Genome-wide analysis of SHORTROOT targets in the Arabidopsis root stele

Asymmetric cell division (ACD) and positional signals play critical roles in the tissue patterning during multicellular organ development. In the Arabidopsis root meristem, two major phloem cell types arise via ACDs of distinct origins: one for companion cells and the other for proto- and metaphloem sieve elements. The molecular mechanisms underlying each of these processes have been reported, however, how these two are coordinated has remained elusive. Here, we report that SHORTROOT (SHR) is the key coordinator of two ACD processes for phloem development. SHR moving into the endodermis regulates ACD for companion cells by turning on microRNA165/6. ACD that generates phloem sieve elements is mediated by SHR moving into phloem precursors. To find the downstream targets of SHR in the stele, pCRE1::erGFP was introduced into wild type and shr-2 backgrounds to express GFP in the stele cells in the root meristem. We then collected GFP-expressing stele cells from the wild type, shr-2 and pCRE1::SHRΔNLELDV:nlsGFP; shr-2 through Fluorescence Activated Cell Sorter (FACS). RNAs were extracted from the sorted cells of each line and processed for generating labeled probes to be hybridized onto GeneChip Arabidopsis Tiling 1.0R Array (Affymetrix). We then examined the influence of SHR on phloem enriched genes in the tiling array data. In this analysis, we found 224 genes that are down-regulated in shr-2 in comparison to the wild type and then restore expression in pCRE1::SHRΔNLELDV:nlsGFP; shr-2. A total of 7 data (2 replicates of pCRE1::erGFP;shr-2 mutant, 2 replicates of pCRE1::SHRΔNLELDV:nlsGFP; shr-2, 3 replicates of pCRE1::erGFP in the wild type Col-0) are submitted. Three genotypes were germinated and grown for 6 days on MS agar media with 1% of Sucrose. Protoplasts from these roots were isolated and sorted through FACS, and then RNA transcripts collected from GFP expressing protoplasts were profiled on the Arabidopsis Tiling 1.0R Array.