incurvata13, a novel allele of AUXIN RESISTANT6, reveals a specific role for auxin and the SCF complex in Arabidopsis embryogenesis, vascular specification, and leaf flatness.

Auxin plays a pivotal role in plant development by modulating the activity of SCF ubiquitin ligase complexes. Here, we positionally cloned Arabidopsis (Arabidopsis thaliana) incurvata13 (icu13), a mutation that causes leaf hyponasty and reduces leaf venation pattern complexity and auxin responsiveness. We found that icu13 is a novel recessive allele of AUXIN RESISTANT6 (AXR6), which encodes CULLIN1, an invariable component of the SCF complex. Consistent with a role for auxin in vascular specification, the vascular defects in the icu13 mutant were accompanied by reduced expression of auxin transport and auxin perception markers in provascular cells. This observation is consistent with the expression pattern of AXR6, which we found to be restricted to vascular precursors and hydathodes in wild-type leaf primordia. AXR1, RELATED TO UBIQUITIN1-CONJUGATING ENZYME1, CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME5A, and CULLIN-ASSOCIATED NEDD8-DISSOCIATED1 participate in the covalent modification of CULLIN1 by RELATED TO UBIQUITIN. Hypomorphic alleles of these genes also display simple venation patterns, and their double mutant combinations with icu13 exhibited a synergistic, rootless phenotype reminiscent of that caused by loss of function of MONOPTEROS (MP), which forms an auxin-signaling module with BODENLOS (BDL). The phenotypes of double mutant combinations of icu13 with either a gain-of-function allele of BDL or a loss-of-function allele of MP were synergistic. In addition, a BDL:green fluorescent protein fusion protein accumulated in icu13, and BDL loss of function or MP overexpression suppressed the phenotype of icu13. Our results demonstrate that the MP-BDL module is required not only for root specification in embryogenesis and vascular postembryonic development but also for leaf flatness.

The core of the SCF complex includes three major components: RBX1  (Berná et al., 1999;Serrano-Cartagena et al., 1999;Serrano-Cartagena et al., 2000). Ten of the initial set of ICU genes have been cloned, allowing their functional classification in three different pathways, related to chromatin-mediated cellular memory (Barrero et al., 2007), microRNA biogenesis and action (Jover-Gil et al., 2005;Jover-Gil et al., 2012) and auxin signaling (Pérez-Pérez et al., 2010). Two of the genes in the last group are SHY2 (SHORT HYPOCOTYL 2, also called IAA3) and AXR3 (AUXIN RESISTANT3, also called IAA17) (Pérez-Pérez et al., 2010). For example, icu6, a semi-dominant allele of AXR3, causes reduced size of leaf adaxial pavement cells and abnormal expansion of palisade mesophyll cells. Hence, the differential growth of the adaxial and abaxial leaf tissues of icu6 is thought to be a consequence of the limited space available for the internal mesophyll, which is defined by the epidermal layers (Pérez-Pérez et al., 2010). We also studied mutants exhibiting aberrant leaf venation patterns (Candela et al., 1999;Alonso-Peral et al., 2006;Candela et al., 2007;Robles et al., 2010), three of which carried loss-of-function alleles of HEMIVENATA (HVE), the gene encoding CAND1 (Alonso-Peral et al., 2006;Candela et al., 2007), which is known to physically interact with AXR6.
Here, we report the characterization of icu13, a novel recessive allele of AXR6 that causes leaf hyponasty as a consequence of a strong reduction of AXR6 protein levels. Our genetic and molecular results support the hypothesis that AXR6 acts through the MP-BDL module, which is crucial for embryo basal patterning and vascular specification in the early stages of leaf development.
Hence, altering the amount of active CUL1, via missregulation of rubylation and derubylation, phenocopies the strong defects of mp and bdl mutants. As the leaf incurvature of icu13 can be rescued by either overexpression of MP or loss-offunction of BDL, we suggest that the MP-BDL module is also responsible for leaf flatness.

