Technology development and data set generation 

We pioneered the use of fluorescence activated cell sorting (FACS) to perform cell-type specific expression analysis in the root. Recently, we completed a third-generation analysis of gene expression using Illumina short-read sequencing and PacBio long-read sequencing. In collaboration with Uwe Ohler (Berlin) we found evidence that alternative splicing is particularly important in fine-tuning developmental processes in the root. Quantitative proteomics revealed a strong correlation between abundance of the major splice isoform of a gene and the resulting protein. Using the SOLiD sequencing technology coupled with FACS we were able to identify new natural antisense transcripts. In collaboration with Molly Megraw (Oregon State) we used a similar approach to identify transcription start sites in the Arabidopsis genome. Finally, in collaboration with Asaph Aharoni (Weizmann, Israel) we performed a metabolomics analysis and identified cell-type specific metabolites.

The technology for analyzing translation has dramatically changed with the advent of ribosome profiling or Ribo-Seq. We systematically explored experimental parameters to optimize ribosome profiles. We then identified over 20 small open reading frames that could encode new signaling molecules. Most have cell type-specific RNA expression patterns and are conserved evolutionarily.

Having identified transcription factors and signaling molecules involved in the path to differentiation, we collaborated with Joe Ecker (HHMI, Salk) to explore the role of epigenetics. We isolated small RNAs and performed bi-sulfite sequencing on different root cell types. Surprisingly, there was very little difference in DNA methylation or small RNA distribution among the cell types, with one exception, the columella root cap, which had dramatically more CHH methylation than any other tissue. The columella root cap differentiates rapidly and it has been suggested that chromosome decondensation may facilitate rapid differentiation. Thus, increased production of CHH methylation may reduce the possibility of transposable element activity during the rapid differentiation phase.

Another focus of our work is to develop imaging modalities that allow us to quantify key aspects of plant development. We have pioneered the use of fluorescence correlation spectroscopy (FCS) in plants.  We are among the first to use light sheet microscopy to follow molecular dynamics in plants. We also developed a microfluidics device, the RootArray, which holds 64 seedlings each with a fluorescent transgene that can be imaged with confocal microscopy.


Cell sorting / Omics analysis


Root Imaging