Functional genomics strategies for stress resistance in arabidopsis
2003
Maza, M.
To identify genes for stress tolerance in plants using Arabidopsis as a model, forward and reverse genetics strategies were used primarily with gain-of-function experiments. Screening systems were devised to identify mutants and the genes involved in stress resistance. In a Forward genetics strategy, activation tagging was used to identify gain-of-function mutants that can circumvent the problems of genome redundancy in Arabidopsis and display dominant mutants. A population of transposon based activation tagged lines was used to develop a set of media based screens to positively select for mutants that are resistant to different stress parameters. Mutants were screened for resistance to Methotrexate, sucrose, salt and paraquat at concentrations that normally inhibit seedling germination and growth. Towards a Reverse genetics strategy to identify tagged genes, a preselection of Activation tagged mutant lines was made for lines that exhibit poor growth or fitness, that were used as a basis to identify genes that might be overexpressing essential pathways like stress resistance. A database of transposon flanking sites was generated that allowed the identification and selection of candidate mutants/genes for stress tolerance, that were further analyzed by phenotypic screens. Constructs overexpressing a candidate Arabidopsis gene under control of the CaMV35S promoter and tomato EREBP gene under control of the stress inducible rd29A promoter were transformed into Arabidopsis. The transformants were characterized and the phenotypes were assessed by stress resistance assays. The genes identified would be useful to engineer stress tolerance in crop plants.
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