A novel conserved motif is present in the HD-Zip I transcription factor MTHB1 from Medicago truncatula

ARIEL, FD; GRUBER, V; DIET, A; CRESPI, M; CHAN, RL

Lisboa

Conferencia; 6th European Conference on Grain Legumes; 2007

Grain Legumes Integrating Project

The HD-Zip family of transcription factors is unique to plants. These proteins exhibit a singular combination of a homeodomain with a leucine zipper. They can be classified into four subfamilies, according to distinctive features, including DNA binding specificity, gene structure, additional motifs and physiological functions. Members from subfamily I exhibit a conserved homeodomain-leucine zipper, but no other common motif, and are generally involved in responses to abiotic stresses (1). We have isolated by RT-PCR the cDNA of MTHB1 from Medicago truncatula, whose expression in roots increases in response to high concentrations of NaCl. The protein was expressed as a GST-fusion and purified by affinity chromatography. Electrophoretic mobility shift assays revealed that MTHB1 recognises in vitro the sequence CAAT(N)ATTG with the highest affinity, as other characterised members of this subfamily. Transgenic Arabidopsis plants over-expressing MTHB1 were obtained showing a distinguishable phenotype compared with non transformed plants. Interestingly, sequence alignment among a subset of HD-Zip I proteins from different plant species showed that MTHB1 exhibits a non-described motif conserved in the C-terminus. Arabidopsis HD-Zip I proteins were classified into several groups according to their gene structure (2). However, none of them exhibit this domain. Comparison among proteins from different species, including legumes, may serve to reveal additional conserved domains in this TF family. Regarding the Arabidopsis HD-Zip family, MTHB1 bears closest resemblance to ATHB7 and ATHB12, in spite of their different C-terminus motif. Recombinant ATHB7 was similarly purified and shown to bind to the same target sequence of MTHB1 while recombinant ATHB12 was unable to recognise this DNA probe. An analysis in silico was carried out to identify potential post-translational modification motifs that would be responsible for these alternative regulation mechanisms. On the other hand, transgenic Arabidopsis plants constitutively expressing MTHB1 exhibit a different phenotype from the one observed for ATHB7 and ATHB12 (3). Unfortunately, none of the C-terminus motif-containing proteins have been over-expressed so as to compare their phenotypes. A functional analysis of this new domain and the identification of interacting proteins may reveal why proteins with the same DNA binding specificity are able to generate alternative phenotypes in plants. Parallel analysis in M. truncatula is now being undertaken to understand the signal transduction pathway in which MTHB1 is involved in response to abiotic stresses. (1) Ariel F., Manavella P., Dezar C. and Chan R. (2007) Trends in Plant Science (next September issue, to be updated). (2) Henriksson E., Olsson A., Johannesson H., Johansson H., Hanson J., Engstrom P., Soderman E. (2005) Plant Physiology. 139(1), 509-518. (3) Olsson A., Engström P., Söderman E. (2004) Plant Molecular Biology. 55(5), 663-677.