Wayne T. Avigne

Sugar Levels Modulate Differential Expression of Maize Sucrose Synthase Genes.

The two genes encoding sucrose synthase in maize (Sh1 and Sus1) show markedly different responses to changes in tissue carbohydrate status. This enzyme is widely regarded as pivotal to sucrose partitioning, import, and/or metabolism by developing plant organs. Excised maize root tips were incubated for varying periods in different sugars and a range of concentrations. The Sh1 mRNA was maximally expressed under conditions of limited carbohydrate supply (~0.2% glucose). In contrast, Sus1 transcript levels were low or nondetectable under sugar-depleted conditions and peaked at 10-fold greater glucose concentrations (2.0%). Responses to other metabolizable sugars were similar, but L-glucose and elevation of osmolarity with mannitol had little effect. Plentiful sugar supplies thus increased expression of Sus1, whereas reduced sugar availability enhanced Sh1. At the protein level, shifts in abundance of subunits encoded by Sh1 and Sus1 were much less pronounced but corresponded to changes in respective mRNA levels. Although total enzyme activity did not show net change, cellular localization of sucrose synthase protein was markedly altered. In intact roots, sucrose synthase was most prevalent in the stele and apex. In contrast, sugar depletion favored accumulation in peripheral cells, whereas high sugar levels resulted in elevated expression in all cell types. The differential response of the two sucrose synthase genes to sugars provides a potential mechanism for altering the pattern of enzyme distribution in response to changing carbohydrate status and also for adjusting the sucrose-metabolizing capacity of importing cells relative to levels of available photosynthetic products.

A Similar Dichotomy of Sugar Modulation and Developmental Expression Affects Both Paths of Sucrose Metabolism: Evidence from a Maize Invertase Gene Family.

Invertase and sucrose synthase catalyze the two known paths for the first step in carbon use by sucrose-importing plant cells. The hypothesis that sugar-modulated expression of these genes could provide a means of import adjustment was initially suggested based on data from sucrose synthases alone; however, this hypothesis remained largely conjectural without critical evidence for invertases. Toward this end, a family of maize invertases was cloned and characterized. Here, we show that invertases are indeed sugar modulated and, surprisingly, like the sucrose synthase genes, fall into two classes with contrasting sugar responses. In both families, one class of genes is upregulated by increasing carbohydrate supply (Sucrose synthase1 [Sus1] and Invertase2 [Ivr2]), whereas a second class in the same family is repressed by sugars and upregulated by depletion of this resource (Shrunken1 [Sh1] and Invertase1 [Ivr1]). The two classes also display differential expression during development, with sugar-enhanced genes (Sus1 and Ivr2) expressed in many importing organs and sugar-repressed, starvation-tolerant genes (Sh1 and Ivr1) upregulated primarily during reproductive development. Both the Ivr1 and Ivr2 invertase mRNAs are abundant in root tips, very young kernels, silk, anthers, and pollen, where a close relationship is evident between changes in message abundance and soluble invertase activity. During development, patterns of expression shift as assimilate partitioning changes from elongating silks to newly fertilized kernels. Together, the data support a model for integrating expression of genes differentially responsive to carbohydrate availability (i.e., feast and famine conditions) with developmental signals. The demonstration that similar regulatory patterns occur in both paths of sucrose metabolism indicates a potential to influence profoundly the adjustment of carbon resource allocation.

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.

Mu-seq: Sequence-Based Mapping and Identification of Transposon Induced Mutations

Mutations tagged by transposon insertions can be readily mapped and identified in organisms with sequenced genomes. Collections of such mutants allow a systematic analysis of gene function, and can be sequence-indexed to build invaluable resources. Here we present Mu-seq (Mutant-seq), a high-throughput NextGen sequencing method for harnessing high-copy transposons. We illustrate the efficacy of Mu-seq by applying it to the Robertson’s Mutator system in a large population of maize plants. A single Mu-seq library, for example, constructed from 576 different families (2304 plants), enabled 4, 723 novel, germinal, transposon insertions to be detected, identified, and mapped with single base-pair resolution. In addition to the specificity, efficiency, and reproducibility of Mu-seq, a key feature of this method is its adjustable scale that can accomodate simultaneous profiling of transposons in thousands of individuals. We also describe a Mu-seq bioinformatics framework tailored to high-throughput, genome-wide, and population-wide analysis of transposon insertions.

Genetic and molecular analyses of UniformMu transposon insertion lines.

The UniformMu transposon population is a large public resource for reverse genetics and functional genomics of maize. Users access the collection of UniformMu genetic stocks that are freely distributed by the Maize Cooperation Stock Center using online tools maintained at MaizeGDB.org. Genetic and molecular analyses of UniformMu stocks (UFMu insertion lines) typically require development of genotyping assays that use a gene-specific polymerase chain reaction (PCR) to follow segregation of transposon insertions in genes of interest. Here we describe methods for accessing the resource and recommended protocols for genotyping of transposon insertion alleles.