A molecular circuit that creates the phloem pattern

The vascular system of plants consists of xylem, phloem and procambium in a specific pattern. The phloem consists of sieve elements, the apparatus for bulk flow of photo-assimilates, and companion cells, which mediate transport of photo-assimilates between the sieve elements and surrounding cells and support the biological activities of the sieve element cells. The regulatory mechanisms of vascular development are being uncovered. Here we show that PHLOEM EARLY DOFs (PEARs) and related genes (collectively phloem-Dofs) not only regulate the number of procambium cell files, but also positively regulate phloem differentiation. Overexpression of phloem-Dofs induced cells that expressed either sieve element or companion cell marker genes, which are mutually exclusive. Conversely, disruption of phloem-Dofs caused loss of phloem. Phloem-Dofs induce CLAVATA3/EMBRYO SURROUNDING REGION-RELATED25 (CLE25) and CLE26 peptides, which in turn inhibit expression of phloem-Dofs and phloem formation, forming a negative feedback loop. Disruption of multiple genes for either phloem-expressed CLEs, BARELY ANY MERISTEM (BAM)-class receptors, or their coreceptors, CLAVATA3 INSENSITIVE RECEPTOR KINASEs (CIKs), caused excess formation of phloem cell files. We further show that phloem-Dofs are under positive self and mutual regulation. These positive and negative feedback loops create the proper phloem pattern. Dof genes expressed preferentially expressed in the phloem were overexpressed by using estrogen-inducible expression vector, ER8. Arabidopsis lines produced after transformation with ER8Dof1.1, ER8Dof2.2, and ER8PEAR were treated with or without estriol for 16 hours, and RNA samples were prepared from roots, and subjected to a microarray experiments.