Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress
Salsomaggiore Terme, Italy - 26/29 September, 2001
A CHIMAERIC REPORTER GENE TO MONITOR AUXIN DISTRIBUTION IN PLANT TISSUES
ROSSI M.*, MAZZUCATO A.*, CACCIA R.*, SALAMINI F.**, SORESSI G.P.*
* Dipartimento di Agrobiologia e Agrochimica, Sezione di Genetica, Università degli Studi della Tuscia, Via S.C. de Lellis snc, 01100 Viterbo, Italy
** Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné Weg 10, 50829 Köln, Germany
auxin, GUS, mutants, reporter genes, tomato
The plant growth regulator auxin mediates an enormous range of developmental and growth responses. Despite the considerable body of research devoted to study auxin biosynthesis, translocation and transduction, details on the mechanisms underlying the function of the “growth hormone” remains unclear. Among the reasons that have slowed progress in the understanding of auxin-related regulatory controls, is the difficulty of correlating particular phenotypes (developmental phases or mutant phenotypes) with changes in hormone levels in plant tissues. Moreover, mass spectroscopy, the most rigorous procedure now available to quantify hormone levels in plants, does not unravel the eventual hormone compartmentation in different tissues and/or cell types of the same organ. In this research, we describe the construction of a chimaeric gene where the auxin-inducible promoter of A. tumefaciens gene 5 is cloned upstream of the GUS reporter gene. We demonstrate that this construct is amenable for monitoring auxin amounts and distribution at the plant cell and tissue level.
The promoter sequence of the T-DNA gene 5 has been amplified by PCR from the pGV153 plasmid and TA cloned in pGEM-T. A site mutagenised fragment has been inserted into the HindIII-BamHI cloning sites of the pBI101 plasmid, upstream of the GUS reporter gene (pro5::GUS). Competent A. tumefaciens EHA105 cells have been transformed with the chimaeric construct. For plant transformation, we have used plants belonging to the tomato cv. Chico III. Primary transformants were selfed and T2 progenies were microcutted and rooted in kanamicin to select plants segregating a single copy of the transgene. T2 kanamicin-resistant plantlets were treated by root submersion with 0, 10-7 and 10-5 M IAA for 6 hours. The GUS assay was carried out immediately after the treatment on root apexes, hypocotyls and cotyledon petioles. After 12 hours, strong GUS staining was observed in the elongation zone of the root tip, at the sites of lateral roots primordia and in the vascular tissue of hypocotyls and cotyledon petioles. Although a darker GUS signal was present at increasing auxin treatment concentrations, staining was also reported from non-treated roots, indicating that endogenous levels of the hormone are sufficient to drive the gene 5 promoter at this stage. No staining was observed in organs of untransformed control plants.
Because we reported GUS staining at sites where auxin accumulation is likely to occur in roots and young vegetative organs accordingly to the literature and the staining intensity was sensitive to exogenous IAA, we conclude that the pro5::GUS construct is able to detect auxin in plant tissues. Such biotechnological tool may prove to be highly valuable to monitor auxin distribution in situ in the perspective of shedding light on the role of auxin in different plant phenotypes. A crossing scheme is on going in order to transfer the pro5::GUS construct to several tomato mutant genotypes putatively involved in auxin metabolism and/or translocation.