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 - 1.20

A SMALL HEAT SHOCK GENE IS POSITIVELY REGULATED BY ECTOPIC EXPRESSION OF A MAIZE GENE CODING FOR A bHLH TRANSCRIPTION FACTOR

F. DAMIANI, N. TOSTI, M. RAGANO-CARACCIOLO, F. PAOLOCCI

C.N.R. Istituto Genetica Vegetale sez. di Perugia

condensed tannins, cDNA-AFLP, real-time PCR, gene expression, Lotus corniculatus

The production of bloat-safe alfalfa is recognised as one of the main goal of breeding of such species. In fact the most of proteic fraction of such crop is wasted for degradative processes occurring either in the rumen or during silage.

The presence of moderate amount of condensed tannins is claimed as the solution of such problem. However, notwithstanding the remarkable progresses of last years (Devic et al., 1999; Xié et al., 2003; Tanner et al., 2003) the process leading to the synthesis of such compounds is not completely described and some genes necessary for their synthesis need to be identified, yet.

At this purpose several approaches have been attempted. In our laboratory we produced, through genetic transformation with Sn, a maize gene coding for a bHLH transcription factor, some high contrasting phenotypes (Paolocci et al., 1999; Robbins et al., 2003) of Lotus corniculatus, where tannins level in leaves was highly enhanced (E) or strongly depleted (D). With such materials a strategy for isolating the genes involved in tannin synthesis consisted on the comparison of cDNA–AFLP pattern of two enhanced plants (E), two suppressed plants (D) and a control plant (C).

216 primer combinations out of the 256 possible (two selective bases for each primer) were carried out.

A total of 201 putative differentially expressed fragments were recovered from the gel and 77 of them were sequenced. Only 43 (55.8%) sequenced bands matched with known genes. In this paper we report the results of the study on a gene coding for a small Heat Shock Protein (HSP), that gene resulted highly expressed in plants rich of tannin while almost absent in the control plant and in the suppressed plants.

The differential expression of such gene was confirmed through northern reverse and RT-PCR analysis. Full messenger and promoter region of the gene were recovered trough Genome Walking.

Real Time PCR was performed on the selected gene utilising ABI PRISM 5700 SDS on the different cDNA of the same plants and compared with that of dfr and Sn. The first gene is strongly up-regulated in the E plants and Sn is expressed in the E plants only. A silencing mechanism has been proposed to explain the production of D plants (Damiani et al., 1999).

HSP resulted up-regulated (8.9 and 17.2 fold, for the two enhanced tannin plants) with respect to the control plant (C) The D plants had a different trend between them: one showed a decrease level of the HSP gene level (0.59), while the other maintain substantially the level of the C plant (1.3). The level of the up regulation was greater then those of dfr in both the E plants. As expected Sn expression was detected only in E plants.

From this study comes out that Sn that transactivates dfr, can also affect, directly or indirectly, HSP; however, while dfr over-expression is consistent with the increase of tannin level, it seems hard to find a direct correlation between HSP expression and tannin increase. Experiments of transformation with chimeric HSP constructs are going to be performed to clarify this point. However, as general comment, we can hypothetise that the bHLH gene enhances generic mechanisms of resistance to stresses among which tannins and heat shock proteins represent different outcomes.