Isolation And Characterization Of Low-sulfur Tolerant Mutants Of Arabidopsis Thaliana

Sulfur is an essential element for plant growth and development as well as for defense against biotic and abiotic stresses. But little is known about the genetic determinants for sulfate utilization efficiency, because of the technical difficulties imposed by low sulfur demand of plants.Here we report the isolation and characterization of two low-sulfur tolerant mutants, sue3 and sue4 using a high-throughput genetic screen from an activation-tagging library of approximately 55,000 individual lines where a "sulfur-free" solid medium was devised to give the selection pressure necessary to suppress the growth of the wild type seedlings. Both mutants showed improved tolerance to low sulfur conditions and markedly increased root systems. Potentially these mutants have enhanced sulfate utilization efficiency. The mutant phenotype of both sue3 and sue4 was specific to sulfate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulfur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis.Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulfur tolerance and potentially with enhanced sulfur utilization efficiency. The two loci identified in sue3 and sue4 should assist understanding the pertinent molecular mechanisms involved in low sulfur tolerance. To investigate further the function of SUE4, the expression patteren and protein localization was analyzed via promoter-GUS reporter and GFP tagging, respectively. It was found that SUE4 showed a similar expression pattern to PIN1. SUE4 protein localization also overlapped with PIN1. This led us to test whether SUE4 and PIN1 interact. Further analysis with bacterial two hybrid and BiFC demonstrated that SUE4 interacted with the auxin efflux carrier PIN1 in vivo. This interaction likely altered IAA distribution in roots and resulted in altered root architecture of the mutant.