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Thomas P Brutnell

Publications and source records attributed to Thomas P Brutnell.

18 recordsLinked to original sources
PubMed2007

A multi-treatment experimental system to examine photosynthetic differentiation in the maize leaf.

BACKGROUND: The establishment of C4 photosynthesis in maize is associated with differential accumulation of gene transcripts and proteins between bundle sheath and mesophyll photosynthetic cell types. We have physically separated photosynthetic cell types in the leaf blade to characterize differences in gene expression by microarray analysis. Additional control treatments were used to account for transcriptional changes induced by cell preparation treatments. To analyse these data, we have developed a statistical model to compare gene expression values derived from multiple, partially confounded, treatment groups. RESULTS: Differential gene expression in the leaves of wild-type maize seedlings was characterized using the latest release of a maize long-oligonucleotide microarray produced by the Maize Array Project consortium. The complete data set is available through the project web site. Data is also available at the NCBI GEO website, series record GSE3890. Data was analysed with and without consideration of cell preparation associated stress. CONCLUSION: Empirical comparison of the two analyses suggested that consideration of stress helped to reduce the false identification of stress responsive transcripts as cell-type enriched. Using our model including a stress term, we identified 8% of features as differentially expressed between bundle sheath and mesophyll cell types under control of false discovery rate of 5%. An estimate of the overall proportion of differentially accumulating transcripts (1-pi0) suggested that as many as 18% of the genes may be differentially expressed between B and M. The analytical model presented here is generally applicable to gene expression data and demonstrates the use of statistical elimination of confounding effects such as stress in the context of microarray analysis. We discuss the implications of the high degree of differential transcript accumulation observed with regard to both the establishment and engineering of the C4 syndrome.

Botany↗
PubMed2006

Subfunctionalization of PhyB1 and PhyB2 in the control of seedling and mature plant traits in maize.

Phytochromes are the primary red/far-red photoreceptors of higher plants, mediating numerous developmental processes throughout the life cycle, from germination to flowering. In seed plants, phytochromes are encoded by a small gene family with each member performing both distinct and redundant roles in mediating physiological responses to light cues. Studies in both eudicot and monocot species have defined a central role for phytochrome B in mediating responses to light in the control of several agronomically important traits, including plant height, transitions to flowering and axillary branch meristem development. Here we characterize Mutator-induced alleles of PhyB1 and a naturally occurring deletion allele of PhyB2 in Zea mays (maize). Using single and double mutants, we show that the highly similar PhyB1 and PhyB2 genes encode proteins with both overlapping and non-redundant functions that control seedling and mature plant traits. PHYB1 and PHYB2 regulate elongation of sheath and stem tissues of mature plants and contribute to the light-mediated regulation of PhyA and Cab gene transcripts. However, PHYB1 and not PHYB2 contributes significantly to the inhibition of mesocotyl elongation under red light, whereas PHYB2 and to a lesser extent PHYB1 mediate the photoperiod-dependent floral transition. This sub functionalization of PHYB activities in maize has probably occurred since the tetraploidization of maize, and may contribute to flowering time variation in modern-day varieties.

Alleles↗
PubMed2006

Generating novel allelic variation through Activator insertional mutagenesis in maize.

The maize transposable element Activator (Ac) has been exploited as an insertional mutagen to disrupt, clone, and characterize genes in a number of plant species. To develop an Ac-based mutagenesis platform for maize, a large-scale mutagenesis was conducted targeting the pink scutellum1 locus. We selected 1092 Ac transposition events from a closely linked donor Ac, resulting in the recovery of 17 novel ps1 alleles. Multiple phenotypic classes were identified corresponding to Ac insertions in the 5'-UTR and coding region of the predicted Ps1 gene. To generate a stable allelic series, we employed genetic screens and identified 83 germinally heritable ps1 excision alleles. Molecular characterization of these excision alleles revealed a position-dependent bias in excision allele frequencies and the predominance of 7- and 8-bp footprint products. In total, 19 unique ps1 excision alleles were generated in this study, including several that resulted in weak mutant phenotypes. The analysis of footprint alleles suggests a model of Ac excision in maize that is consistent with recent in vitro studies of hAT element excision. Importantly, the genetic and molecular methods developed in this study can be extended to generate novel allelic variation at any Ac-tagged gene in the genome.

