Thomas P. Brutnell

Natural Genetic Variation in Lycopene Epsilon Cyclase Tapped for Maize Biofortification

Dietary vitamin A deficiency causes eye disease in 40 million children each year and places 140 to 250 million at risk for health disorders. Many children in sub-Saharan Africa subsist on maize-based diets. Maize displays considerable natural variation for carotenoid composition, including vitamin A precursors α-carotene, β-carotene, and β-cryptoxanthin. Through association analysis, linkage mapping, expression analysis, and mutagenesis, we show that variation at the lycopene epsilon cyclase (lcyE) locus alters flux down α-carotene versus β-carotene branches of the carotenoid pathway. Four natural lcyE polymorphisms explained 58% of the variation in these two branches and a threefold difference in provitamin A compounds. Selection of favorable lcyE alleles with inexpensive molecular markers will now enable developing-country breeders to more effectively produce maize grain with higher provitamin A levels.

The developmental dynamics of the maize leaf transcriptome

We have analyzed the maize leaf transcriptome using Illumina sequencing. We mapped more than 120 million reads to define gene structure and alternative splicing events and to quantify transcript abundance along a leaf developmental gradient and in mature bundle sheath and mesophyll cells. We detected differential mRNA processing events for most maize genes. We found that 64% and 21% of genes were differentially expressed along the developmental gradient and between bundle sheath and mesophyll cells, respectively. We implemented Gbrowse, an electronic fluorescent pictograph browser, and created a two-cell biochemical pathway viewer to visualize datasets. Cluster analysis of the data revealed a dynamic transcriptome, with transcripts for primary cell wall and basic cellular metabolism at the leaf base transitioning to transcripts for secondary cell wall biosynthesis and C4 photosynthetic development toward the tip. This dataset will serve as the foundation for a systems biology approach to the understanding of photosynthetic development.

Reference Genome Sequence Of The Model Plant Setaria

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ∼400-Mb assembly covers ∼80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species that demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).

Rare genetic variation at Zea mays crtRB1 increases Β-carotene in maize grain

Breeding to increase β-carotene levels in cereal grains, termed provitamin A biofortification, is an economical approach to address dietary vitamin A deficiency in the developing world. Experimental evidence from association and linkage populations in maize (Zea mays L.) demonstrate that the gene encoding β-carotene hydroxylase 1 (crtRB1) underlies a principal quantitative trait locus associated with β-carotene concentration and conversion in maize kernels. crtRB1 alleles associated with reduced transcript expression correlate with higher β-carotene concentrations. Genetic variation at crtRB1 also affects hydroxylation efficiency among encoded allozymes, as observed by resultant carotenoid profiles in recombinant expression assays. The most favorable crtRB1 alleles, rare in frequency and unique to temperate germplasm, are being introgressed via inexpensive PCR marker-assisted selection into tropical maize germplasm adapted to developing countries, where it is most needed for human health.

The iPlant Collaborative: Cyberinfrastructure for Plant Biology

The iPlant Collaborative (iPlant) is a United States National Science Foundation (NSF) funded project that aims to create an innovative, comprehensive, and foundational cyberinfrastructure in support of plant biology research (PSCIC, 2006). iPlant is developing cyberinfrastructure that uniquely enables scientists throughout the diverse fields that comprise plant biology to address Grand Challenges in new ways, to stimulate and facilitate cross-disciplinary research, to promote biology and computer science research interactions, and to train the next generation of scientists on the use of cyberinfrastructure in research and education. Meeting humanitys projected demands for agricultural and forest products and the expectation that natural ecosystems be managed sustainably will require synergies from the application of information technologies. The iPlant cyberinfrastructure design is based on an unprecedented period of research community input, and leverages developments in high-performance computing, data storage, and cyberinfrastructure for the physical sciences. iPlant is an open-source project with application programming interfaces that allow the community to extend the infrastructure to meet its needs. iPlant is sponsoring community-driven workshops addressing specific scientific questions via analysis tool integration and hypothesis testing. These workshops teach researchers how to add bioinformatics tools and/or datasets into the iPlant cyberinfrastructure enabling plant scientists to perform complex analyses on large datasets without the need to master the command-line or high-performance computational services.

Setaria viridis: A Model for C4 Photosynthesis

C4 photosynthesis drives productivity in several major food crops and bioenergy grasses, including maize (Zea mays), sugarcane (Saccharum officinarum), sorghum (Sorghum bicolor), Miscanthus x giganteus, and switchgrass (Panicum virgatum). Gains in productivity associated with C4 photosynthesis include improved water and nitrogen use efficiencies. Thus, engineering C4 traits into C3 crops is an attractive target for crop improvement. However, the lack of a small, rapid cycling genetic model system to study C4 photosynthesis has limited progress in dissecting the regulatory networks underlying the C4 syndrome. Setaria viridis is a member of the Panicoideae clade and is a close relative of several major feed, fuel, and bioenergy grasses. It is a true diploid with a relatively small genome of ~510 Mb. Its short stature, simple growth requirements, and rapid life cycle will greatly facilitate genetic studies of the C4 grasses. Importantly, S. viridis uses an NADP-malic enzyme subtype C4 photosynthetic system to fix carbon and therefore is a potentially powerful model system for dissecting C4 photosynthesis. Here, we summarize some of the recent advances that promise greatly to accelerate the use of S. viridis as a genetic system. These include our recent successful efforts at regenerating plants from seed callus, establishing a transient transformation system, and developing stable transformation.

