Undergraduate Research Intern University of Florida Gainesville, Florida
Body of Abstract: Glucosinolates are defense compounds found in Brassicales. For glucosinolate biosynthesis, C-S lyase activity is necessary. SUPERROOT1 (SUR1/HLS3/ALF1/RTY1, At2g20610) is the only characterized glucosinolate C-S lyase encoding gene in Arabidopsis. Disruption of SUR1 leads to severe dwarfism, resulting from the redirection of indole-3-acetaldoxime (IAOx), a precursor of indole glucosinolates, to indole-3-acetic acid (IAA) the potent auxin. As sur1 mutants do not develop any reproductive organs, it has been challenging to evaluate function in seeds - the glucosinolate-enriched organ. To evaluate SUR1 function in glucosinolate biosynthesis in seeds, we generated sur1 mutants in Arabidopsis IAOx-deficient mutant background. In Arabidopsis, CYP79B2 and CYP79B3 convert tryptophan to IAOx. As the double mutant of CYP79B2 and CYP79B3, cyp79b2cyp79b3 (b2b3), is IAOx-free, we expected that b2b3sur1 triple mutants would be fertile and suitable to study C-S lyases functioning in glucosinolate biosynthesis.
Using the CRISPR system, we established four types of sur1 mutations in b2b3 background, which we named b2b3sur1 #2, #9, #15, and #24. Insertion or deletion mutations in #2, #9, and #15 lines result in early stop codons leading to premature termination of SUR1, whereas the 9 base-pair deletion mutation in #24 leads to a three amino acid deletion of SUR1. b2b3sur1 triple mutants (#2, #9, #15) exhibited abnormal growth and developmental alterations while b2b3 and #24 are indistinguishable from wild type, suggesting the altered growth and development in the triple mutants result from the defect of SUR1 rather than off-target effects of CRISPR. As the triple mutants are fertile, we analyzed metabolite profiles in various organs of these mutants, including seeds. Our metabolite analysis indicated that other C-S lyases play roles in glucosinolate biosynthesis in Arabidopsis seeds. We also found a crosstalk between glucosinolate biosynthesis and the phenylpropanoid pathway. RNAseq analysis unveiled a complex metabolite network linking specialized metabolism and growth and development.