Maureen A. Clancy

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.

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.

A Transcript Accounting from Diverse Tissues of a Cultivated Strawberry

Strawberry (Fragaria spp.) is a valuable fruit crop as well as an outstanding system for studying functional genomics in plants. The goal of this study was to substantially increase and analyze the available expressed sequence information in the genus by examining the transcriptome of the cultivated strawberry (Fragaria × ananassa Duchesne). To maximize transcript diversity and discovery, plants representing an octoploid strawberry cultivar were subjected to a broad range of treatments. Plant materials were pooled by tissue type. cDNA pools were sequenced by the Roche‐454 GS‐FLX system and assembled into over 32,000 contigs. Predictions of cellular localization and function were made by associating assembled contigs to annotated homologs, and the tissue pool tags provided a means to assess the overall expression pattern for any given transcript. Contigs comprised of reads originating from only one organ type and those present equally in all plant organs were both identified. Bacterial and fungal sequences found in the strawberry samples provide a metagenomic survey of the microbial community of a greenhouse strawberry plant. This study utilized an innovative assembly strategy on pooled tissues, thus providing a foundation for developing tissue‐specific tools, an opportunity to identify alleles for marker‐assisted selection, a reference of strawberry gene annotations, and a basis for comparative transcriptomics between cultivated strawberry, its diploid ancestors, and the wider Rosaceae family.

Validation of reference transcripts in strawberry (Fragaria spp.)

Contemporary methods to assay gene expression depend on a stable set of reference transcripts for accurate quantitation. A lack of well-tested reference genes slows progress in characterizing gene expression in high-value specialty crops. In this study, a set of strawberry (Fragaria spp.) constitutively expressed reference genes has been identified by merging digital gene expression data with expression profiling. Constitutive reference candidates were validated using quantitative PCR and hybridization. Several transcripts have been identified that show improved stability across tissues relative to traditional reference transcripts. Results are similar between commercial octoploid strawberry and the diploid model. Our findings also show that while some never-before-used references are appropriate for most applications, even the most stable reference transcripts require careful assessment across the diverse tissues and fruit developmental states before being adopted as controls.

A Strawberry KNOX Gene Regulates Leaf, Flower and Meristem Architecture

The KNOTTED-LIKE HOMEODOMAIN (KNOX) genes play a central role in maintenance of the shoot apical meristem. They also contribute to the morphology of simple and compound leaves. In this report we characterize the FaKNOX1 gene from strawberry (Fragaria spp.) and demonstrate its function in trasgenic plants. The FaKNOX1 cDNA was isolated from a cultivated strawberry (F.×ananassa) flower EST library. The sequence is most similar to Class I KNOX genes, and was mapped to linkage group VI of the diploid strawberry genome. Unlike most KNOX genes studied, steady-state transcript levels were highest in flowers and fruits. Transcripts were also detected in emerging leaf primordia and the apical dome. Transgenic strawberry plants suppressing or overexpressing FaKNOX1 exhibited conspicuous changes in plant form. The FaKNOX1 RNAi plants presented a dwarfed phenotype with deeply serrated leaflets and exaggerated petiolules. They also exhibited a high level of cellular disorganization of the shoot apical meristem and leaves. Overexpression of FaKNOX1 caused dwarfed stature with wrinkled leaves. These gain- and loss-of-function assays in strawberry functionally demonstrate the contributions of a KNOX domain protein in a rosaceous species.

Identification of Novel Growth Regulators in Plant Populations Expressing Random Peptides

New peptide-based plant growth regulators may be identified by screening populations of plants with random peptide sequences that cause reproducible effects on biology, including plant growth inhibition, aberrant light response, and hastened developmental transitions. The use of chemical genomics approaches allows the identification of small molecules that integrate into biological systems, thereby changing discrete processes that influence growth, development, or metabolism. Libraries of chemicals are applied to living systems, and changes in phenotype are observed, potentially leading to the identification of new growth regulators. This work describes an approach that is the nexus of chemical genomics and synthetic biology. Here, each plant in an extensive population synthesizes a unique small peptide arising from a transgene composed of a randomized nucleic acid sequence core flanked by translational start, stop, and cysteine-encoding (for disulfide cyclization) sequences. Ten and 16 amino acid sequences, bearing a core of six and 12 random amino acids, have been synthesized in Arabidopsis (Arabidopsis thaliana) plants. Populations were screened for phenotypes from the seedling stage through senescence. Dozens of phenotypes were observed in over 2,000 plants analyzed. Ten conspicuous phenotypes were verified through separate transformation and analysis of multiple independent lines. The results indicate that these populations contain sequences that often influence discrete aspects of plant biology. Novel peptides that affect photosynthesis, flowering, and red light response are described. The challenge now is to identify the mechanistic integrations of these peptides into biochemical processes. These populations serve as a new tool to identify small molecules that modulate discrete plant functions that could be produced later in transgenic plants or potentially applied exogenously to impart their effects. These findings could usher in a new generation of agricultural growth regulators, herbicides, or defense compounds.

Analysis of Block of cell proliferation 1 (BOP1) activity in strawberry and Arabidopsis.

Block of cell proliferation (BOP) proteins are conserved among eukaryotes, and studies in mammals and yeast have described their role in ribosome biogenesis and cell cycle regulation. A BOP1 orthologue was identified in plants, and loss-of-function analyses in tobacco cells confirmed similar activities. This report characterizes a role for BOP1 activity in planta. Two transgenic plant species were used: the diploid strawberry (Fragaria vesca) and Arabidopsis thaliana. FvBOP1 silencing showed changes in pre-rRNA processing, and demonstrated FvBOP1s role in growth and physiology throughout different stages of plant development. In the strawberry, repression of FvBOP1 activity decreased plant fitness prior to flowering, followed by plant death after the reproductive transition, indicating that BOP1 activity is required for transition back to vegetative growth after flowering. A T-DNA null allele of the AtBOP1 gene is lethal, and a 50% decrease in transcript accumulation is sufficient to cause severe developmental defects linked to defective cell division. The conserved protein BOP1 is essential for viability. Lower transcript levels result in defects in rRNA processing and developmental abnormalities that are consistent with its predicted role in ribosome biogenesis.