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1.
Plant Physiol ; 193(2): 1456-1478, 2023 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-37339339

RESUMO

Molecular mechanisms that distinguish the synthesis of semi-crystalline α-glucan polymers found in plant starch granules from the synthesis of water-soluble polymers by nonplant species are not well understood. To address this, starch biosynthetic enzymes from maize (Zea mays L.) endosperm were isolated in a reconstituted environment using yeast (Saccharomyces cerevisiae) as a test bed. Ninety strains were constructed containing unique combinations of 11 synthetic transcription units specifying maize starch synthase (SS), starch phosphorylase (PHO), starch branching enzyme (SBE), or isoamylase-type starch debranching enzyme (ISA). Soluble and insoluble branched α-glucans accumulated in varying proportions depending on the enzyme suite, with ISA function stimulating distribution into the insoluble form. Among the SS isoforms, SSIIa, SSIII, and SSIV individually supported the accumulation of glucan polymer. Neither SSI nor SSV alone produced polymers; however, synergistic effects demonstrated that both isoforms can stimulate α-glucan accumulation. PHO did not support α-glucan production by itself, but it had either positive or negative effects on polymer content depending on which SS or a combination thereof was present. The complete suite of maize enzymes generated insoluble particles resembling native starch granules in size, shape, and crystallinity. Ultrastructural analysis revealed a hierarchical assembly starting with subparticles of approximately 50 nm diameter that coalesce into discrete structures of approximately 200 nm diameter. These are assembled into semi-crystalline α-glucan superstructures up to 4 µm in length filling most of the yeast cytosol. ISA was not essential for the formation of such particles, but their abundance was increased dramatically by ISA presence.


Assuntos
Endosperma , Sintase do Amido , Saccharomyces cerevisiae , Zea mays/genética , Proteínas de Plantas/química , Amido , Glucanos , Sintase do Amido/química
2.
Proc Natl Acad Sci U S A ; 115(1): E24-E33, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29255019

RESUMO

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.


Assuntos
Endosperma/enzimologia , Metabolismo Energético/fisiologia , Proteínas de Plantas/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Zea mays/enzimologia , Endosperma/genética , Mutação , Proteínas de Plantas/genética , Piruvato Ortofosfato Diquinase/genética , Amido/biossíntese , Amido/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zea mays/genética
3.
Plant J ; 96(3): 595-606, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30062763

RESUMO

Enzymological and starch analyses of various ADP-glucose pyrophosphorylase (AGPase) null mutants point to fundamental differences in the pathways for starch synthesis in the maize leaf, embryo, ovary and endosperm. Leaf starch is synthesized via the AGPase encoded by the small and large subunits shown previously to be expressed at abundant levels in the leaf, whereas more than one AGPase isoform functions in the embryo and in the ovary. Embryo starch content is also dependent on genes functioning in the leaf and in the endosperm. AGPase encoded by shrunken-2 and brittle-2 synthesizes ~75% of endosperm starch. The gene, agpsemzm, previously shown to encode the small subunit expressed in the embryo, and agpllzm, the leaf large subunit gene, are here shown to encode the endosperm, plastid-localized AGPase. Loss of this enzyme does not reduce endosperm starch. Rather, the data suggest that AGPase-independent starch synthesis accounts for ~25% of endosperm starch. Three maize genes encode the small subunit of the AGPase. Data here show that the triple mutant lacking all three small subunits is lethal in early seed development but can be viable in both male and female gametes. Seed and plant viability is restored by any one of the three small subunit genes, including one previously thought to function only in the cytosol of the endosperm. Data herein also show the functionality of a fourth gene encoding the large subunit of this enzyme. Although adenosine diphosphate glucose pyrophosphorylase is shown here to be essential for maize viability, strong evidence for starch synthesis in the endosperm that is independent of this enzyme is also presented. Starch synthesis is distinct in the maize embryo, ovary, leaf and endosperm, and is coordinated among the various tissues.


Assuntos
Glucose-1-Fosfato Adenililtransferase/metabolismo , Amido/metabolismo , Zea mays/enzimologia , Endosperma/enzimologia , Endosperma/genética , Flores/enzimologia , Flores/genética , Glucose-1-Fosfato Adenililtransferase/genética , Especificidade de Órgãos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastídeos/enzimologia , Sementes/enzimologia , Sementes/genética , Zea mays/genética
4.
Plant Cell ; 24(6): 2352-63, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22751213

RESUMO

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.


