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 α-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.
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.
Summary
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.
Significance Statement
While 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.
Abstract
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.
Expressing up to 11 maize starch biosynthesis enzymes in yeast results in the production of insoluble and soluble glucose polymers that resemble plant starch molecules in many respects.
We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred ...background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences.
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.
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.
In maize, starch mutants have facilitated characterization of key genes involved in endosperm starch biosynthesis such as
(
) and
(
). While many starch biosynthesis enzymes have been characterized, ...the mechanisms of certain genes (including
) are yet undefined, and very little is understood about the regulation of starch biosynthesis. As a model, we utilize commercially important sweet corn mutations,
and
, 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,
, and
) 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
gene led to minimal changes in the endosperm transcriptome. Responses to loss of
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,
, and
transcription factor-like (AP2/ERF) transcription factors. Additionally, our results provide insight into the mechanism of
, suggesting a potential role in regulating GA signaling via GRAS transcription factor
.
In this work we test the hypothesis that yield of rice (Oryza sativa L.) can be enhanced by increasing endosperm activity of ADP-glucose pyrophosphorylase (AGP), a key enzyme in starch biosynthesis. ...The potential for increases in yield exist because rice initiates more seeds than are taken to maturity and possesses excess photosynthetic capacity that could be utilized if there were more demand for assimilate. Following an approach already shown to be successful in wheat, experiments were designed to increase demand for assimilate by increasing the capacity for starch synthesis in endosperm. This was accomplished by transforming rice with a modified maize AGP large subunit sequence (Sh2r6hs) under control of an endosperm-specific promoter. This altered subunit confers upon AGP decreased sensitivity to allosteric inhibition by inorganic phosphate (Pi) and enhanced heat stability, potentially leading to higher AGP activity in vivo. The Sh2r6hs transgene increased AGP activity in developing endosperm by 2.7-fold in the presence of Pi. Increases in AGP activity in transgenic seeds compared with controls were maximal between 10—15 days after anthesis. Starch content of individual seeds at harvest was not increased, but seed weight per plant and total plant biomass were each increased by more than 20%. Increased endosperm AGP activity thus stimulates setting of additional seeds and overall plant growth rather than increasing yield of seeds already set. Results demonstrate that deregulation of endosperm AGP increases overall plant sink strength, leading to larger, more productive plants in a manner similar to that in wheat having similar genetic modification.
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