Alleles↗
PubMed2006

In planta transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis.

BACKGROUND: Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg chelatase mutants have been isolated from higher plants, including semi-dominant alleles of ChlI, the gene encoding the I subunit of the enzyme. To investigate the function of higher plant CHLI, bacterial orthologues have been engineered to carry analogous amino acid substitutions to the higher plant mutations and the phenotypes examined through in vitro characterization of heterologously produced proteins. Here, we demonstrate the utility of a transient expression system in Nicotiana benthamiana for rapidly assaying mutant variants of the maize CHLI protein in vivo. RESULTS: Transient expression of mutant maize ChlI alleles in N. benthamiana resulted in the formation of chlorotic lesions within 4 d of inoculation. Immunoblot analyses confirmed the accumulation of maize CHLI protein suggesting that the chlorosis observed resulted from an interaction between maize CHLI and endogenous components of the N. benthamiana chlorophyll biosynthetic pathway. On the basis of this assay, PCR-based cloning techniques were used to rapidly recombine polymorphisms present in the alleles studied allowing confirmation of causative lesions. A PCR-based mutagenesis was conducted and clones assayed by transient expression. A number of novel allelic variants of maize ZmChlI were generated and analyzed using this assay, demonstrating the utility of this technique for fine mapping. CONCLUSION: Transient expression provides a convenient, high-throughput, qualitative assay for functional variation in the CHLI protein. Furthermore, we suggest that the approach used here would be applicable to the analysis of other plastid-localized proteins where gain-of-function mutations will result in readily observable mutant phenotypes.

Journal Article↗
PubMed2006

The maize Oil yellow1 (Oy1) gene encodes the I subunit of magnesium chelatase.

Semi-dominant Oil yellow1 (Oy1) mutants of maize (Zea mays) are deficient in the conversion of protoporphyrin IX to magnesium protoporphyrin IX, the first committed step of chlorophyll biosynthesis. Using a candidate gene approach, a cDNA clone was isolated that was predicted to encode the I subunit of magnesium chelatase (ZmCHLI) and mapped to the same genetic interval as Oy1. Allelic variation was identified at ZmCHLI between wild-type plants and plants carrying semi-dominant alleles of Oy1. These differences revealed putative amino acid substitutions that could account for the alterations in protein function. Candidate lesions were tested by introduction of homologous changes into the Synechocystis magnesium chelatase I gene (SschlI) and characterization of the activity of mutant protein variants in an in vitro enzyme activity assay. The results of these analyses suggest that SsChlI protein variants containing the substitutions identified in the dominant Oy1 maize alleles lack activity necessary for magnesium chelation and confer a semi-dominant phenotype via competitive inhibition of wild-type SsChlI.

Amino Acid Sequence↗
PubMed2005

Ac-immobilized, a stable source of Activator transposase that mediates sporophytic and gametophytic excision of Dissociation elements in maize.

We have identified and characterized a novel Activator (Ac) element that is incapable of excision yet contributes to the canonical negative dosage effect of Ac. Cloning and sequence analysis of this immobilized Ac (Ac-im) revealed that it is identical to Ac with the exception of a 10-bp deletion of sequences at the left end of the element. In screens of approximately 6800 seeds, no germinal transpositions of Ac-im were detected. Importantly, Ac-im catalyzes germinal excisions of a Ds element resident at the r1 locus resulting in the recovery of independent transposed Ds insertions in approximately 4.5% of progeny kernels. Many of these transposition events occur during gametophytic development. Furthermore, we demonstrate that Ac-im transactivates multiple Ds insertions in somatic tissues including those in reporter alleles at bronze1, anthocyaninless1, and anthocyaninless2. We propose a model for the generation of Ac-im as an aberrant transposition event that failed to generate an 8-bp target site duplication and resulted in the deletion of Ac end sequences. We also discuss the utility of Ac-im in two-component Ac/Ds gene-tagging programs in maize.

