DEVELOPMENT 3755 RESEARCH ARTICLE INTRODUCTION The venation patterns of plant leaves provide two-dimensional models for dissecting the generative mechanisms of branched structures. Although detailed descriptions and consistent theoretical models account for venation pattern formation (Meinhardt, 1976; Meinhardt, 1984; Mitchison, 1980; Mitchison, 1981), the mechanism by which such structures are built is still not fully understood at the genetic level. Considerable experimental evidence suggests that indole acetic acid (IAA, the main auxin) transport is involved in the patterning of plant vasculature. The spatial distribution of IAA correlates well with the sites of vascular differentiation in shoot apices and young leaf primordia (Avsian- Kretchmer et al., 2002), and the inhibition of auxin polar transport causes a variety of developmental defects in leaves, including an abnormal vascular network (Mattsson et al., 1999; Sieburth, 1999). These observations support the so-called ‘canalization hypothesis’, which states that the patterning of plant vascular systems depends on a positive feedback loop acting on the transport of auxin (Sachs, 1991a; Sachs, 1991b). Mutants with aberrant venation patterns in leaves or cotyledons have been isolated using Arabidopsis thaliana as a model (Carland and McHale, 1996; Carland et al., 1999; Deyholos et al., 2000; Koizumi et al., 2000; Steynen and Schultz, 2003). Some of the corresponding genes have already been cloned, including COTYLEDON VASCULAR PATTERN1 (CVP1), which encodes a sterol methyltransferase involved in the biosynthesis of sterol and brassinosteroid compounds (Carland et al., 2002), and CVP2, which encodes an inositol polyphosphate 5' phosphatase and confirms a role for inositol 1,4,5-trisphosphate (IP3)-mediated signal transduction in vein ontogeny (Carland and Nelson, 2004). In a search for naturally occurring variations of the leaf venation pattern in Arabidopsis, we found that the spontaneous hemivenata- 1 (hve-1) allele reduces venation complexity in leaves and cotyledons (Candela et al., 1999). Here, we analyze the developmental effects of hve-1 and two other recessive alleles of HVE, which we positionally cloned and found to encode a CAND1 protein. Our results demonstrate a role for ubiquitin-mediated auxin signaling in Arabidopsis vein patterning. MATERIALS AND METHODS Plant material and growth conditions Seeds of most Arabidopsis thaliana (L.) Heynh. lines were supplied by the Nottingham Arabidopsis Stock Centre (NASC) and the Arabidopsis Biological Resource Center (ABRC). These included the Wassilewskija-2 (Ws-2; NASC Accession Number N1601), Col-0 (N1092) and Eifel-5 (Ei- 5; N1128) wild-type accessions, the N599479 and N610969 T-DNA insertion lines from the Salk Institute Genome Analysis Laboratory (Alonso et al., 2003), and the pin1 cay (N324) and axr1-12 (Leyser et al., 1993) mutants. The pin1 cay mutant was isolated by G. Röbbelen and later named cay by Serrano-Cartagena et al. (Serrano-Cartagena et al., 1999). Allelism of cay and pin1-1 was suggested by their similar phenotypes and confirmed in complementation crosses. We found that pin1 cay is a null allele carrying a G-to-A substitution that produces a truncated protein lacking 489 amino acids. lop1-65 and cvp2-1 (Carland and McHale, 1996; Carland et al., 1999) were kindly provided by Francine Carland; mp T370 (Przemeck et al., 1996) by Thomas Berleth; and the ATHB-8-GUS (Baima et al., 1995), CAND1- GUS (Chuang et al., 2004) and DR5-GUS (Ulmasov et al., 1997) transgenic lines by Simona Baima, William Gray and Thomas Guilfoyle, respectively. We obtained mp T370 /mp T370 ;hve-3/hve-3 and mp T370 /mp T370 ;HVE/- seedlings, all of which were lethal and failed to develop roots and produce leaves. In our hands, the induction of adventitious roots by hypocotyl basal The HVE/CAND1 gene is required for the early patterning of leaf venation in Arabidopsis María Magdalena Alonso-Peral 1, *, Héctor Candela 1, * ,† , Juan Carlos del Pozo 2 , Antonio Martínez-Laborda 3 , María Rosa Ponce 1 and José Luis Micol 1,‡ The hemivenata-1 (hve-1) recessive allele was isolated in a search for natural variations in the leaf venation pattern of Arabidopsis thaliana, where it was seen to cause extremely simple venation in vegetative leaves and cotyledons, increased shoot branching, and reduced root waving and fertility, traits that are reminiscent of some mutants deficient in auxin signaling. Reduced sensitivity to exogenous auxin was found in the hve-1 mutant, which otherwise displayed a wild-type response to auxin transport inhibitors. The HVE gene was positionally cloned and found to encode a CAND1 protein. The hve-1 mutation caused mis-splicing of the transcripts of the HVE/CAND1 gene and a vein phenotype indistinguishable from that of hve-2 and hve-3, two putatively null T-DNA alleles. Inflorescence size and fertility were more affected by hve-2 and hve-3, suggesting that hve-1 is hypomorphic. The simple venation pattern of hve plants seems to arise from an early patterning defect. We found that HVE/CAND1 binds to CULLIN1, and that the venation patterns of axr1 and hve mutants are similar, which suggest that ubiquitin-mediated auxin signaling is required for venation patterning in laminar organs, the only exception being cauline leaves. Our analyses of double mutant and transgenic plants indicated that auxin transport and perception act independently to pattern leaf veins, and that the HVE/CAND1 gene acts upstream of ATHB-8 at least in higher order veins, in a pathway that involves AXR1, but not LOP1, PIN1, CVP1 or CVP2. KEY WORDS: Arabidopsis, TIP120, CAND1, Venation pattern formation, Natural variation Development 133, 3755-3766 (2006) doi:10.1242/dev.02554 1 División de Genética and Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Alicante, Spain. 2 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Departamento de Biotecnología, Carretera de la Coruña Km. 7, 28040 Madrid, Spain. 3 División de Genética, Universidad Miguel Hernández, Campus de San Juan, 03550 Alicante, Spain. *These authors contributed equally to this work † Present address: Plant Gene Expression Center, University of California, Berkeley, Albany, CA 94710, USA ‡ Author for correspondence (e-mail: jlmicol@umh.es) Accepted 24 July 2006