Arabidopsis nuclear copper binding protein, LCAS is required for normal vascular formation and anther dehiscence

Abstract

A male sterile mutant, lcas(Leaf Curling to Abaxial Side) with a defect in anther dehiscence was identified in Arabidopsis thaliana population, mutagenized with T-DNA insertion. Anther dehiscence in mutant`s anther is not taken placed after flowering in compare with wild type Col-0 plant, but mechanical force can release the mature pollen which can fertilize mutant`s itself and fertilized eggs show normal growth but slightly retarded maturation in compared with that of wild type. This mutation is tagged by T-DNA harboring BAR gene which confers resistance to BASTA(main gradient is phoshinothricin), a herbicide, and the segregation ratio of mutant phenotype to wild type is 1(mutant 189): 3(wild type 608), and all mutants show Basta resistance, so this mutation is recessive . The causative gene is cloned by TAIL-PCR method and this gene`s expression is disappeared in mutant background. Full genomic fragment, encompassing LCAS gene`s promoter, coding region and terminator could complemented the mutant phenotype, but mutated form of genomic fragment failed in rescue. LCAS is predicted to encodes an nuclear metal binding protein. In transient particle bombarding assay, GUS::LCAS protein is localized in nucleus of onion epidermis cell, and in divalent metal ion charged affinity column test, GST::LCAS proteins have copper ion affinity but mutated form of GST::mLCAS lost it`s affinity to copper ion. therefore LCAS is a nuclear copper binding protein which is required for normal anther dehiscence. It`s expression was detected mainly in Pistil, vascular bundle and inner connective tissue of anther, phloem of leaf and root and stem in LCAS::GUS transgenic plant. LCAS`s expression is repressed by CuSO4, and in mutant`s background, Myb26is up-regulated in compare with that of wild type. This mutant shows greatly retarded growth, increased branching, leaf curling, early termination of flower bud formation and triple cauline leaves. In mutant and over expressor`s background,...