Assuntos
Plantas Geneticamente Modificadas/genética , Sementes/crescimento & desenvolvimento , Zea mays/crescimento & desenvolvimento , Zea mays/genética , Flores/genética , Dosagem de Genes , Regulação da Expressão Gênica de Plantas , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/genética , Temperatura , Transgenes
5.
Arch Biochem Biophys ; 568: 28-37, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25600571

RESUMO

Iterative saturation mutagenesis (ISM) has been used to improve the thermostability of maize endosperm ADP-glucose pyrophosphorylase (AGPase), a highly-regulated, rate-limiting and temperature-sensitive enzyme essential for starch biosynthesis. The thermo-sensitivity of heterotetrameric AGPase has been linked to grain loss in cereals and improving this property might therefore have direct impacts on grain yield. Nine amino acids were selected for site-saturation mutagenesis on the basis of elevated B-factors in the crystal structure of the closest available homolog (a small subunit homotetramer of potato AGPase). After each round of mutagenesis, iodine staining and antibody capture activity assays at varying temperatures were used to select the optimum positions and amino acid changes for the next rounds of mutagenesis. After three iterations, the signals from whole-colony iodine staining were saturated and a heat stable AGPase variant was obtained. Kinetic studies of the heat stable mutant showed that it also had an unexpected increased affinity for the activator, 3-PGA. This is particularly valuable as both the temperature stability and allosteric properties of AGPase significantly influence grain yield.


Assuntos
Endosperma/enzimologia , Estabilidade Enzimática , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Zea mays/enzimologia , Cristalografia por Raios X , Endosperma/química , Endosperma/genética , Regulação da Expressão Gênica de Plantas , Glucose-1-Fosfato Adenililtransferase/química , Temperatura Alta , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Zea mays/química , Zea mays/genética
6.
Theor Appl Genet ; 128(3): 445-52, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25504539

RESUMO

KEY MESSAGE: The mutant that originally defined the shrunken - 2 locus of maize is shown here to be the product of a complex chromosomal rearrangement. The maize shrunken-2 gene (sh2) encodes the large subunit of the heterotetrameric enzyme, adenosine diphosphate glucose pyrophosphorylases and a rate-limiting enzyme in starch biosynthesis. The sh2 gene was defined approximately 72 years ago by the isolation of a loss-of-function allele conditioning a shrunken, but viable seed. In subsequent years, the realization that this allele, termed zsh2-R or sh2-Reference, causes an extremely high level of sucrose to accumulate in the developing seed led to a revolution in the sweet corn industry. Now, the vast majority of sweet corns grown throughout the world contain this mutant allele. Through initial Southern analysis followed by genomic sequencing, the work reported here shows that this allele arose through a complex set of events involving at least three breaks of chromosome 3 as well as an intra-chromosomal inversion. These findings provide an explanation for some previously reported, unexpected observations concerning rates of recombination within and between genes in this region.


Assuntos
Alelos , Glucose-1-Fosfato Adenililtransferase/genética , Recombinação Genética , Zea mays/genética , Cromossomos de Plantas , DNA de Plantas/genética , Rearranjo Gênico , Genes de Plantas , Biblioteca Genômica , Análise de Sequência de DNA , Zea mays/enzimologia
7.
Arch Biochem Biophys ; 543: 1-9, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24378757

RESUMO

ADP-glucose pyrophosphorylase (AGPase) controls the rate-limiting step in starch biosynthesis and is regulated at various levels. Cereal endosperm enzymes, in contrast to other plant AGPases, are particularly heat labile and transgenic studies highlight the importance of temperature for cereal yield. Previously, a phylogenetic approach identified Type II and positively selected amino acid positions in the large subunit of maize endosperm AGPase. Glycogen content, kinetic parameters and heat stability were measured in AGPases having mutations in these sites and interesting differences were observed. This study expands on our earlier evolutionary work by determining how all Type II and positively selected sites affect kinetic constants, heat stability and catalytic rates at increased temperatures. Variants with enhanced properties were identified and combined into one gene, designated Sh2-E. Enhanced properties include: heat stability, enhanced activity at 37 °C, activity at 55 °C, reduced Ka and activity in the absence of activator. The resulting enzyme exhibited all improved properties of the various individual changes. Additionally, Sh2-E was expressed with a small subunit variant with enhanced enzyme properties resulting in an enzyme that has exceptional heat stability, a high catalytic rate at increased temperatures and significantly decreased Km values for both substrates in the absence of the activator.


