Settles Lab Research Interests
Signaling in Seed Development
The endosperm and embryo develop from the two products of double fertilization. In higher plants, each pollen grain contains two sperm cells. Upon fertilization, one sperm fuses with the egg cell of the female gametophyte to form the embryo. The second sperm fuses to the diploid central cell to give rise to the triploid endosperm. The endosperm and embryo then develop coordinately in a species-specific manner. In cereals, the endosperm persists at the end of seed development and forms a starchy storage tissue that is the basis for much of our food and animal feeds. The endosperm differentiates into at least four cell-types that have distinct roles in nutrient transport and storage as well as nutrient utilization upon seed germination. We are interested in answering the following questions about seed development:
- What are the signals that allow the endosperm and embryo to communicate with each other during seed formation?
- How do these signals regulate meristem formation and organogenesis within the embryo?
- Are similar signaling mechanisms used to determine the different cell types within the endosperm as those used for endosperm-embryo communication and organogenesis in the embryo?
To address these questions, we are focusing on a class of maize seed mutants that we term rough endosperm (rgh) mutants. The rgh mutants are characterized by having an etched, pitted, or crazed surface to the seed. These mutants have dramatic effects on endosperm and embryo development that suggest roles for organogenesis in the embryo as well as cell differentiation within the endosperm. We are using genetic tools and the visible phenotypes of rgh mutants to identify loci that have distinct roles in seed development. For example, we have found a rgh locus that is involved in endosperm-embryo communication using chromosome translocations. In addition, we have identified a separate rgh locus that causes defects in carotenoid and chlorophyll accumulation as well as a loss of seed dormancy. This Rgh gene is likely to be involved in a plastid function required for both endosperm and embryo development.
The embryo and endosperm derive from the two products of double fertilization. (Schematics modified from Kiesselbach, 1949)
Ears of corn segregating for rgh seed mutants. Functional Genomics
To facilitate our studies of seed development, the lab is also developing genomics tools focused on maize. We are working in several collaborative projects to generate:
- Transposon mutagenesis resources
- Transposon flanking sequence tags
- Endosperm expressed sequence tags
- Non-destructive chemical phenotyping of maize kernels
We have developed the UniformMu transposon-tagging population to simplify molecular analysis of maize seed mutants. UniformMu carries active Robertson's Mutator transposons that can move within the genome to cause mutations. The UniformMu population has been crossed for multiple generations into a single genetic inbred line of maize, W22, which simplifies the identification of parental transposon insertions.
We use MuTAIL-PCR to identify the transposons within UniformMu individuals. MuTAIL-PCR is a specialized molecular technique that amplifies the genomic DNA next to the Mutator transposons. By cloning and sequencing this DNA, we can identify where a transposon is located within the maize genome. More details about UniformMu mutants and MuTAIL-PCR are available at www.uniformmu.org.
In addition, we are collaborating with Rutger's University, Iowa State University, and the University of Arizona to discover the genes that are expressed during maize endosperm development. Both endosperm Expressed Sequence Tags and UniformMu Transposon Flanking Sequence Tags are available at endosperm.info.
Finally, we are using single-kernel near infrared reflectance (NIR) spectroscopy as a method for non-destructive analysis of the UniformMu seed mutants. In collaboration with the USDA-GMPRC, we are screening the UniformMu population with NIR technology to find mutants that have effects on the chemical composition of the seed.
UniformMu is an inbred Mutator population. We have isolated >2,200 independent seed mutants from UniformMu.
We have sequenced >34,000 MuTAIL flanking sequence tags from UniformMu seed mutants.
We use NIR spectroscopy to analyze the composition of UniformMu seed mutants.
