosphorylase larger subunit in rice and maize

ADP ?C glucose pyrophosphorylase (AGPase) is a key enzyme for starch synthesis in plants. Heterotetrameric plant AGPase is encoded by two genes, Shrunken-2 (Sh2) and Brittle-2 (Bt2). The Sh2 gene encodes regulatory larger subunit and the Bt2 gene encodes smaller subunit having catalytic properties. A specific mutation in Sh2 gene involving insertion of six nucleotides, without changing the reading frame, resulted in the insertion of two additional amino acid residues serine and tyrosine at specific position at carboxyl end and also in an increase in seed weight up to 11?C17%. No increase in seed weight with the same insertion in larger subunit of AGPase enzyme in rice was observed even though the rice and maize subunits have 93% of sequence similarity. In this study, the predicted 3D-structures of larger subunit of normal as well as mutated AGPase in maize and rice, were analyzed and superposed. The segment of six amino acid residues long secondary structure just before the site of insertion of additional amino acids (serine and tyrosine) got reduced in case of mutated maize but not in mutated rice. Therefore, the six residue sequence and corresponding subtle secondary structural difference might be the key factors for functional disparity of engineered larger subunits in the two crops.     

The potato tuber,maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.     

Both subunits of ADP-glucose pyrophosphorylase are regulatory

The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.     

Relative turnover numbers of maize endosperm and potato tuber ADP-glucose pyrophosphorylases in the absence and presence of 3-phosphoglyceric acid

Adenosine diphosphate glucose pyrophosphorylase (AGPase; EC 2.7.7.27) synthesizes the starch precursor, ADP-glucose. It is a rate-limiting enzyme in starch biosynthesis and its activation by 3-phosphoglyceric acid (3PGA) and/or inhibition by inorganic phosphate (Pi) are believed to be physiologically important. Leaf, tuber and cereal embryo AGPases are highly sensitive to these effectors, whereas endosperm AGPases are much less responsive. Two hypotheses can explain the 3PGA activation differences. Compared to leaf AGPases, endosperm AGPases (i) lack the marked ability to be activated by 3PGA or (ii) they are less dependent on 3PGA for activity. The absence of purified preparations has heretofore negated answering this question. To resolve this issue, heterotetrameric maize ( Zea mays L.) endosperm and potato ( Solanum tuberosum L.) tuber AGPases expressed in Escherichia coli were isolated and the relative amounts of enzyme protein were measured by reaction to antibodies against a motif resident in both small subunits. Resulting reaction rates of both AGPases are comparable in the presence but not in the absence of 3PGA when expressed on an active-protein basis. We also placed the potato tuber UpReg1 mutation into the maize AGPase. This mutation greatly enhances 3PGA sensitivity of the potato AGPase but it has little effect on the maize AGPase. Thirdly, lysines known to bind 3PGA in potato tuber AGPase, but missing from the maize endosperm AGPase, were introduced into the maize enzyme. These had minimal effect on maize endosperm activity. In conclusion, the maize endosperm AGPase is not nearly as dependent on 3PGA for activity as is the potato tuber AGPase.     

The two AGPase subunits evolve at different rates in angiosperms, yet they are equally sensitive to activity-altering amino acid changes when expressed in bacteria

The rate of protein evolution is generally thought to reflect, at least in part, the proportion of amino acids within the protein that are needed for proper function. In the case of ADP-glucose pyrophosphorylase (AGPase), this premise led to the hypothesis that, because the AGPase small subunit is more conserved compared with the large subunit, a higher proportion of the amino acids of the small subunit are required for enzyme activity compared with the large subunit. Evolutionary analysis indicates that the AGPase small subunit has been subject to more intense purifying selection than the large subunit in the angiosperms. However, random mutagenesis and expression of the maize (Zea mays) endosperm AGPase in bacteria show that the two AGPase subunits are equally predisposed to enzyme activity-altering amino acid changes when expressed in one environment with a single complementary subunit. As an alternative hypothesis, we suggest that the small subunit exhibits more evolutionary constraints in planta than does the large subunit because it is less tissue specific and thus must form functional enzyme complexes with different large subunits. Independent approaches provide data consistent with this alternative hypothesis.     

ADP-glucose pyrophosphorylase is located in the plastid in developing tomato fruit

The subcellular location of activity and protein of ADP-glucose pyrophosphorylase (AGPase) in developing tomato (Lycopersicon esculentum) fruit was determined following a report that the enzyme might be present inside and outside the plastids in this organ. Plastids prepared from crude homogenates of columella and pericarp, the starch-accumulating tissues of developing fruit, contained 8% to 18% of the total activity of enzymes known to be confined to plastids, and 0.2% to 0.5% of the total activity of enzymes known to be confined to the cytosol. The proportion of the total activity of AGPase in the plastids was the same as that of the enzymes known to be confined to the plastid. When samples of plastid and total homogenate fractions were subjected to immunoblotting with an antiserum raised to AGPase, most or all of the protein detected was plastidial. Taken as a whole, these data provide strong evidence that AGPase is confined to the plastids in developing tomato fruit.     

Isolation and characterization of <Emphasis Type="Italic">Brittle2</Emphasis> promoter from <Emphasis Type="Italic">Zea Mays</Emphasis> and its comparison with <Emphasis Type="Italic">Ze19</Emphasis> promoter in transgenic tobacco plants

ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression in embryos, which is different from similar promoters tested in maize.     

Intracellular immunolocalization of adenosine 5′-diphosphoglucose pyrophosphorylase in developing endosperm cells of maize (Zea mays L.)

We present immuno-electron microscopic evidence to show that ADPglucose pyrophosphorylase (AGP, EC 2.7.7.27) encoded by the Sh2 (shrunken 2) and the Bt2 (brittle 2) genes is localized to amyloplasts in developing endosperm of maize. The three AGP antibodies, including the two maize antisera, each raised against the Sh2encoded large or the Bt2-encoded small subunit and the spinach leaf protein, showed strong immuno-gold signals on developing starch grains in amyloplasts. The maize antibodies, but not the spinach, detected additional cross-reactivity sites to endosperm cell wall. Similar endosperm sections of the sh2-null mutant, lacking the Sh2-encoded subunit, yielded a drastic reduction in immuno signal on both starch grains and cell wall with the Sh2 anti-serum. However, the Bt2 and the spinach antisera showed no detectable difference between the sh2-null and the wild-type genotypes, except that the spinach antisera showed no reactivity to the cell wall in either of the two genotypes. Because the Bt2 epitope was readily detectable on the sh2-null starch grains, we suggest that the Bt2 subunit of this heteromeric enzyme is able to target itself to the organelle. The amyloplastic localization of the AGP protein in our studies is given additional significance by recent molecular data which indicate that full-length cDNA clones of the Sh2 and Bt2 genes show no cleavable transit-peptide signal sequence in their deduced aminoacid sequences. The observed disparity between the molecular and immunocytochemical data described here is discussed in the context of other proteins engaged in intracellular translocation with and without the known signal sequences.