Assuntos
Endosperma/enzimologia , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/metabolismo , Temperatura Alta , Filogenia , Engenharia de Proteínas , Zea mays/enzimologia , Substituição de Aminoácidos , Aminoácidos , Biocatálise , Estabilidade Enzimática , Evolução Molecular , Glucose-1-Fosfato Adenililtransferase/antagonistas & inibidores , Glucose-1-Fosfato Adenililtransferase/genética , Cinética , Mutagênese Sítio-Dirigida , Mutação , Fosfatos/farmacologia , Desnaturação Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
8.
Arch Biochem Biophys ; 537(2): 210-6, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23906662

RESUMO

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.


Assuntos
Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/classificação , Fosfatos/química , Tubérculos/enzimologia , Plantas Geneticamente Modificadas/enzimologia , Solanum tuberosum/enzimologia , Zea mays/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/química , Estabilidade Enzimática , Solanum tuberosum/genética
9.
Plant Physiol ; 152(1): 85-95, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19889875

RESUMO

Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined--in both the small and large subunits--bound 3-PGA more weakly than the wild type (A(50) increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly.


Assuntos
Endosperma/enzimologia , Glucose-1-Fosfato Adenililtransferase/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Glucose-1-Fosfato Adenililtransferase/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , Subunidades Proteicas
10.
Plant Physiol ; 152(2): 1056-64, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20018600

RESUMO

ADP-glucose pyrophosphorylase catalyzes the synthesis of ADP-glucose (ADP-Glc) from Glc-1-phosphate (G-1-P) and ATP. Kinetic studies were performed to define the nature of the reaction, both in the presence and absence of allosteric effector molecules. When 3-phosphoglycerate (3-PGA), the putative physiological activator, was present at a saturating level, initial velocity studies were consistent with a Theorell-Chance BiBi mechanism and product inhibition data supported sequential binding of ATP and G-1-P, followed by ordered release of pyrophosphate and ADP-Glc. A sequential mechanism was also followed when 3-PGA was absent, but product inhibition patterns changed dramatically. In the presence of 3-PGA, ADP-Glc is a competitive inhibitor with respect to ATP. In the absence of 3-PGA--with or without 5.0 mm inorganic phosphate--ADP-Glc actually stimulated catalytic activity, acting as a feedback product activator. By contrast, the other product, pyrophosphate, is a potent inhibitor in the absence of 3-PGA. In the presence of subsaturating levels of allosteric effectors, G-1-P serves not only as a substrate but also as an activator. Finally, in the absence of 3-PGA, inorganic phosphate, a classic inhibitor or antiactivator of the enzyme, stimulates enzyme activity at low substrate by lowering the K(M) values for both substrates.


Assuntos
Endosperma/enzimologia , Glucose-1-Fosfato Adenililtransferase/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/enzimologia , Difosfatos/metabolismo , Ácidos Glicéricos/metabolismo , Dados de Sequência Molecular , Especificidade por Substrato
11.
Front Plant Sci ; 12: 800326, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35211133

RESUMO

In maize, starch mutants have facilitated characterization of key genes involved in endosperm starch biosynthesis such as large subunit of AGPase Shrunken2 (Sh2) and isoamylase type DBE Sugary1 (Su1). While many starch biosynthesis enzymes have been characterized, the mechanisms of certain genes (including Sugary enhancer1) are yet undefined, and very little is understood about the regulation of starch biosynthesis. As a model, we utilize commercially important sweet corn mutations, sh2 and su1, to genetically perturb starch production in the endosperm. To characterize the transcriptomic response to starch mutations and identify potential regulators of this pathway, differential expression and coexpression network analysis was performed on near-isogenic lines (NILs) (wildtype, sh2, and su1) in six genetic backgrounds. Lines were grown in field conditions and kernels were sampled in consecutive developmental stages (blister stage at 14 days after pollination (DAP), milk stage at 21 DAP, and dent stage at 28 DAP). Kernels were dissected to separate embryo and pericarp from the endosperm tissue and 3' RNA-seq libraries were prepared. Mutation of the Su1 gene led to minimal changes in the endosperm transcriptome. Responses to loss of sh2 function include increased expression of sugar (SWEET) transporters and of genes for ABA signaling. Key regulators of starch biosynthesis and grain filling were identified. Notably, this includes Class II trehalose 6-phosphate synthases, Hexokinase1, and Apetala2 transcription factor-like (AP2/ERF) transcription factors. Additionally, our results provide insight into the mechanism of Sugary enhancer1, suggesting a potential role in regulating GA signaling via GRAS transcription factor Scarecrow-like1.

