Plant & Animal Genome VII Conference

W64

USE OF RTPCR AND FLUORESCENCE TECHNOLOGY TO QUANTIFY AUXIN-INDUCED GENE EXPRESSION DURING GRAVICURVATURE OF TOMATO HYPOCOTYLS.

Oregon State University, Department of Botany, 2082 Cordley Hall, Corvallis, OR 97331 USA

The focus of our research is the signal transduction pathway that mediates plant responses to gravity. To elucidate the underlying mechanisms we are using a tomato mutant, lazy-2, which has shoots that show a phytochrome-dependent reversal of the direction of gravitropic curvature.
A central goal in our work is to test the Cholodny-Went theory which states that the hormone auxin is redistributed unequally towards the lower side of the stem of a gravistimulated plant. In order to further understand the role that auxin transport plays in gravitropism, we have developed a technique that utilizes molecular tools to dissect the pathway of auxin movement. We have used auxin-responsive genes of the,LeIAA gene family to monitor auxin activity during gravicurvature. 10-day-old, light-grown seedlings were gravistimulated, and the hypocotyls were bisected at specific time points. Total RNA was extracted and reverse transcribed to make cDNA and RT-PCR was performed to detect auxin-induced differences in gene expression between the upper and lower halves.
To analyze expression data from PCR reactions, we have used an improved version of a technique known as multiplex titration RT-PCRs (MTRP) developed in our lab (Nebenfuhr and Lomax, 1998). Briefly, a dilution series of the template for the PCR reaction is used to determine the range in which the PCR progress is at a linear rate. As an internal control we included primers which amplify a constitutively expressed gene in addition to gene-specific primers for the auxin-upregulated LeIAA 11 gene. A fluoroimager coupled with FMBIO densitometry software was used to measure the intensity of the staining of the PCR product and obtain quantitative data.
We have observed increased expression of LeIAA 11 on the lower side of the upward-bending wild-type stems. However, it appears that an exact reversal of this pattern in downward bending lazy-2 stems does not take place. This leads us to believe that the gravitropic response may occur in two independent phases as previously suggested by MacDonald and Hart (1987). The first phase is auxin-dependent, whereas the second phase may be auxin-independent. (Supported by NASA award No. NAGS-6573 to T.L.L. and a Doktorandenstipendium of the Studienstiftung des deutschen Volkes to A.M.).