ABSTRACT
As in higher plants, the development of the moss Physcomitrella patens is regulated by environmental signals and phytohormones. At the protonema level transition from chloronema to caulonema cells is under auxin control. The formation on second sub-apical caulonema cells of buds that will give rise to the leafy gametophore requires cytokinins. Using [3H]azidoCPPU (1-(2-azido-6-chloropyrid-4-yl)-3-(4-[3H])phenylurea), a photoactivatable cytokinin agonist, we have specifically photolabelled a soluble 34 kDa protein of P. patens. Urea derivatives were very efficient competitors of photolabelling while purine-type cytokinins were poor competitors. The protein UBP34 was purified by affinity chromatography and the sequences of six internal peptides obtained. A cDNA encoding UBP34 was cloned by screening a P. patens protonema cDNA library with a probe amplified by PCR using degenerate primers designed from the peptide sequences. The UBP34 amino acid sequence shows an average sequence identity of 42% with both intracellular PR proteins and the BetV1-related family of plant allergens. Recombinant UBP34 expressed in Escherichia coli was confirmed to bind azidoCPPU.
Subject(s)
Bryopsida/genetics , Phenylurea Compounds/chemistry , Plant Proteins/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cytokinins/agonists , Cytokinins/metabolism , DNA, Complementary/chemistry , DNA, Complementary/genetics , Molecular Sequence Data , Phenylurea Compounds/pharmacology , Photoaffinity Labels , Plant Proteins/chemistry , Plant Proteins/isolation & purification , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Analysis, Protein , Sequence Homology, Amino Acid , TritiumABSTRACT
In this issue we described a dwarf mutant in Arabidopsis thaliana, sax1, which is affected in brassinosteroid biosynthesis. This primary defect is responsible for alterations in hormone sensitivity of sax1 plants characterized by the hypersensitivity of root elongation to abscisic acid and auxin and the insensitivity of hypocotyl growth to gibberellins and ethylene (Ephritikhine et al., 1999; Plant J. 18, 303-314). In this paper, we report the further characterization of the sax1 mutant aimed at identification of the mutated step in the brassinosteroid biosynthesis pathway. Rescue experiments with various intermediates of the pathway showed that the sax1 mutation alters a very early step catalyzing the oxidation and isomerization of 3 beta-hydroxyl, delta 5,6 precursors to 3-oxo, delta 4,5 steroids. The mapping of the mutation, the physiological properties of the mutant and the rescue experiments indicate that sax1 defines a new locus in the brassinosteroid biosynthesis pathway. The SAX1 protein is involved in brassinosteroid-dependent growth of seedlings in both light and dark conditions.
