A new reporter construct to monitor IAA dynamics during tomato development
Mazzucato, A., Olimpieri, I., Rossi, M., Caccia, R., Soressi, G.P.
Studying the function of plant growth regulators (PGRs) has historically been hindered by the difficulty of detecting their very low concentrations at the tissue level. Recently, new solutions were offered by the introduction of improved instruments of analysis, of immunological methods, and of tissue-specific PGR genetic engineering. In parallel, plant transformation has offered new tools to detect hormone distribution in plant organs and tissues by using constructs where reporter genes are driven by PGR-inducible promoters.
The plant growth hormone auxin (typified by indoleacetic acid, IAA) affects several primary cellular processes and is responsible of lateral organ patterning, differentiation and elongation. In addition, IAA mediates apical dominance, stimulates the differentiation of vascular tissue, induces root initiation and lateral root development, mediates the tropistic responses, and finally exerts various effects on leaf and fruit abscission and fruit set, development and ripening. Therefore, the characterization of mutants, whose phenotype is putatively determined by altered IAA levels or perception may shed light on the role played by this PGR in different aspects of plant developmental biology. In this perspective, the tomato is a favoured experimental species, because a plethora of more or less characterized monogenic mutants have been described to date, that cover all aspects of plant growth and development (http://tgrc.ucdavis.edu/; http://zamir.sgn.cornell.edu/mutants/).
A number of constructs suitable to report IAA kinetics in plant tissues have been proposed using natural (GH3 gene promoter from soybean, Hagen et al., 1991; PS-IAA4/5 gene promoter from pea, Ballas et al., 1995) as well as synthetic (DR5 promoter, Ulmasov et al. 1997) auxin-response elements fused to reporter proteins (e.g. GUS and GFP). All these constructs have been useful to detect IAA dynamics in higher and even lower plants, although their efficiency and sensitivity was variable depending on the species and the tissue assayed (Oono et al., 1998; Brierfreund et al., 2003; Aloni et al., 2006).
In this study, we used the IAA-inducible promoter of the Agrobacterium tumefaciens gene 5 (Koncz and Shell, 1986; Körber et al., 1991) to construct a tomato transgenic line expressing the b-glucuronidase (GUS) reporter in an IAA-dependent manner. A 1060 bp-long 5’ fragment upstream to the gene 5 start transcription codon (p5) was amplified from the pGV0153 plasmid (Koncz and Shell, 1986) and ligated to the GUS coding sequence. The p5::GUS fusion product was cloned in E. coli, transferred to A. tumefaciens and used to transform tomato (cv. Chico III) cotyledons as described (Caccia et al., 1999). PCR-positive primary transformants were assayed for GUS staining in root tips and a plant with good expression was selected for further experiments. Inspection of its progeny revealed that genetic transformation targeted a single insertion site and allowed to select a plant homozygous for the reporter gene (assessed by further progeny testing).
To demonstrate the IAA-sensitivity of the construct, we treated two-weeks old seedlings by deepening them overnight in solutions containing different IAA concentrations and carried out the GUS assay the day after. As shown in Fig. 1B-E, root apexes showed GUS staining around the tip (quiescent centre and columella root cap initials) with an intensity that increased with IAA concentration in the treatment up to a maximum reached at 10-5 M (Fig. 1D). This behaviour corresponded to the range of functionality obtained with other IAA-responsive reporter constructs (Oono et al., 1998; Aloni et al., 2006).
Another site of IAA accumulation in the root was the region of initiation (Fig. 1F) and emergence (Fig. 1G) of lateral roots. In leaflets (Fig. 1H), as in sepals (Fig. 1I) and petals (Fig. 1J), IAA was detected in the vascular tissue, although with different, distinctive patterns. In the mature anther, the reporter gene was expressed in the region corresponding to the pollen sacs (Fig. 1K), whereas in the ovary only the basal vascular bundles possessed clear IAA accumulation (Fig. 1L). Accordingly to its connective function towards all the floral whorls, a strong signal was observed throughout the receptacle vascular tissues (Fig. 1M).
The p5::GUS Chico III line has been crossed with several tomato mutants with primary defects in cotyledon (pct), stem (ls, mon, oli, pro, sd), leaf (cb, Cu, marm, Me, tf), flower (an, ex, ps-2, sl-2, uf), and fruit (f, pat, pat-2) development. The F2 populations, segregating the mutations together with the p5::GUS construct, will be used to study IAA dynamic in the mentioned phenotypes, thus allowing a deeper understanding of its role in plant growth and development.
Acknowledgements
We acknowledge C. Koncz and F. Salamini for the gift of the pGV0153 plasmid, and P. Mosconi and R. Magro for technical assistance. The authors also thank the Tomato Genetic Resource Center, Davis (CA) for providing some of the mutant seed stocks.
References
Aloni, R., Aloni, E., Langhans, M. and Ullrich, C.I. 2006. Role of auxin in regulating Arabidopsis flower development. Planta 223:315-328.
Ballas, N., Wong, L.M., Ke, M. and Theologis, A. 1995. Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5. Proc. Natl. Acad. Sci. U.S.A. 92:3483–3487.
Bierfreund, N.M., Reski, R. and Decker, E.L. 2003. Use of an inducible reporter gene system for the analysis of auxin distribution in the moss Physcomitrella patens. Plant Cell Rep 21:1143–1152.
Caccia, R., Delledonne, M., Balestra, G.M., Varvaro, L. and Soressi, G.P. 1999. Plant-bacterial pathogen interaction modified in transgenic tomato plants expressing the Gox gene encoding glucose oxidase. In: Scarascia Mugnozza G.T., Porceddu E. and Pagnotta M. (eds)."Genetics and Breeding for Crop Quality and Resistance", Kluwer, The Netherlands, pp. 119-125.
Hagen, G., Martin, G., Li, Y. and Guilfoyle, T.J. 1991. Auxin-induced expression of the soybean GH3 promoter in transgenic tobacco plants. Plant Mol. Biol. 17:567-79.
Koncz, C. and Shell, J. 1986. The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204:383-396.
Körber, H., Strizhov, N., Staiger, D., Feldwisch, J., Olsson, O., Sandberg, G., Palme, K., Schell, J. and Koncz, C. 1991. T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants. EMBO J. 10:3983–3991.
Oono, Y., Chen, Q.G., Overvoorde, P.J., Köhler, C. and Theologis, A. 1998. age1 mutants of Arabidopsis exhibited altered auxin-regulated gene expression. Plant Cell 10:1649–1662.
Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T.J. 1997. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9:1963–1971.
Figure 1. Histochemical analysis of GUS activity in the root apex of a control, untransformed plant (A) and in several organs of Chico III plants transgenic for the p5::GUS reporter construct (B-M). Bright field microscope images of root apexes from Chico III p5::GUS plants treated with 0 (B), 10-7 (C), 10-5 (D) and 10-3 (E) M IAA. Stereomicroscope images of the region of initiation (F) and emergence (G) of lateral roots, of a young leaflet (H), and of a sepal (I), a petal (J), a stamen (K), a ovary (L), and a receptacle (M) dissected from a flower at anthesis.