L. Curtis Hannah

The Maize Genome Contains a Helitron Insertion

The maize mutation sh2-7527 was isolated in a conventional maize breeding program in the 1970s. Although the mutant contains foreign sequences within the gene, the mutation is not attributable to an interchromosomal exchange or to a chromosomal inversion. Hence, the mutation was caused by an insertion. Sequences at the two Sh2 borders have not been scrambled or mutated, suggesting that the insertion is not caused by a catastrophic reshuffling of the maize genome. The insertion is large, at least 12 kb, and is highly repetitive in maize. As judged by hybridization, sorghum contains only one or a few copies of the element, whereas no hybridization was seen to the Arabidopsis genome. The insertion acts from a distance to alter the splicing of the sh2 pre-mRNA. Three distinct intron-bearing maize genes were found in the insertion. Of most significance, the insertion bears striking similarity to the recently described DNA helicase–bearing transposable elements termed Helitrons. Like Helitrons, the inserted sequence of sh2-7527 is large, lacks terminal repeats, does not duplicate host sequences, and was inserted between a host dinucleotide AT. Like Helitrons, the maize element contains 5′ TC and 3′ CTRR termini as well as two short palindromic sequences near the 3′ terminus that potentially can form a 20-bp hairpin. Although the maize element lacks sequence information for a DNA helicase, it does contain four exons with similarity to a plant DEAD box RNA helicase. A second Helitron insertion was found in the maize genomic database. These data strongly suggest an active Helitron in the present-day maize genome.

The Complexities of Starch Biosynthesis in Cereal Endosperms

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.

Splicing of the Maize Sh1 First Intron Is Essential for Enhancement of Gene Expression, and a T-Rich Motif Increases Expression without Affecting Splicing

Certain plant and animal introns increase expression of protein-coding sequences when placed in the 5′ region of the transcription unit. The mechanisms of intron-mediated enhancement have not been defined, but are generally accepted to be post- or cotranscriptional in character. One of the most effective plant introns in stimulating gene expression is the 1,028-bp first intron of the Sh1 gene that encodes maize (Zea mays) sucrose synthase. To address the mechanisms of intron-mediated enhancement, we used reporter gene fusions to identify features of the Sh1 first intron required for enhancement in cultured maize cells. A 145-bp derivative conferred approximately the same 20- to 50-fold stimulation typical for the full-length intron in this transient expression system. A 35-bp motif contained within the intron is required for maximum levels of enhancement but not for efficient transcript splicing. The important feature of this redundant 35-bp motif is T-richness rather than the specific sequence. When transcript splicing was abolished by mutations at the intron borders, enhancement was reduced to about 2-fold. The requirement of splicing for enhancement was not because of upstream translation initiation codons contained in unspliced transcripts. On the basis of our current findings, we conclude that splicing of theSh1 intron is integral to enhancement, and we hypothesize that transcript modifications triggered by the T-rich motif and splicing may link the mRNA with the trafficking system of the cell.

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.

Sequence-indexed mutations in maize using the UniformMu transposon-tagging population

BackgroundGene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations.ResultsTransposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill.ConclusionWe show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.

Maize Shrunken-1 intron and exon regions increase gene expression in maize protoplasts

