Roles Of Tomato JERFs Genes In GCC-Box-Mediated Gene Expression

Transcriptional regulation of the expression of stress-responsive genes is a crucial part of the plant response to a range of abiotic and biotic stresses. Research carried out in the past few years has been productive in identifying transcription factors that are important for regulating plant responses to these stresses. ERF proteins are a transcription factor family that is unique to plants. ERF proteins share a conserved DNA binding domain (ERF domain). They are important for regulating plant responses to cold, drought, salt, pathogen infection and ethylene treatment. So the study of ERF transcription factors will reveal some molecular mechanism of plant response to stresses, and are likely to lead to new ways to enhance crop tolerance to disease and environmental stress.Our lab have isolated four genes which encode ERF domain from tomato cDNA expression library using yeast-one-hybrid system. They bind both ethylene responsive element and jasmonate responsive element, so named JERFs (Jasmonate and ethylene responsive element binding factors).To study the transcriptional activation activity of JERFs, I fused the coding regions for JERFs to the LexA DNA binding domain expression vector. The expression vectors were introduced into yeast strain EGY48, which transformed with the autonomously replicating reporter plasmid p8op-lacZ. By colony-lift filter assay of 0 -galactosidase, It was found that JERFs proteins fused to LexA DNA binding domain activated transcription of the lacZ reporter gene. This result indicated that JERFs proteins function as transcription activators in yeast.The expression pattern of the JERFs genes under various abiotic stress conditions was analyzed with RNA gel blot analysis. JERFs genes expression is induced after ethylene, methyl jasmonate, ABA, salt, or cold treatment, with overlapping but distinct induction kinetics. The expression of some tomato GCC-box-containing genes induced after ethylene treatment was also analyzed. For the control probes, the fragment of the tomato ChiB, GluB, NP24 and PRlbl was amplified by polymerase chain reaction (PCR). The first appearance of ChiB, GluB, and NP24 was after that of JERFs. This result suggests that JERFs play roles in the expression ofthese tomato GCC-box-containing genes. The expression pattern of the JERFs genes induced after SA treatment is similar to that induced after EtOH treatment, which is a control experiment. However, the transcription abundance of GluB and NP24 induced after SA treatment is lower than that induced after EtOH treatment. This suggest that SA might have a negative effect on the expression of GCC-box-containing genes, which activated by JERFs.The expression of both JERFs and theirs possible target genes was analyzed in 355V:JERFs tobacco plants and compared with that of control plants transformed with pROK2. For the control probes of tobacco GCC-box-containing genes, the fragment of the tobacco CHN50, GLA, Osmotin and prb-lb was amplified by PCR. Overexpression of JERF2 in transgenic tobacco activated the expression of several GCC-box-containing genes, such as GLA and osmotin, under normal unstressed conditions.These results suggest that JERFs are factors that respond to extracellular signals and modulate GCC-box-mediated gene expression positively.