ABSTRACT
* Auxin is essential for many aspects of plant growth and development, including the determination of lateral organ shapes. * Here, the characterization of a dominant Arabidopsis thaliana mutant spl-D (SPOROCYTELESS dominant), and the roles of SPL in auxin homeostasis and plant development, are reported. * The spl-D mutant displayed a severe up-curling leaf phenotype caused by increased expression of SPOROCYTELESS/NOZZLE (SPL/NZZ), a putative transcription factor gene that was previously linked to sporocyte formation. The spl-D plants also displayed pleiotropic developmental defects including fewer lateral roots, simpler venation patterns, and reduced shoot apical dominance. The leaf and floral phenotypes of spl-D and SPL over-expression lines were reminiscent of yucca (yuc) triple and quadruple mutants, suggesting that SPL may regulate auxin homeostasis. Consistent with this hypothesis, it was found that over-expression of SPL led to down-regulation of the auxin reporter DR5-GUS, and that many auxin-responsive genes were down-regulated in spl-D leaves. Interestingly, the expression of YUC2 and YUC6, two key genes in auxin biosynthesis, was significantly repressed in spl-D plants. * Taken together with the genetic and phenotypic analysis of spl-D/yuc6-D double mutant, these data suggest that SPL may regulate auxin homeostasis by repressing the transcription of YUC2 and YUC6 and participate in lateral organ morphogenesis.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/physiology , Arabidopsis/genetics , Gene Expression Regulation, Plant , Nuclear Proteins/physiology , Oxygenases/genetics , Repressor Proteins/physiology , Arabidopsis/anatomy & histology , Arabidopsis/growth & development , Arabidopsis Proteins/biosynthesis , Cloning, Molecular , Homeostasis , Indoleacetic Acids/metabolism , Mutagenesis, Insertional , Nuclear Proteins/genetics , Oxygenases/biosynthesis , Phenotype , Plant Leaves/anatomy & histology , Plant Leaves/genetics , Plant Leaves/growth & development , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/metabolism , Repressor Proteins/geneticsABSTRACT
A pair of degenerate primers were designed based on NBS (nucleotide binding site, NBS) domain of resistance(R) gene and used to perform PCR with cDNA from the translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa. A clone (N7) characterized with NBS was obtained by sequencing analysis. Two specific primers were designed from the N7 sequence and used to screen a genomic TAC (transformation-competent artificial chromosome, TAC) library of 6VS/6AL consisting of ca. 2 x 10(6) clones. The library was stored as clone pools in twenty-two 96-well plates, each well containing approximately 1000 TAC clones. TAC plasmids were prepared from all the 2112 pools. Using a pooled PCR screening procedure, a positive TAC clone having a 40 kb insert was obtained. The positive clone was confirmed by Southern hybridization with the NBS fragment as a probe. The results indicate that the pooled PCR method is effective for screening of genomic libraries having large number of clones.
