High Level Overview

We are studying 90 hours of early anther development in maize (Zea mays L., “corn”) to elucidate the signaling networks that regulate cell type acquisition and maintenance within anther locules. The question: Without a germ line, how do cells in plants switch from mitosis to meiosis?

What are we doing now?

phasiRNA story

1. CRISPR-mediated elimination of the sequential steps in 24-nt phasiRNA production to test the hypothesis that these small RNAs are required for fertility. Observation: at least one component is essential!
2. Search for phasiRNA binding proteins. See our recent review on ARGONAUTEs for an overview.
3. Zhang, H., R. Xia, B. C. Meyers, and V. Walbot. 2015. Evolution, functions and mysteries of plant ARGONAUTE proteins. Current Op. Plant Biol. doi: 10.1016/j.pbi.2015.06.011
4. Determine if phasiRNAs influence DNA methylation status or are otherwise tied into epigenetic regulation in anthers.
5. Determine the length and structure of the phasiRNA precursor molecules; to date, we've studied the cleaved precursor (cleavage mediated by microRNA2275) that is the substrate for DICER5 slicing into 24-nt phased small RNAs.
6. Analyze phasiRNA cleaved precursor abundances in more mutants.
7. To date all of our work has been in the inbred line W23 - are results similar in other inbreds with regard to timing and distribution of abundances from the 176 known phas 24-nt loci?

Pre-meiotic anther development

1. We've cloned, done microarray and confocal analysis of ms23; we are now analyzing MS23, a bHLH transcription factor, for interaction with other proteins.
2. Defining cell division defects - either anticlinal or periclinal - in 10 early acting male-sterility mutants. Most mutants result in an aberrant number of cells per lobe, but some mutants show compensatory division in one cell type to balance under or over-proliferation in another cell type.
3. We have 23 pre-meiotic mutants, 8 of which are already cloned and at least partially analyzed for expression, in situ localization, and impact on anther development (in detail by confocal microscopy). We are slowly closing in on three additional loci.
4. We are developing a clever new tool for local delivery of MAC1 in growing anthers.
5. We have identified MSCA1 interacting proteins, presumptive targets of the redox relay initiated by hypoxia in anthers.
6. We are analyzing earlier steps in tassel development to connect the extensive knowledge of organ specification to the initial steps of cell specification in anthers-are some of the same factors used again?
7. We are identifying defining characteristics of AR and PMC cells compared to the neighboring soma and pursuing our observation that just specified AR express many (more than 100) genes associated with meiosis, which occurs six days later and after 3-4 mitotic divisions by the AR.
8. We are analyzing some aspects of sugar metabolism in developing anthers as nutrition and sugar signaling may coordinate development. We discovered that the subepidermal endothecial cells contain chloroplasts and accumulate massive amounts of starch prior to meiosis.

If these projects interest you, consider joining us. Contact Professor Walbot (walbot@stanford.edu) for more information.

Follow-up studies and a short review

• Kelliher, T. and V. Walbot. 2014. Germinal cell initials accommodate hypoxia and precociously express meiotic genes. Plant J. 77: 639-652
  doi:10.1111/tpj.12414
  
• Zhang, H., R. Egger, T. Kelliher, D. J. Morrow, J. Fernandes, G-L. Nan, and V. Walbot. 2014. Transcriptomes and proteomes define gene expression progression in pre-meiotic maize anthers. G3 4: 994-1010. Special issue on the Genetics of Sex. doi:10.1534/g3.113.009738
• Kelliher, T., R. Egger, H. Zhang, and V. Walbot. 2014. Unresolved issues in pre-meiotic anther development. Front. Plant Sci. 5: Article 347. doi:10.3389/fpls.2014.00347

Maize phasiRNAs: big bang timing and discrete spatial deployment

The first paper from our collaboration with Blake Meyers and his lab, starting in June 2014 was published recently. We utilized developmental mutants to demonstrate that 21-nt phasiRNAs require a normal epidermis for biogenesis (none found in the ocl4 mutant) while 24-nt phasiRNAs require a normal tapetum (none found in msca1, mac1, or ms23). We draw attention to the likely convergent evolution of grass phasiRNAs found in anthers and mammalian piRNAs found in the testes.

Zhai, J., H. Zhang, S. Arikit, K. Huang, G. Nan, V. Walbot, and B. Meyers. 2015. Spatiotemporal and cell-type dependent biogenesis of phasi-RNAs during male reproduction in Zea mays. Proc. Natl. Acad. Sci. USA 112: 3146-3151. doi:10.1073/pnas.1418918112