Intercellular transport of the phytohormone auxin is a significant factor for plant organogenesis. To investigate molecular mechanisms by which auxin controls organogenesis, we analyzed the macchi-bou 4 (mab4) mutant identified as an enhancer of pinoid (pid). Although mab4 and pid single mutants displayed relatively mild cotyledon phenotypes, pid mab4 double mutants completely lacked cotyledons. We found that MAB4 was identical to ENHANCER OF PINOID (ENP), which has been suggested to control PIN1 polarity in cotyledon primordia. MAB4/ENP encodes a novel protein, which belongs to the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) family thought to function as a signal transducer in phototropism and control lateral translocation of auxin. MAB4/ENP mRNA was detected in the protodermal cell layer of the embryo and the meristem L1 layer at the site of organ initiation. In the mab4 embryo, the abundance of PIN1:GFP was severely decreased at the plasma membrane in the protodermal cell layer. In addition, subcellular localization analyses indicated that MAB4/ENP resides on a subpopulation of endosomes as well as on unidentified intracellular compartments. These results indicate that MAB4/ENP is involved in polar auxin transport in organogenesis.

SUMMARYPIN-FORMED (PIN)-dependent auxin transport is essential for plant development and its modulation in response to the environment or endogenous signals. A NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)-like protein, MACCHI-BOU 4 (MAB4), has been shown to control PIN1 localization during organ formation, but its contribution is limited. The Arabidopsis genome contains four genes, MAB4/ENP/NPY1-LIKE1 (MEL1), MEL2, MEL3 and MEL4, highly homologous to MAB4. Genetic analysis disclosed functional redundancy between MAB4 and MEL genes in regulation of not only organ formation but also of root gravitropism, revealing that NPH3 family proteins have a wider range of functions than previously suspected. Multiple mutants showed severe reduction in PIN abundance and PIN polar localization, leading to defective expression of an auxin responsive marker DR5rev::GFP. Pharmacological analyses and fluorescence recovery after photo-bleaching experiments showed that mel mutations increase PIN2 internalization from the plasma membrane, but affect neither intracellular PIN2 trafficking nor PIN2 lateral diffusion at the plasma membrane. Notably, all MAB4 subfamily proteins show polar localization at the cell periphery in plants. The MAB4 polarity was almost identical to PIN polarity. Our results suggest that the MAB4 subfamily proteins specifically retain PIN proteins in a polarized manner at the plasma membrane, thus controlling directional auxin transport and plant development.

In Arabidopsis, three major regions, which ultimately develop into the two cotyledons, the cotyledon boundaries and the shoot apical meristem (SAM), are formed at the apex of the globular stage embryo. To reveal the molecular mechanism underlying this pattern formation, we isolated a cotyledon-defective mutant from EMS mutagenized lines. This mutant completely lacks cotyledons in the most severe cases, and is allelic to gurke (gk), which was previously reported as a mutant defective in apical patterning of the embryo. To evaluate the morphological effects of the mutation in the GK gene, we investigated the expression patterns in gk embryos of SHOOT MERISTEMLESS (STM), AINTEGUMENTA (ANT) and CUP-SHAPED COTYLEDON1 (CUC1), which are markers of the SAM, cotyledons and cotyledon boundaries, respectively. Expression of all these genes largely overlapped in gk, suggesting a failure to partition the apex of the embryo into the three subregions. Enlargement of the CUC1 expression domain was also observed and may explain the inhibition of cotyledon development in gk. Moreover, we cloned the GK gene, and confirmed that it encodes ACC1, an acetyl-CoA carboxylase which catalyzes malonyl-CoA synthesis. Our results suggest that metabolites derived from malonyl-CoA are required for partitioning of the apical part of the embryo.