Forward genetic screen for auxin-deficient mutants by cytokinin.

Lei Wu,Shao-Dong Wei,Ming Wang,Miroslav Strnad,Pan Luo,Ondřej Novák,Li Wang,Li Zhang,Petra Amakorová,Dong-Wei Di,Cheng-Kai Lu,Guang-Qin Guo,Tian-Zi Zhang

doi:10.1038/srep11923

Abstract

Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

Highlights

Auxin biosynthesis is less well understood than auxin transport and signaling, but includes several tryptophan (Trp)-dependent and -independent pathways[11]
Some genes involved in auxin biosynthesis have been identified by isolating mutants with high auxin contents[16,17,18,19] or characterizing mutants isolated in studies that initially focused on indirectly associated phenomena[13,14,20,21,22]
The results clearly suggest that the screening system is effective for isolating auxin-deficient mutants, and discovering genes that participate in auxin biosynthesis or dynamics

Summary

Introduction

Auxin biosynthesis is less well understood than auxin transport and signaling, but includes several tryptophan (Trp)-dependent and -independent pathways[11]. We hypothesized that a potentially useful marker may be root curling induced by cytokinin (CK), as recently described in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1), arising from auxin deficiencies in the root tip[20]. To test the possibility that this trait could be used to isolate more auxin-deficient mutants, we screened large collections of Arabidopsis T-DNA insertion lines, obtained from the Arabidopsis Biological Resource Center (ABRC), to isolate ckrc-like mutants. The advantage of Agrobacterium T-DNA over classical mutagens is that the plant sequences flanking the insertion site can be isolated . This simplifies the identification of genes corresponding to interesting mutants. The results clearly suggest that the screening system is effective for isolating auxin-deficient mutants, and discovering genes that participate in auxin biosynthesis or dynamics

Methods

Results

Conclusion