Brachypodium as a model for the grasses: Today and the future

Over the past several years, Brachypodium distachyon (Brachypodium) has emerged as a tractable model system to study biological questions relevant to the grasses. To place its relevance in the larger context of plant biology, we outline here the expanding adoption of Brachypodium as a model grass and compare this to the early history of another plant model, Arabidopsis thaliana. In this context, Brachypodium has followed an accelerated path in which the development of genomic resources, most notably a whole genome sequence, occurred concurrently with the generation of other experimental tools (e.g. highly efficient transformation and large collections of natural accessions). This update provides a snapshot of available and upcoming Brachypodium resources and an overview of the community including the trajectory of Brachypodium as a model grass.

Setaria viridis and Setaria italica, model genetic systems for the Panicoid grasses

Setaria italica and its wild ancestor Setaria viridis are diploid C(4) grasses with small genomes of ∼515 Mb. Both species have attributes that make them attractive as model systems. Setaria italica is a grain crop widely grown in Northern China and India that is closely related to the major food and feed crops maize and sorghum. A large collection of S. italica accessions are available and thus opportunities exist for association mapping and allele mining for novel variants that will have direct application in agriculture. Setaria viridis is the weedy relative of S. italica with many attributes suitable for genetic analyses including a small stature, rapid life cycle, and prolific seed production. Setaria sp. are morphologically similar to most of the Panicoideae grasses, including major biofuel feedstocks, switchgrass (Panicum virgatum) and Miscanthus (Miscanthus giganteus). They are broadly distributed geographically and occupy diverse ecological niches. The cross-compatibility of S. italica and S. viridis also suggests that gene flow is likely between wild and domesticated accessions. In addition to serving as excellent models for C(4) photosynthesis, these grasses provide novel opportunities to study abiotic stress tolerance and as models for bioenergy feedstocks.

BUNDLE SHEATH DEFECTIVE2, a Novel Protein Required for Post-Translational Regulation of the rbcL Gene of Maize

The Bundle sheath defective2 (Bsd2) gene is required for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Using a Mutator transposable element as a molecular probe, we identified a tightly linked restriction fragment length polymorphism that cosegregated with the bsd2-conferred phenotype. This fragment was cloned, and sequences flanking the Mutator insertion were used to screen a maize leaf cDNA library. Using a full-length cDNA clone isolated in this screen, we show that an abundant 0.6-kb transcript could be detected in wild-type plants but not in bsd2-m1 plants. This 0.6-kb transcript accumulated to low levels in plants carrying an allele derived from bsd2-m1 that conditions a less severe mutant phenotype. Taken together, these data strongly suggest that we have cloned the Bsd2 gene. Sequence analysis of the full-length cDNA clone revealed a chloroplast targeting sequence and a region of homology shared between BSD2 and the DnaJ class of molecular chaperones. This region of homology is limited to a cysteine-rich Zn binding domain in DnaJ believed to play a role in protein–protein interactions. We show that BSD2 is targeted to the chloroplast but is not involved in general photosynthetic complex assembly or protein import. In bsd2 mutants, we could not detect the Rubisco protein, but the chloroplast-encoded Rubisco large subunit transcript (rbcL) was abundant and associated with polysomes in both bundle sheath and mesophyll cells. By characterizing Bsd2 expression patterns and analyzing the bsd2-conferred phenotype, we propose a model for BSD2 in the post-translational regulation of rbcL in maize.

High Glycolate Oxidase Activity Is Required for Survival of Maize in Normal Air

A mutant in the maize (Zea mays) Glycolate Oxidase1 (GO1) gene was characterized to investigate the role of photorespiration in C4 photosynthesis. An Activator-induced allele of GO1 conditioned a seedling lethal phenotype when homozygous and had 5% to 10% of wild-type GO activity. Growth of seedlings in high CO2 (1%–5%) was sufficient to rescue the mutant phenotype. Upon transfer to normal air, the go1 mutant became necrotic within 7 d and plants died within 15 d. Providing [1-14C]glycolate to leaf tissue of go1 mutants in darkness confirmed that the substrate is inefficiently converted to 14CO2, but both wild-type and GO-deficient mutant seedlings metabolized [1-14C]glycine similarly to produce [14C]serine and 14CO2 in a 1:1 ratio, suggesting that the photorespiratory pathway is otherwise normal in the mutant. The net CO2 assimilation rate in wild-type leaves was only slightly inhibited in 50% O2 in high light but decreased rapidly and linearly with time in leaves with low GO. When go1 mutants were shifted from high CO2 to air in light, they accumulated glycolate linearly for 6 h to levels 7-fold higher than wild type and 11-fold higher after 25 h. These studies show that C4 photosynthesis in maize is dependent on photorespiration throughout seedling development and support the view that the carbon oxidation pathway evolved to prevent accumulation of toxic glycolate.