The principles underlying the formation of veins in the leaf have long intrigued developmental biologists. In Arabidopsis leaves, files of anatomically inconspicuous subepidermal cells that will elongate into vein-forming procambial cells selectively activate ATHB8 gene expression. The biological role of ATHB8 in vein formation and the molecular events that culminate in acquisition of the ATHB8 preprocambial cell state are unknown, but intertwined pathways of auxin transport and signal transduction have been implicated in defining paths of vascular strand differentiation. Here we show that ATHB8 is required to stabilize preprocambial cell specification against auxin transport perturbations, to restrict preprocambial cell state acquisition to narrow fields and to coordinate procambium formation within and between veins. We further show that ATHB8 expression at preprocambial stages is directly and positively controlled by the auxin-response transcription factor MONOPTEROS (MP) through an auxin-response element in the ATHB8 promoter. We finally show that the consequences of loss of ATHB8 function for vein formation are masked by MP activity. Our observations define, at the molecular level, patterning inputs of auxin signaling in vein formation.

The sequence of events underlying the formation of vascular networks in the leaf has long fascinated developmental biologists. In Arabidopsis leaves, vascular-precursor procambial cells derive from the elongation of morphologically inconspicuous ground cells that selectively activate expression of the HD-ZIP III gene ATHB8. Inception of ATHB8 expression operationally defines acquisition of a typically irreversible preprocambial cell state that preludes to vein formation. A view of the constellation of genes whose expression is activated at preprocambial stages would therefore be particularly desirable; however, very few preprocambial gene expression profiles have been identified. Here, we show that expression of three genes encoding members of the DOF family of plant-specific transcription factors is activated at stages overlapping onset of ATHB8 expression. Expression of DOF genes is initiated in wide domains that become confined to sites of vein development. Congruence between DOF expression fields and zones of vein formation persists upon experimental manipulation of leaf vascular patterning, suggesting that DOF expression identifies consistently recurring steps in vein ontogeny. Our results contribute to defining preprocambial cell identity at the molecular level.

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