Transcriptional Control of Auxin-Transport-Dependent Vein Patterning in Arabidopsis thaliana

Abstract

Most multicellular organisms solve the problem of long-distance transport of signals and nutrients by means of tissue networks such as the vascular system of vertebrate embryos and the vein network of plant leaves; therefore, how vascular networks form is a key question in biology. In vertebrates, the formation of the embryonic vascular system relies on direct cell-cell interaction and, at least in part, on cell migration, both of which are precluded in plants by a cell wall that keeps cells apart and in place; therefore, vein networks form differently in plant leaves. How leaf vein networks form is unclear, but available evidence suggests that the polar transport of the plant signal auxin is required for vein patterning. Functions of polar auxin transport in vein patterning in turn depend on functions of the PIN-FORMED1 (PIN1) auxin transporter. At early stages of leaf tissue development, PIN1 polar localization at the plasma membrane of epidermal cells is directed toward single cells along the marginal epidermis of developing leaves. These “convergence points” of epidermal PIN1 polarity are associated with broad domains of PIN1 expression in the inner tissue of the leaf; these broad domains will over time become restricted to the narrow sites where major veins will form. Consistent with those observations, for the past 15 years the prevailing hypotheses of leaf vein patterning have proposed that convergence points of epidermal PIN1 polarity lead to the formation of local peaks of auxin level in the epidermis, and that that auxin is transported by PIN1 from the epidermal convergence points into the inner tissue where it will lead to vein formation. As such, these hypotheses predict that epidermal PIN1 expression is strictly required for vein patterning. I tested this prediction in Arabidopsis thaliana by a combination of targeted gene expression, molecular genetic analysis, and cellular imaging, and found it unsupported: epidermal PIN1 expression is neither required nor sufficient for PIN1-dependent vein patterning, whereas PIN1 expression in the inner tissues of the leaf turns out to be both required and sufficient for PIN1-dependent vein patterning. To identify regulatory inputs upstream of PIN1-dependent vein patterning, I next sought regulatory elements that are necessary for that component of PIN1 expression in the inner tissues of the leaf that is required for PIN1-dependent vein patterning. By means of a combination of promoter deletion, molecular genetic analysis, and cellular imaging, I found that vascular expression of PIN1 is required for PIN1-dependent vein patterning; that such vascular expression of PIN1 depends on the 151-bp region of the PIN1 promoter from -645 to -495; and that that region of the PIN1 promoter contains putative binding sites for members of an uncharacterized plant-specific family of transcription factors. Finally, for the future characterization of such putative upstream regulators of PIN1 expression, I identified and characterized GAL4/GFP enhancer-trap lines for the targeted misexpression of genes of interest in specific cells and tissues of developing leaves. In conclusion, my results refute all vein patterning hypotheses based on polar auxin transport from the epidermis and suggest alternatives for future tests. Further, my results have identified regulatory inputs that are required for PIN1-dependent vein patterning, and have generated the resources to characterize those regulatory inputs.