Base Sequence↗
PubMed2005

Light-regulated overexpression of an Arabidopsis phytochrome A gene in rice alters plant architecture and increases grain yield.

The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T(5)) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6-21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.

Arabidopsis↗
PubMed2005

Cereal phytochromes: targets of selection, targets for manipulation?

Plants respond to shading through an adaptive syndrome termed shade avoidance. In high-density crop plantings, shade avoidance generally increases extension growth at the expense of yield and can be at odds with the agronomic performance of the crop as a whole. Studies in Arabidopsis are beginning to reveal the essential role phytochromes play in regulating this process and to identify genes underlying the response. In this article, we focus on how phytochrome signaling networks have been targeted in cereal breeding programs in the past and discuss the potential to alter these pathways through breeding and transgenic manipulation to develop crops that perform better under typical high density conditions.

Edible Grain↗
PubMed2004

Distribution of Activator (Ac) throughout the maize genome for use in regional mutagenesis.

A collection of Activator (Ac)-containing, near-isogenic W22 inbred lines has been generated for use in regional mutagenesis experiments. Each line is homozygous for a single, precisely positioned Ac element and the Ds reporter, r1-sc:m3. Through classical and molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the maize genome and 41 were precisely placed on the linkage map utilizing multiple recombinant inbred populations. Several PCR techniques were utilized to amplify DNA fragments flanking tr-Ac insertions up to 8 kb in length. Sequencing and database searches of flanking DNA revealed that the majority of insertions are in hypomethylated, low- or single-copy sequences, indicating an insertion site preference for genic sequences in the genome. However, a number of Ac transposition events were to highly repetitive sequences in the genome. We present evidence that suggests Ac expression is regulated by genomic context resulting in subtle variations in Ac-mediated excision patterns. These tr-Ac lines can be utilized to isolate genes with unknown function, to conduct fine-scale genetic mapping experiments, and to generate novel allelic diversity in applied breeding programs.

Chromosome Mapping↗
PubMed2004

The Elm1 (ZmHy2) gene of maize encodes a phytochromobilin synthase.

The light insensitive maize (Zea mays) mutant elongated mesocotyl1 (elm1) has previously been shown to be deficient in the synthesis of the phytochrome chromophore 3E-phytochromobilin (PPhiB). To identify the Elm1 gene, a maize homolog of the Arabidopsis PPhiB synthase gene AtHY2 was isolated and designated ZmHy2. ZmHy2 encodes a 297-amino acid protein of 34 kD that is 50% identical to AtHY2. ZmHY2 was predicted to be plastid localized and was targeted to chloroplasts following transient expression in tobacco (Nicotiana plumbaginifolia) leaves. Molecular mapping indicated that ZmHy2 is a single copy gene in maize that is genetically linked to the Elm1 locus. Sequence analysis revealed that the ZmHy2 gene of elm1 mutants contains a single G to A transition at the 3' splice junction of intron III resulting in missplicing and premature translational termination. However, flexibility in the splicing machinery allowed a small pool of in-frame ZmHy2 transcripts to accumulate in elm1 plants. In addition, multiple ZmHy2 transcript forms accumulated in both wild-type and elm1 mutant plants. ZmHy2 splice variants were expressed in Escherichia coli and products examined for activity using a coupled apophytochrome assembly assay. Only full-length ZmHY2 (as defined by homology to AtHY2) was found to exhibit PPhiB synthase activity. Thus, the elm1 mutant of maize is deficient in phytochrome response due to a lesion in a gene encoding phytochromobilin synthase that severely compromises the PPhiB pool.

Amino Acid Sequence↗
PubMed2004

Structure and expression of maize phytochrome family homeologs.

