Map-based cloning and functional characterization of Arabidopsis LHR1 and SHI7 genes

In an effort to identify novel genetic components that involve in the gene regulation and signaling pathways of plant abiotic stress response and tolerance, two forward genetic mutant screening systems were established by utilizing the stress-inducible promoters of AtHSP18.2 and AtSOT12 genes and the LUC reporter gene. A number of mutants displaying altered luciferase activity in response to abiotic stresses were isolated. In this study, two mutants named lhr1 and shi7 were subjected to map-based cloning and further molecular and physiological characterizations. The lhr1 mutant contains a single recessive mutation which leads to reduced expression of the LUC reporter gene under both normal and heat stress conditions comparing with wild type. Map-based cloning of the lhr1 mutation identified a single nucleotide missense change in a gene encoding a polyamine uptake transporter. LHR1 protein contains 12 transmembrane domains and belongs to an amino acid permease family. The lhr1 mutant shows tolerance to paraquat and cycloheximide, hypersensitivity to ABA and NaCl in seed germination, and altered expression of several abotic stress-responsive genes. Further study showed LHR1 is engaged in the transport of extracellular paraquat and polyamines into cytoplasm. The shi7 mutant has a single recessive mutation causing higher LUC expression after both NaCl and sorbitol treatments when compared with wild type. A single nucleotide missense mutation in a gene encoding an ARF-GAP DOMAIN protein was identified in the shi7 mutant by Map-based cloning. The SHI7 protein contains 828 amino acid residues and consists of four conserved domains including an ARF-GAP domain. The shi7 mutant displays root hair biogenesis deficiency in newly formed maturation zone of root, but shows no apparent morphological phenotypes under tested stress conditions. Both genetic and molecular complementation confirmed that the mutant phenotype of shi7 is caused by the mutation in the SHI7 gene. Both LHR1 and SHI7 genes play important roles in abiotic stress response in Arabidopsis. The molecular mechanisms by which these two genes modulate stress-responsive gene expression were further studied, and the direction of future research were also discussed.