Abstract A DNA cassette containing the first intron of the maize Shrunken-1 (Sh1) and adjacent exon sequences stimulates reporter gene expression in maize protoplasts up to 100-fold. Because this highly active cassette is being used to elevate gene expression in numerous genetic engineering experiments and because of the paucity of information concerning the mechanism(s) governing increased expression, we examined how placement and orientation of this cassette, intron sequence and length, and interactions of intron and exon sequences affect expression levels. The data reveal two significant features: (1) substantial alterations within the intron portion of the cassette do not affect its stimulatory effect and (2) the stimulation appears to occur at more than one level of gene expression. Specifically, we show that the magnitude of intron-mediated stimulation is not increased by the tandem addition of a complete cassette to a construct already containing the Sh1 first intron cassette nor is stimulation affected by an 87% increase or a 63% decrease in the size of the intron. Placement of an additional cassette within a Sh1 first intron cassette does not significantly alter the stimulatory effect; however, the orientation of sequences is important, since placement of an inverted cassette within another cassette abolishes the stimulatory effect. A significant finding is that the exon portion of the Sh1 intron 1 cassette boosts gene expression approximately 7-fold in the absence of the intron, while exon sequences from the alcohol dehydrogenase-1 (Adh1) gene inhibit gene expression. Placement of Adh1 exon sequences adjacent to the Sh1 first intron, however, elevates gene expression above the level observed with the Sh1 first intron cassette. These mixing experiments strongly suggest that the exon and intron components of the Sh1 first intron cassette act at different steps in gene expression.

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.

A shrunken-2 Transgene Increases Maize Yield by Acting in Maternal Tissues to Increase the Frequency of Seed Development

This work examines the function of a maize heat-stable, less inhibitor–sensitive form of ADP-glucose pyrophosphorylase, which increases maize yield by increasing seed number. This work shows that this increase requires high temperature during early seed development and results from transgene function in maternal tissues to increase the probability that an ovary will produce a seed. 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.

Editor’s Choice: Crop Genome Plasticity and Its Relevance to Food and Feed Safety of Genetically Engineered Breeding Stacks

Genetically engineered (GE) stacks, combinations of two or more single transgenic events (i.e. single-locus insertions) that have been produced by crossing sexually compatible parents, are an important and growing sector of the crop seed market. Stacked traits covered 26% of the global transgenic crop area in 2011 and were the fastest growing trait group, with a 31% increase in the area planted compared with 2010 (James, 2011). Stacked traits already dominate the market in some regions. For example, 95% of the cotton (Gossypium spp.) grown in Australia during 2011 had both herbicide tolerance and insect resistance traits (James, 2011). Worldwide, at least 12 countries are now growing stacked varieties, of which nine are developing countries (James, 2011). The rapid adoption of GE stacks has focused attention on whether the safety of such products differs from that of the individual events.

The Maize Viviparous8 Locus, Encoding a Putative ALTERED MERISTEM PROGRAM1-Like Peptidase, Regulates Abscisic Acid Accumulation and Coordinates Embryo and Endosperm Development

We describe a mutant of Zea mays isolated from a W22 inbred transposon population, widows peak mutant1 (wpk1), with an altered pattern of anthocyanin synthesis and aleurone cell differentiation in endosperm. In addition, a failure of the developing mutant embryo to form leaf initials is associated with decreased expression of a subset of meristem regulatory genes that includes Abphyl1 and Td1. We show that the viviparous8 (vp8) mutant has a similar pleiotropic phenotype in the W22 inbred background in contrast to the viviparous embryo phenotype exhibited in the standard genetic background, and we confirmed that wpk1 is allelic to vp8. Further genetic analysis revealed that the standard vp8 stock contains an unlinked, partially dominant suppressor of the vp8 mutation that is not present in W22. Consistent with the early-onset viviparous phenotype of vp8, expression of several embryonic regulators, including LEC1/B3 domain transcription factors, was reduced in the mutant embryo. Moreover, reduced abscisic acid (ABA) content of vp8/wpk1 embryos was correlated with altered regulation of ABA biosynthesis, as well as ABA catabolic pathways. The ABA biosynthetic gene Vp14 was down-regulated in the nonsuppressed background, whereas the ZmABA8′oxA1a ABA 8′-hydroxylase gene was strongly up-regulated in both genetic backgrounds. Molecular analysis revealed that Vp8 encodes a putative peptidase closely related to Arabidopsis thaliana ALTERED MERISTEM PROGRAM1. Because the Vp8 regulates meristem development as well as seed maturation processes, including ABA accumulation, we propose that VP8 is required for synthesis of an unidentified signal that integrates meristem and embryo formation in seeds.