Genetic identification of salt tolerance determinants in Arabidopsis thaliana

Yuzuki Manabe, Purdue University

Abstract

Salinity is a major abiotic stress which reduces crop productivity. Determination of the molecular components involved in salinity stress signaling and understanding the mechanisms of adaptation to salinity is essential to genetic improvement of crop plant salt tolerance. Forward genetics, using the model plant Arabidopsis thaliana, has been and will continue to be a powerful method used to identify the determinants of salt tolerance. A T-DNA insertional population of A. thaliana was generated and screened by a root-bending assay for seedlings exhibiting altered sensitivity to NaCl. Ninety-five putative salt-sensitive mutants were identified through the screening of 94,400 transformants. Those putative mutants were categorized based on their phenotype according to the presence and absence of NaCl. Mutants of Category I exhibit almost normal length of root growth without NaCl and respond to NaCl. Category II mutants exhibit root growth defects under normal conditions but still respond to NaCl. Category III mutants are morphological mutants. Further characterization was performed mainly on Category I mutants. This further characterization included identification of the T-DNA insertion, measurement of mRNA accumulation to confirm the interference of transcription by the T-DNA, diagnostic PCR to test the linkage between genotype and phenotype, in silico search for reverse genetic mutants to further confirm that the altered expression of the gene is responsible for causing the phenotype observed and physiological analysis to dissect the salt-sensitive phenotype. The T-DNA insertions were located at, or close to, an array of genes including Kinase-associated Phosphatase 2/2 (KAPP), Heat shock Protein 70, and Salt Overly Sensitive1. A Category I mutant, root attenuated growth1-1 (rag1-1), is a loss of function mutant of KAPP. rag1-1 seedlings exhibit a salt-sensitive phenotype manifested by reduction in primary root growth, swelling in the root elongation zone, and lateral root branching. RAG1/KAPP is a negative regulator of receptor-like kinase (RLK) signaling pathways including those involved in brassinosteroid sensing (BRI1), flower development (CLAVATA), innate immunity against bacterial pathogens (FLS2), somatic embryogenesis (AtSERK1), systemic signaling in herbivore defense (SR160), floral abscission (HAESA), and self-incompatibility (SRK). However, its role in salt adaptation has not been implicated prior to this research.

Degree

Ph.D.

Advisors

Bressan, Purdue University.

Subject Area

Molecular biology|Genetics|Plant sciences

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