12.
Nat Commun ; 12(1): 1227, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623026

RESUMO

Sweet corn is one of the most important vegetables in the United States and Canada. Here, we present a de novo assembly of a sweet corn inbred line Ia453 with the mutated shrunken2-reference allele (Ia453-sh2). This mutation accumulates more sugar and is present in most commercial hybrids developed for the processing and fresh markets. The ten pseudochromosomes cover 92% of the total assembly and 99% of the estimated genome size, with a scaffold N50 of 222.2 Mb. This reference genome completely assembles the large structural variation that created the mutant sh2-R allele. Furthermore, comparative genomics analysis with six field corn genomes highlights differences in single-nucleotide polymorphisms, structural variations, and transposon composition. Phylogenetic analysis of 5,381 diverse maize and teosinte accessions reveals genetic relationships between sweet corn and other types of maize. Our results show evidence for a common origin in northern Mexico for modern sweet corn in the U.S. Finally, population genomic analysis identifies regions of the genome under selection and candidate genes associated with sweet corn traits, such as early flowering, endosperm composition, plant and tassel architecture, and kernel row number. Our study provides a high-quality reference-genome sequence to facilitate comparative genomics, functional studies, and genomic-assisted breeding for sweet corn.


Assuntos
Evolução Molecular , Genética Populacional , Genoma de Planta , Zea mays/genética , Alelos , Elementos de DNA Transponíveis/genética , Loci Gênicos , Haplótipos/genética , Anotação de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Análise de Sequência de DNA , Zea mays/anatomia & histologia
13.
Plant Mol Biol ; 74(4-5): 353-65, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20725765

RESUMO

Plant oil content and composition improvement is a major goal of plant breeding and biotechnology. The Puroindoline a and b (PINA and PINB) proteins together control whether wheat seeds are soft or hard textured and share a similar structure to that of plant non-specific lipid-transfer proteins. Here we transformed corn (Zea mays L.) with the wheat (Triticum aestivum L.) puroindoline genes (Pina and Pinb) to assess their effects upon seed oil content and quality. Pina and Pinb coding sequences were introduced into corn under the control of a corn Ubiquitin promoter. Three Pina/Pinb expression positive transgenic events were evaluated over two growing seasons. The results showed that Pin expression increased germ size significantly without negatively impacting seed size. Germ yield increased 33.8% while total seed oil content was increased by 25.23%. Seed oil content increases were primarily the result of increased germ size. This work indicates that higher oil content corn hybrids having increased food or feed value could be produced via puroindoline expression.


Assuntos
Óleo de Milho/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/metabolismo , Zea mays/genética , Plantas Geneticamente Modificadas/anatomia & histologia , Sementes/anatomia & histologia , Sementes/genética , Triticum/genética , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
15.
Curr Opin Biotechnol ; 19(2): 160-5, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18400487

RESUMO

Starch serves not only as an energy source for plants, animals, and humans but also as an environmentally friendly alternative for fossil fuels. Here, we describe recent findings concerning the synthesis of this important molecule in the cereal endosperm. Results from six separate transgenic reports point to the importance of adenosine diphosphate glucose pyrophosphorylase in controlling the amount of starch synthesized. The unexpected cause underlying the contrast in sequence divergence of its two subunits is also described. A major unresolved question concerning the synthesis of starch is the origin of nonrandom or clustered alpha-1,6 branch-points within the major component of starch, amylopectin. Developing evidence that several of the starch biosynthetic enzymes involved in amylopectin synthesis occur in complexes is reviewed. These complexes may provide the specificity for the formation of nonrandom branch-points.


