Map-based Cloning And Functional Analysis Of The Auxin-related Gene BIG In Arabidopsis

We screened abaxial epidermis of rosette leaves from EMS-mutagenized seeds of Arabidopsis by light microscopy, a stomata development mutant, named j588, was isolated. The j588 mutant exhibited one-fold to two-fold increases in stomata density, smaller pavement cell areas and shorter petiole length. In addition, j588 mutant was dwarf, with small leavers, and defective in lateral root formation, fewer secondary veins, and more secondary inflorescences. The application of NAA can rescue the mutant phenotype of lateral formation to wide-type and reduced stomata density. j588 becomes extremely sensitive to NPA.By map-based cloning technique, we mapped the BIG gene which is required for normal auxin efflux. BIG gene encodes a member of the Calossin/Pushover family of proteins,560 kD. It contains two cysteine-rich putative zinc-finger domains (CRD-1, CRD-2) and a Zinc-binding domain (ZZ). BIG are highly conserved in Oryza, Vitis, Medicago, Mus and humans. By GUS staining and Real-Time RT-PCR show that BIG is widely expressed in root meristemoid, lateral roots, leaf primordial, juvenile leaf blades and vascular tissues. GUS expression also was detected in the cotyledon epidermal cell, stomata and young trichomes. With the help of DR5-GUS and CYCB1-GUS reporter lines, it demonstrated that the auxin expression level was reduced and auxin distribution was changed in j588.Auxin plays an essential role in the regulation of lateral root formation, apical dominance, venation patterns, cell expansion, leaf size and other developmental phenotypes. Double mutants of big-j588;pin1 and big-j588;axr1 display severe defects in auxin-dependent growth of lateral root formation, apical dominance, venation patterns, cell expansion. We proposed that BIG involved in AXR1-mediated auxin response, PIN1-mediated polar auxin transport and TIR1-mediated ubiquitin-proteasome pathway.