To begin the study of phytochrome signaling in maize, we have cloned and characterized the phytochrome gene family from the inbred B73. Through DNA gel blot analysis of maize genomic DNA and BAC library screens, we show that the PhyA, PhyB, and PhyC genes are each duplicated once in the genome of maize. Each gene pair was positioned to homeologous regions of the genome using recombinant inbred mapping populations. These results strongly suggest that the duplication of the phytochrome gene family in maize arose as a consequence of an ancient tetraploidization in the maize ancestral lineage. Furthermore, sequencing of Phy genes directly from BAC clones indicates that there are six functional phytochrome genes in maize. Through Northern gel blot analysis and a semiquantitative reverse transcriptase polymerase chain reaction assay, we determined that all six phytochrome genes are transcribed in several seedling tissues. However, expression from PhyA1, PhyB1, and PhyC1 predominate in all seedling tissues examined. Dark-grown seedlings express higher levels of PhyA and PhyB than do light-grown plants but PhyC genes are expressed at similar levels under light and dark growth conditions. These results are discussed in relation to phytochrome gene regulation in model eudicots and monocots and in light of current genome sequencing efforts in maize.

Base Sequence↗
PubMed2003

Photomorphogenic responses in maize seedling development.

As an emerging maize (Zea mays) seedling senses light, there is a decrease in the rate of mesocotyl elongation, an induction of root growth, and an expansion of leaves. In leaf tissues, mesophyll and bundle sheath cell fate is determined, and the proplastids of each differentiate into the dimorphic chloroplasts typical of each cell type. Although it has been inferred from recent studies in several model plant species that multiple photoreceptor systems mediate this process, surprisingly little is known of light signal transduction in maize. Here, we examine two photomorphogenic responses in maize: inhibition of mesocotyl elongation and C4 photosynthetic differentiation. Through an extensive survey of white, red, far-red, and blue light responses among a diverse collection of germplasm, including a phytochrome-deficient mutant elm1, we show that light response is a highly variable trait in maize. Although all inbreds examined appear to have a functional phytochrome signal transduction pathway, several lines showed reduced sensitivity to blue light. A significant correlation was observed between light response and subpopulation, suggesting that light responsiveness may be a target of artificial selection. An examination of C4 gene expression patterns under various light regimes in the standard W22 inbred and elm1 indicate that cell-specific patterns of C4 gene expression are maintained in fully differentiated tissues independent of light quality. To our knowledge, these findings represent the first comprehensive survey of light response in maize and are discussed in relation to maize breeding strategies.

Base Sequence↗
PubMed2003

Activator mutagenesis of the pink scutellum1/viviparous7 locus of maize.

The transposable elements Activator/Dissociation (Ac/Ds) were first discovered in maize, yet they have not been used extensively in their native host for gene-tagging experiments. This can be attributed largely to the low forward mutation rate and the propensity for closely linked transpositions associated with Ac and its nonautonomous deletion derivative Ds. To overcome these limitations, we are developing a series of nearly isogenic maize lines, each with a single active Ac element positioned at a well-defined location. These Ac elements are distributed at 10- to 20-centimorgan intervals throughout the genome for use in regional mutagenesis. Here, we demonstrate the utility of this Ac-based gene-tagging approach through the targeted mutagenesis of the pink scutellum1/viviparous7 (ps1/vp7) locus. Using a novel PCR-based technique, the Ps1 gene was cloned and Ac elements positioned precisely in each of the seven alleles recovered. The Ps1 gene is predicted to encode lycopene beta-cyclase and is necessary for the accumulation of both abscisic acid and the carotenoid zeaxanthin in mature maize embryos. This study demonstrates the utility of an Ac mutagenesis program to efficiently generate allelic diversity at closely linked loci in maize.

Abscisic Acid↗
PubMed2003

The globby1-1 (glo1-1) mutation disrupts nuclear and cell division in the developing maize seed causing alterations in endosperm cell fate and tissue differentiation.

