Maize endosperm ADP-glucose pyrophosphorylase SHRUNKEN2 and BRITTLE2 subunit interactions

ADP-glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 (Sh2) and Brittle2 (Bt2) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants (sim) also identified motifs essential for protein interactions.     

QTLs for enzyme activities and soluble carbohydrates involved in starch accumulation during grain filling in maize

ADPglucose, the essential substrate for starch synthesis, is synthesized in maize by a pathway involving at least invertases, sucrose synthase, and ADPglucose pyrophosphorylase, as shown by the starch-deficient mutants, mn1, sh1, and bt2 or sh2, respectively. To improve understanding of the relationship between early grain-filling traits and carbohydrate composition in mature grain, QTLs linked to soluble invertase, sucrose synthase, and ADPglucose pyrophosphorylase activities and to starch, sucrose, fructose, and glucose concentrations were investigated. In order to take into account the specific time-course of each enzyme activity during grain filling, sampling was carried out at three periods (15, 25, and 35 d after pollination) on 100 lines from a recombinant inbred family, grown in the field. The MQTL method associated with QTL interaction analysis revealed numerous QTLs for all traits, but only one QTL was consistently observed at the three sampling periods. Some chromosome zones were heavily labelled, forming clusters of QTLs. Numerous possible candidate genes of the starch synthetic pathway co-located with QTLs. Four QTLs were found close to the locus Sh1 (bin 9.01) coding for the sucrose synthase. In order to confirm the importance of this locus, the CAPS polymorphism of the Sh1 gene was analysed in 45 genetically unrelated maize lines from various geographical origins. The DNA polymorphism was significantly associated with phenotypic traits related to grain filling (starch and amylose content, grain matter, and ADPglucose pyrophosphorylase activity at 35 DAP). Thus, the Sh1 locus could provide a physiologically pertinent marker for maize selection.     

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.     

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.     

Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm

ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles α-glucose-1-P produced per minute per milligram protein. Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30°C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostain was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.     

Isolation and Characterization of a Starchless Mutant of Arabidopsis thaliana (L.) Heynh Lacking ADPglucose Pyrophosphorylase Activity

A mutant of Arabidopsis thaliana lacking ADPglucose pyrophosphorylase activity (EC 2.7.7.27) was isolated (from a mutagenized population of plants) by screening for the absence of leaf starch. The mutant grows as vigorously as the wild type in continuous light but more slowly than the wild type in a 12 hours light/12 hours dark photoperiod. Genetic analysis showed that the deficiency of both starch and ADPglucose pyrophosphorylase activity were attributable to a single, nuclear, recessive mutation at a locus designated adg1. The absence of starch in the mutant demonstrates that starch synthesis in the chloroplast is entirely dependent on a pathway involving ADPglucose pyrophosphorylase. Analysis of leaf extracts by two-dimensional polyacrylamide gel electrophoresis followed by Western blotting experiments using antibodies specific for spinach ADPglucose pyrophosphorylase showed that two proteins, present in the wild type, were absent from the mutant. The heterozygous F₁ progeny of a cross between the mutant and wild type had a specific activity of ADPglucose pyrophosphorylase indistinguishable from the wild type. These observations suggest that the mutation in the adg1 gene in TL25 might affect a regulatory locus.     

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 extraplastidial AGPase activity and the larger of the two proteins recognized by the antibody to the Bt2 subunit.     

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.     

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.     

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.     

Nucleotides and Nucleotide Sugars in Developing Maize Endosperms (Synthesis of ADP-Glucose in brittle-1)

As part of an in vivo study of carbohydrate metabolism during development of Zea mays L. kernels, quantities of nucleotides and nucleotide sugars were measured in endosperm extracts from normal, the single-mutant genotypes shrunken-1 (sh1), shrunken-2 (sh2), and brittle-1 (btl}, and the multiple-mutant genotypes sh1bt1, sh2bt1, and sh1sh2bt1. Results showed that bt1 kernels accumulated more than 13 times as much adenosine 5[prime] diphospho-glucose (ADP-Glc) as normal kernels. Activity of starch synthase in bt1 endosperm was equal to that in endosperm extracts from normal kernels. Thus the ADP-Glc accumulation in bt1 endosperm cells was not due to a deficiency in starch synthase. ADP-Glc content in extracts of sh1bt1 endosperms was similar to that in bt1, but in extracts of the sh2bt1 mutant kernels ADP-Glc content was much reduced compared to bt1 (about 3 times higher than that in normal). Endosperm extracts from sh1sh2bt1, kernels that are deficient in both ADP-Glc pyrophosphorylase (AGPase) and sucrose synthase, had quantities of ADP-Glc much lower than in normal kernels. These results clearly indicate that AGPase is the predominant enzyme responsible for the in vivo synthesis of ADP-Glc in bt1 mutant kernels, but Suc synthase may also contribute to the synthesis of ADP-Glc in kernels deficient in AGPase.