Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 1.08

FUNCTIONAL ANALYSIS OF RICE GSTs

G. DE TOMA, M. SARI GORLA, C. FROVA

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano

Glutathione transferases (GSTs), transcriptional profiling, structure-function relationship

Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes ubiquitously distributed in all aerobic organisms. These proteins have evolved multiple functions in the metabolism of endogenous as well as foreign toxic compounds, and have important roles in cellular protection against a wide range of biotic and abiotic stresses. In plants they detoxify herbicides, organic pollutants and natural toxins and are involved in counteracting oxidative injury from various sources, including heavy metals. Most plant species harbour tens of GST genes: the estimates in Arabidopsis, maize and soybean are 48, 42 and 25 members respectively. In rice, we have isolated and cloned 58 putative GST genes, belonging to the four major plant classes: Phi (F), Tau (U), Zeta (Z) and Theta (T).

However, so far little is known about the specific function of individual members. With the aim to functionally characterise rice GSTs, their expression patterns were analysed by 1) systematic screening of rice EST collections and 2) DNA macroarray analysis. By comparing whole genome data with ESTs, about 55% of rice GST genes appear to be expressed. Inspection of EST libraries indicates that 67% of the individual cDNAs correspond to Tau class GSTs, 26% to Phi, 5% to Zeta and 1.4% to Theta. Individual rate of expression, estimated by the number of EST hits, shows that 19% are poorly expressed (1-3 hits), but 10% are very highly expressed (15 or more hits). Analysis in selected tissues indicates the lowest expression in leaves and the highest in callus.

As for macroarray analysis, we previously reported (SIGA 2002) that several rice GST genes are transcriptionally induced by either H₂O₂ or Cadmium or both. Interestingly, 2 Tau class genes (U9 and U3), which share a very high sequence identity, showed different transcriptional profiles in response to the two stresses. We thus further investigated these two genes also at the protein level: close sequence inspection shows that non-synonimous aminoacid substitutions are clustered in the co-substrate binding domain (an a helix made up by a ten aminoacid stretch), likely changing the two GSTs specificities. This hypothesis is supported by simulations performed through the SWISS-MODEL 3D program, which show that in GSTU3 this a helix structure is lost due to the specific aminoacid substitutions.

Macroarray transcriptional profiling of GST genes in response to two herbicides (the chloroacetanilide Pretilachlor and the thiocarbammate Molinate) and the safener Fenchlorim is currently under way.