LETTER The Vascular Prepattern Enhancer Trap Marks Early Vascular Development in Arabidopsis David R. Holding † and Patricia S. Springer* Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, California Received 5 April 2002; Accepted 10 May 2002 Summary: Vascular development is a fundamental com- ponent of leaf morphogenesis, and the mechanisms that control vascular patterning are poorly understood. We report here the identification of an enhancer trap line, Vascular Prepattern (VPP), that acts as a marker for early vascular development. GUS reporter gene expression in VPP was detected in provascular cells from the earliest stages of primary midvein formation in leaf primordia and subsequently coincided with the early specification of higher order veins. GUS expression in VPP also marks the quiescent center cells of the root apical meristem at all stages of root development. VPP provides a marker for early vascular development and will be a useful tool for studying vascular patterning. genesis 33:155–159, 2002. © 2002 Wiley-Liss, Inc. Key words: arabidopsis; vascular patterning; molecular marker; gene trap INTRODUCTION Vascular development proceeds through a regular series of events that have been well documented in many plant species (Esau, 1965). Provascular cells are initially se- lected from a uniform field of ground meristem cells and undergo a number of morphological changes, including alterations in cell shape and cell division planes, to be- come procambial cells. Procambial cells develop along narrow linear paths in a regular and highly predictable pattern. The final stages of maturation involve the differ- entiation of procambial cells into xylem or phloem. The terms “provascular” and “procambial” have often been used interchangeably in the literature to refer to a stage of development at which vascular precursor cells are axialized and morphologically distinct (Esau, 1965). However, some authors have used the provascular des- ignation to refer to a prior stage of development (Steeves and Sussex, 1989; Xia and Steeves, 2000). We will follow the latter convention and use the term provascular to refer to a stage of vascular development prior to a time when morphological changes are visible. Key events in pattern formation occur before vascular precursor cells are histologically distinguishable from surrounding cells. The molecular mechanisms that con- trol the initial events of vascular patterning are not un- derstood, and a lack of markers for early vascular devel- opment has been a major limitation to the study of vascular patterning. Here we report the identification of an enhancer trap line, Vascular Prepattern (VPP), in which reporter gene expression marks provascular cells. In a screen of gene and enhancer trap transposants (Holding and Springer, unpublished research), trans- posant line ETR273 was identified that displayed a retic- ulate pattern of GUS activity in leaves. GUS activity transiently marked regions undergoing vascular differen- tiation and disappeared from veins prior to maturity (Fig. 1). A similar pattern was seen in forming vascular tissue in floral organs (data not shown). GUS activity was not observed in vascular bundles of the root, hypocotyls, or inflorescence stem (data not shown). We examined GUS activity in the cotyledons and first leaves of ETR273 over a time course. The simple cotyle- don venation is largely patterned during embryogenesis (Kinsman and Pyke, 1998; Sieburth, 1999). GUS activity was detected in cotyledons up to 3 days postimbibition (DPI) in the preaxial cells of developing secondary veins, declining as cells became elongate (Fig. 1a, b). GUS activity was not detected in first leaves of imbibed seeds at 1 DPI. At 2 DPI, the radical was just emerging and the cotyledons had not yet broken the seed coat. At this stage, GUS activity marked a small number of cells in the center of the first two leaf primordia as they emerged from the shoot apical meristem (data not shown). At 3 DPI, the first two leaf primordia were extending out from the meristem and GUS activity was detected in a few subepidermal cells at the leaf tip (Fig. 1c). Leaves that were slightly more advanced displayed GUS activity † Current address: Department of Plant Sciences, University of Arizona, Tucson, AZ 85721. * Correspondence to: Patricia S. Springer, Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-0124. E-mail: patricia.springer@ucr.edu Grant sponsors: The National Science Foundation and The University of California Agricultural Experiment Station. Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/gene.10103 © 2002 Wiley-Liss, Inc. genesis 33:155–159 (2002)