Positional cloning of icu13 and detection of its mRNA and protein products
For positional cloning of the recessive icu13 mutation, a mapping population of 481 F2 phenotypically mutant plants derived from an icu13 × Ler cross was used for linkage analysis as previously described (Ponce et al., 1999;Ponce et al., 2006). The oligonucleotides used for SSLP marker scoring are listed in Supplemental Table S1. The icu13 mutation mapped to a 139 kb interval on top of chromosome 4, encompassing 38 annotated genes, one of which was AXR6 (AUXIN RESISTANT6; Fig. 1A, B). We sequenced the AXR6 transcription unit in the icu13 mutant and its corresponding wild-type En-2, finding in exon 15 a C→T transition that creates a new splicing donor site, as confirmed by comparison of the sequences of PCR amplification products obtained from En-2 and icu13 cDNAs (Supplemental Fig. S1). Two different AXR6 splice variants are present in icu13 seedlings: one variant differs from the wild-type En-2 mRNA only in a silent point mutation (GGC→GGT, both codons encoding glycine), but the other variant lacks the last five nucleotides of the 15 th exon as a consequence of the new splicing donor site (GC→GT) introduced by the mutation. Translation of the latter mRNA is predicted to produce a truncated protein product; whereas the AXR6 wild-type protein includes 738 amino acids, the mutant protein is predicted to contain only 492, 19 of which, at its Cterminus, are different from those of the wild type (Supplemental Fig. S2).
To quantify the relative levels of the two splice variants produced by the icu13 allele, we performed qRT-PCR amplifications using two primer pairs: one icu13, we tried to detect its predicted truncated protein product (Supplemental Fig. S2) by Western blotting using the available α -CUL1 antibody (Gray et al., 1999). Although we could not detect any band with the size predicted for the truncated protein (57 kDa), we confirmed that both wild-type inactive (CUL1) and wild-type rubylated (CUL1-RUB) protein species were strongly decreased in icu13 seedling extracts, compared to wild type (Fig. 1E).
We found that the complexity of the vascular network was reduced in icu13 and eta1 first-and third-node leaves compared with their wild types.
Third-, fourth-and higher-order veins were missing from icu13 and eta1 venation patterns in these leaves (Fig. 2D). The venation density and number of 1 0 responses by examining the expression of the DR5rev:GFP reporter (Friml et al., 2003) in primary root meristems. DR5rev:GFP expression in icu13 and eta1 roots, in contrast to expression in wild type, was restricted to the distal region of the meristem (quiescent center and columella) and excluded from the vasculature (Supplemental Fig. S4). These results are consistent with a reduction in auxin responses in the icu13 and eta1 mutants, a reduction that can be explained by impaired SCF TIR1/AFB1-3 function.

Phenotypic characterization of heterozygotes for AXR6 alleles
Twelve mutations of AXR6 have already been described, including loss-and gain-of-function alleles (Fig. 1B). To study their phenotypes in combination with icu13, we selected four mutants, a semi-dominant, gain-of-function allele, axr6-2 (Hobbie et al., 2000), and three recessive alleles, two of which are null (cul1-1 and cul1-2; Shen et al., 2002) and one that is likely hypomorphic (eta1;Quint et al., 2005). We studied the embryo and seedling phenotypes of plants heterozygous for all possible combinations of these five alleles of AXR6 (Fig. 3).

Distribution of the AXR6 protein
To study AXR6 protein distribution and subcellular localization, we used an 1 3 AXR6:GFP fusion expressed under the control of the AXR6 promoter. We obtained two T2 families in the Col-0 background and four in the En-2 background, all of them expressing the AXR6 pro :AXR6:GFP construct.
AXR6:GFP was detected early in embryogenesis, from the dermatogen stage onwards ( Fig. 5G-K). The pattern of early embryonic AXR6:GFP expression was ubiquitous, similar to that seen for AXR6 pro :GUS. However, increased AXR6:GFP signal was detected in provascular tissues from the heart stage onwards (

Genetic interactions of icu13 and eta1 with loss-of-function alleles of genes required for SCF Function
To investigate whether precise regulation of SCF function is essential for leaf development and vein patterning, we examined the interaction of icu13 with mutations affecting genes that regulate the covalent modification of CULLIN1 by The axr1-12 and hve-2 mutants display reduced venation pattern 1 4 complexity and leaf size, compared to their wild type counterparts (Dharmasiri et al., 2003;Alonso-Peral et al., 2006;Robles et al., 2010). We found a T-DNA insertion in the first intron of RCE1 that reduced its expression to 6% of the wildtype level (Supplemental Fig. S6), and we named this allele rce1-10. Leaves of homozygous rce1-10 plants were slightly hyponastic (Fig. 6A) and normally sized, but exhibited reduced venation density and number of branching points veins per mm 2 ( Fig. 6B; Table I). Leaves of the csn5a-2 mutant were of reduced size but with a venation density and number of branching points per mm 2 similar to those of Col-0. The number of free-ending veins per venation length, however, was significantly decreased in csn5a-2 mutant leaves compared with those of Col-0 ( Fig. 6B; Table I).
However, no clear pattern emerged from our phenotypic classification that would allow us to make conclusions on differences in the strength of the genetic interactions found. We further dissected siliques from F2 plants that were homozygous for icu13 and heterozygous for either axr1-12, rce1-10, csn5a-2 or phenotypes that we found were in some cases more severe than those usually observed in mp, bdl, axr6-1 and axr6-2 homozygotes ( Fig. 6I; 6L). In summary, we found genetic interactions between loss-of-function alleles of AXR6 and four genes required to regulate SCF activity by the rubylation-derubylation pathway, 1 5 suggesting that both activation and inhibition of CUL1 by RUB attachment and release, respectively, are essential for proper SCF function.