Cereal endosperm tissues account for most of the world's calorific intake, yet the regulation of monocot seed development remains poorly understood. The maize endosperm originates with a series of free-nuclear divisions, followed by cellularisation and subsequent formation of a range of functional cellular domains. We describe the isolation and characterisation of a mutation that induces aberrant globular embryo and endosperm morphology, globby1-1 (glo1-1). Our data indicate that glo1-1 plays a role in nuclear division and cytokinesis in the developing seed. Pattern formation in the embryo is severely impaired with development arresting at premature stages, while in the endosperm, the effects of the glo1-1 mutation are manifest at the free-nuclear or syncytial stage. During cellularisation, and at later stages of development, aberrant cell division and localised domains of cell proliferation are apparent in glo1-1 endosperms. As a consequence, cell fate acquisition and subsequent differentiation of endosperm tissues are affected to varying degrees of severity. To date, it has been hypothesised that BETL cell fate is specified in the syncytium and that cell files subsequently develop in response to a gradient of signal(s) derived from the maternal pedicel region. Based on our findings, however, we propose that specification of BETL cells is an irreversible event that occurs within a narrow window of syncytial development, and that BETL cell identity is subsequently inherited in a lineage-dependent manner. Additionally, our data suggest that acquisition of aleurone cell fate does not solely rely upon signalling from the maternal surrounding tissue to the periphery of the endosperm, as previously thought, but that other factor(s) present within the endosperm are involved.

Cell Differentiation↗
PubMed2003

Transposon tagging using Activator (Ac) in maize.

The transposable element Activator (Ac) has been used in several plant species as a tool for gene isolation and characterization. However, it has not been widely utilized in its native host maize, in part, because of a relatively low germinal transposition rate. The propensity of Ac to move to linked sites provides an opportunity to overcome this limitation when Ac elements are distributed at regular intervals throughout the genome. This chapter details the use of such a system in maize through simple genetic manipulations. A detailed protocol is also provided to clone DNA flanking Ac insertions.

Base Sequence↗
PubMed2002

Elongated mesocotyl1, a phytochrome-deficient mutant of maize.

To begin the functional dissection of light signal transduction pathways of maize (Zea mays), we have identified and characterized the light-sensing mutant elm1 (elongated mesocotyl1). Seedlings homozygous for elm1 are pale green, show pronounced elongation of the mesocotyl, and fail to de-etiolate under red or far-red light. Etiolated elm1 mutants contain no spectrally active phytochrome and do not deplete levels of phytochrome A after red-light treatment. High-performance liquid chromatography analyses show that elm1 mutants are unable to convert biliverdin IX alpha to 3Z-phytochromobilin, preventing synthesis of the phytochrome chromophore. Despite the impairment of the phytochrome photoreceptors, elm1 mutants can be grown to maturity in the field. Mature plants retain aspects of the seedling phenotype and flower earlier than wild-type plants under long days. Thus, the elm1 mutant of maize provides the first direct evidence for phytochrome-mediated modulation of flowering time in this agronomically important species.

Biliverdine↗
PubMed2001

Transposon tagging in maize.

Through recent government- and industry-sponsored efforts, several forward and reverse genetic screening programs have emerged over the past few years to aid in the genetic dissection of gene function in maize. Despite a US maize crop valued at $18.4 billion last year (http://www.ncga.com/03world/main/US_crop_value_2000.html) and rich genetic history, maize has taken a back seat to Arabidopsis thaliana as the model genetic system for plants over the past decade. With a fully sequenced genome, short generation time and small size, studies of Arabidopsis have provided plant scientists with a molecular framework for hormonal, developmental and environmental signaling pathways in plants. As investigations into Arabidopsis continue, our capacity to engineer biochemical pathways and alter plant physiological responses will become increasingly sophisticated. Nevertheless, approximately 130 million years have passed since monocot and higher eudicot lineages diverged. Thus, our ability to engineer agronomically important monocot grasses such as maize, rice and wheat will become increasingly limited by our lack of understanding of the physiological and morphological differences that have evolved in the monocots and higher eudicots. The sophisticated transposon collections now being generated for maize are but one of several recent projects (http://www.nsf.gov/bio/pubs/awards/genome01.htm) to provide grass researchers with essential tools for genome analysis. Because grain crops are such a closely related group, it is hoped that many of the findings made in one grass will be directly applicable to understanding the biology of another. The goal of this review is to highlight the recent developments in maize transposon-based gene characterization programs and provide a critical examination of the advantages and disadvantages each system offers.

DNA Transposable Elements↗