Isolation and characterization of EARLI1, a vernalization-responsive gene in Arabidopsis thaliana

In order to gain a clearer understanding of the molecular processes that are induced by vernalization, a subtractive hybridization technique was employed to isolate vernalization-responsive genes from very-late flowering Col- FRI -SF2 Arabidopsis thaliana seedlings. An up-regulated cDNA fragment was isolated that corresponds to the EARLI1 gene which has previously been shown to be responsive to aluminum induction. We have found that EARLI1 is expressed in all vegetative tissues of 7-day-old vernalized seedlings grown under short days and that long day photoperiod has an additive effect on vernalization-induced EARLI1 expression. EARLI1 is also up regulated in early-flowering Nossen and Columbia ecotypes in response to vernalization. However, in the very early-flowering Landsberg ecotype, EARLI1 is expressed equally in unvernalized and vernalized plants. There are no significant ecotype-specific polymorphisms within a 1300-by region upstream from the EARLI1 start codon in Columbia, Nossen and Landsberg plants, which show greater than 95% nucleotide identity. EARLI1 is transiently induced by cold shock but not by exogenous abscisic acid, a stress phytohormone. In contrast to other cold shock genes EARLI1 expression is maintained at least 20 days after vernalization treatment and RNA levels progressively increase with increasing periods of vernalization. Sequence analysis of EARLI1 upstream DNA predicts putative abscisic acid, G-box, light regulated and an ethylene response element. In addition, a canonical low-temperature response element lays 2000bp upstream of and oriented away from EARLI1 . Transgenic plants containing an EARLI1 -genomic -500 bp::GUS reporter gene fusion did not exhibit detectable GUS activity. However, a -1500 by fusion exhibited spatial expression in the root elongation zone, root/hypocotyl transition zone, cotyledons, premature leaves, trichomes, dihescence zone, sepals, stamens and carpel tips in unvernalized Col-FRI -Sf2 plants. One of these transgenic plants also exhibited differential GUS expression in response to cold shock and touch but not to vernalization. A central proline-rich and predicted C-terminal transmembrane domain suggests EARLI1 may function in cell wall modification