Assuntos
Grão Comestível/metabolismo , Amido/biossíntese , Amilopectina/biossíntese , Amilopectina/metabolismo , Grão Comestível/genética , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Modelos Biológicos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/genética , Sementes/metabolismo , Amido/metabolismo
16.
BMC Evol Biol ; 8: 232, 2008 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-18700010

RESUMO

BACKGROUND: ADP-glucose pyrophosphorylase (AGPase), which catalyses a rate limiting step in starch synthesis, is a heterotetramer comprised of two identical large and two identical small subunits in plants. Although the large and small subunits are equally sensitive to activity-altering amino acid changes when expressed in a bacterial system, the overall rate of non-synonymous evolution is approximately 2.7-fold greater for the large subunit than for the small subunit. Herein, we examine the basis for their different rates of evolution, the number of duplications in both large and small subunit genes and document changes in the patterns of AGPase evolution over time. RESULTS: We found that the first duplication in the AGPase large subunit family occurred early in the history of land plants, while the earliest small subunit duplication occurred after the divergence of monocots and eudicots. The large subunit also had a larger number of gene duplications than did the small subunit. The ancient duplications in the large subunit family raise concern about the saturation of synonymous substitutions, but estimates of the absolute rate of AGPase evolution were highly correlated with estimates of omega (the non-synonymous to synonymous rate ratio). Both subunits showed evidence for positive selection and relaxation of purifying selection after duplication, but these phenomena could not explain the different evolutionary rates of the two subunits. Instead, evolutionary constraints appear to be permanently relaxed for the large subunit relative to the small subunit. Both subunits exhibit branch-specific patterns of rate variation among sites. CONCLUSION: These analyses indicate that the higher evolutionary rate of the plant AGPase large subunit reflects permanent relaxation of constraints relative to the small subunit and they show that the large subunit genes have undergone more gene duplications than small subunit genes. Candidate sites potentially responsible for functional divergence within each of the AGPase subunits were investigated by examining branch-specific patterns of rate variation. We discuss the phenotypes of mutants that alter some candidate sites and strategies for examining candidate sites of presently unknown function.


Assuntos
Evolução Molecular , Duplicação Gênica , Genes de Plantas , Glucose-1-Fosfato Adenililtransferase/genética , Magnoliopsida/genética , Substituição de Aminoácidos , Variação Genética , Magnoliopsida/enzimologia , Fenótipo , Filogenia , Seleção Genética , Alinhamento de Sequência
18.
Front Plant Sci ; 9: 1849, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619417

RESUMO

ADP-glucose pyrophosphorylase (AGPase) is an important enzyme in starch synthesis and previous studies showed that the heat lability of this enzyme is a determinant to starch synthesis in the maize endosperm and, in turn, seed yield. Here, amino acids in the AGPase endosperm small subunit with high B-factors were mutagenized and individual changes enhancing heat stability and/or kinetic parameters in an Escherichia coli expression system were chosen. Individual mutations were combined and analyzed. One triple mutant, here termed Bt2-BF, was chosen for further study. Combinations of this heat stable, 3-PGA-independent small subunit variant with large subunits also heat stable yielded complex patterns of heat stability and kinetic and allosteric properties. Interestingly, two of the three changes reside in a protein motif found only in AGPases that exhibit high sensitivity to 3-PGA. While not the 3-PGA binding site, amino acid substitutions in this region significantly alter 3-PGA activation kinetics.

19.
Sci Rep ; 8(1): 13032, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158664

RESUMO

Crop improvement programs focus on characteristics that are important for plant productivity. Typically genes underlying these traits are identified and stacked to create improved cultivars. Hence, identification of valuable traits for plant productivity is critical for plant improvement. Here we describe an important characteristic for maize productivity. Despite the fact mature maize ears are typically covered with kernels, we find that only a fraction of ovaries give rise to mature kernels. Non-developed ovaries degenerate while neighboring fertilized ovaries produce kernels that fill the ear. Abortion occurs throughout the ear, not just at the tip. We show that the fraction of aborted ovaries/kernels is genetically controlled and varies widely among maize lines, and low abortion genotypes are rare. Reducing or eliminating ovary abortion could substantially increase yield, making this characteristic a new target for selection in maize improvement programs.


Assuntos
Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Zea mays/fisiologia , Endogamia , Reprodução , Zea mays/genética
20.
G3 (Bethesda) ; 8(1): 291-302, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29167273

RESUMO

Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S) of Zea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclear restorer-of-fertility (restorer) mutations rescue pollen function. To better understand these genetic interactions, we screened Activator-Dissociation (Ac-Ds), Enhancer/Suppressor-mutator (En/Spm), and Mutator (Mu) transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered from Ac-Ds and En/Spm stocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-S Mu transposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously described rfl2-1 mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development.


Assuntos
Elementos de DNA Transponíveis , Regulação da Expressão Gênica de Plantas , Mutação , Infertilidade das Plantas/genética , Sementes/genética , Zea mays/genética , Núcleo Celular/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , Pólen/metabolismo , Polinização/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
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