Aux/IAA genes
The main role of the SCF TIR1/AFB1-3 complex is the ubiquitin-mediated destabilization of Aux/IAA repressors, which are negative regulators of auxin signaling (Gray et al., 2001;Dharmasiri et al., 2005a;Dharmasiri et al., 2005b).  Table S6). All plants of the eight double mutant genotypes analyzed were dwarfed and exhibited compact rosettes, with leaves of reduced size and increased hyponasty compared to their single mutant siblings (Fig. 7A), phenotypes that we interpreted as additive. Leaves of the shy2-10 and axr2-1 mutants were reduced in size, but their vascular phenotypes were similar to wild type, the only exception being an increased number of free-ending veins per mm of venation (Table I) Table I). All the double mutant combinations including a loss-of-function allele of AXR6 and a gain-of-function allele of SHY2, AXR2 or AXR3 were viable and fertile, in contrast to the interactions that we observed between loss-of-function alleles of AXR6 and 1 6 either directly (strong loss-and gain-of-function alleles of AXR6; Fig. 3) or indirectly (mutations affecting the regulators of SCF activity; Fig. 6). In our standard growth conditions, bdl/BDL heterozygotes (Fig. 8B) displayed a leaf incurvature similar to that seen in icu13 and eta1 homozygotes. In light of our finding that icu13 and eta1 do not interact with gain-of-function alleles of some Aux/IAA genes (Fig. 7), the dose-dependent phenotype of the bdl allele suggested to us that the leaf and vascular phenotypes of icu13 and eta1 primarily arise from the stabilization of BDL, whose levels are known to be precisely titrated during embryogenesis for proper specification of embryonic basal structures (Hamann et al., 2002). In several F2 families derived from selfed AXR6/icu13;bdl/BDL and AXR6/eta1;bdl/BDL plants, we found more rootless, early-lethal seedlings than expected, a significant proportion of which were heterozygous for the bdl mutation and homozygous for either icu13 (21%; BDL is known to interact with and inhibit the MP transcription factor, which is required for basal patterning of embryos and vascular development (Hamann et al., 2002;Hardtke et al., 2004;Weijers et al., 2006). To confirm that the genetic interaction found between AXR6 and BDL is mediated by MP, we We reasoned that the icu13 phenotype might be caused by the inactivation of MP caused by the stabilization of its Aux/IAA repressor, BDL, as a consequence of partial inactivation of the SCF TIR1/AFB1-3 complex (Gray et al., 2001;Dharmasiri et al., 2005a;Dharmasiri et al., 2005b). To test our hypothesis, we obtained 10 icu13 and 9 En-2 transgenic lines expressing the BDL pro :BDL:GFP construct, which were used to study BDL stabilization. We found faint expression of the BDL:GFP protein in the central region of primary roots in young En-2 seedlings (Fig. 8R), which is indicative of rapid protein turnover via 26S proteasome degradation (Gray et al., 2001). Consistent with a reduced function of SCF TIR1/AFB1-3 in the icu13 mutants (see above), we found accumulation of BDL:GFP in the vascular domain of the root meristem in icu13 plants (Fig. 8T). To confirm that BDL:GFP accumulation in icu13 is earlier and independent of proteasome function, we incubated young seedlings in the presence of MG132, a proteasome-inhibitor (Jensen et al., 1995). We found that a short MG132 treatment increased the accumulation of BDL:GFP in wildtype roots ( Fig. 8S; V), whereas no significant differences were found between 1 8 degradation.
Taken together, the genetic interactions found between AXR6, BDL and MP suggest that the regulatory pathway involving these three genes is mainly responsible for the specification of embryonic basal structures and for the specification of postembryonic vascular tissues, and that tight regulation of BDL activity through auxin-mediated SCF regulation is required. T2 families, we observed segregation for a mild leaf hyponasty phenotype, clearly distinguishable from that of the icu13 homozygotes (Fig. 9A). In all cases (n=13), plants with this phenotype were icu13/icu13 and carried the 35S pro :MP construct, likely as homozygotes. We confirmed further this partial rescue in T3 35S pro :MP families which exhibitied a mild leaf incurvature and found that all were transgenic for the 35S pro :MP construct and homozygous for the icu13 mutation (Fig. 9B).

Loss of function in
As BDL accumulated to higher levels in icu13 (Fig. 8R-V), we hypothesized that a stronger phenotypic suppression might be achieved with a lack-of-function allele of BDL. We crossed icu13 and eta1 to the iaa12-1 mutant (Overvoorde et al., 2005; this work), which bears a T-DNA insertion in the second exon of BDL abolishing its expression (Supplemental Fig. S8). In the F2 progeny, the icu13/icu13;iaa12-1/iaa12-1 double mutants exhibited only weak leaf hyponasty (Fig. 9C)     arrest at the globular stage with additional divisions of suspensor cells as also seen in axr6-2/cul1-1 and axr6-2/cul1-2 heterozygotes, to cell overproliferation of the embryo and the suspensor, producing root-like structures. The latter phenotype was also found in double homozygotes of icu13 and axr1-12, rce1-10, csn5a-2 or hve-2. Such strong defects were never seen in bdl and mp       (7) (7) (2) (4)