Quantification of Indole-3-Acetic Acid in Dark-Grown Seedlings of the Diageotropica and Epinastic Mutants of Tomato (Lycopersicon esculentum Mill.)

Abstract

ABSTRACIEndogenous indoleacetic acid (IAA) levels were examined in 7-dayold, dark-grown tomato seedlings (Lycopersicon esculentum Mill. cv VFN8), and in two single-gene mutants, Epinastic and diageotropica. Gas chromatography-mass spectrometry was employed to quantify IAA using '3C6-Ibenzene ringlindoleacetic acid as internal standard. IAA concentrations ranged from 89 to 134 nanograms per gram dry weight and were not significantly different for the three genotypes. Ethylene overproduction by dark-grown Epi seedlings is not likely to result from increased IAA. Assuming similar recovery percentages for each genotype, indole-3-ethanol, a purported storage form of IAA, was identified by GC-MS and found to be more prevalent in the parent tomato, VFN8, with only trace amounts observed in Epi. No IEt was detected by high performance liquid chromatography/fluorescence in dgt (detection limit >100 picograms).The Epi2 tomato mutant is characterized by extreme leaf epinasty, thickened stems and petioles, a shortened and highly branched root system, and a very compact and erect growth habit (7,22 In contrast, the dgt mutant, which is derived from the same parent line as Epi, has reduced auxin sensitivity with respect to hypocotyl elongation (9) and ethylene production (3,8, 30). This suggests that like Epi, dgt also possesses altered target cell responses.We believe these mutants represent a useful genetic system for investigating the mechanisms of auxin perception and action. However, in order to fully interpret the responses to exogenous auxin application, it is imperative to determine the endogenous IAA levels ofthe three genotypes. Therefore, we have investigated the levels of LAA in shoot apices of 7-d-old, dark-grown seedlings of VFN8, and its two mutants, dgt and Epi. Free IAA levels were found to be similar among the three genotypes suggesting that the epinastic phenotype was not the result of IAA induced ethylene production. A second fluorescent compound which eluted just after IAA during HPLC purification did appear to differ in relative amounts between the three genotypes. This compound was identified by GC-MS as indole-3-ethanol.MATERIALS AND METHODS Plant Material. Tomato (Lycopersicon esculentum Mill.) seeds of cultivar VFN8 and the two mutants Epi and dgt were surfacesterilized for 45 min in 40% commercial bleach and rinsed with 3 L of distilled water. Seeds were sown onto moist filter paper in 11 x 11 x 3 cm plastic germination boxes, covered, and placed in the dark at 24C. On d 7, SAs were harvested under a greensafe light and immediately frozen in liquid nitrogen. The tissue was then transferred to a 300 mL freeze-drying flask which was placed in the dark into a modified, refrigerated, recirculating bath (Lauda K-4/R) at -9°C. The flask was then connected to a 780 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

The cyclophilin DIAGEOTROPICA has a conserved role in auxin signaling

“…Ethylene production is also reduced in auxin-treated dgt tomato plants (Bradford and Yang, 1980;Kelly and Bradford, 1986). However, the rate of polar indole-3-acetic acid (IAA) transport in dgt hypocotyls and roots is similar to that of the WT parent (Daniel et al, 1989;Muday et al, 1995) and levels of IAA in dgt shoot apices were shown to be normal (Fujino et al, 1988). These results generally support the hypothesis that tomato dgt is altered in auxin perception or response.…”

“…Phenotypic studies in tomato together with direct measurements of auxin concentration and transport suggested a role for DGT in auxin signaling rather than in auxin biosynthesis or transport (Fujino et al, 1988;Daniel et al, 1989;Muday et al, 1995). The tomato dgt mutants were established as a useful tool to study auxin signaling in tomato and in angiosperms in general.…”

NaCI Reduces Indole-3-Acetic Acid Levels in the Roots of Tomato Plants Independent of Stress-Induced Abscisic Acid

“…Most of the studies in tomato have focused on two mutants, dgt and epinastic (Ursin and Bradford, 1989;Muday et al, 1995). However, the phenotypic characteristics of each mutant are not a consequence of differences in the endogenous levels of IAA (Fujino et al, 1988). Tal and Imber (1970) proposed that another ABAdeficient mutant of RR, flacca, was higher in auxins as measured by the wheat-coleoptile bioassay.…”

“…The dgt mutant exhibits horizontal growth habit, thin stems, hyponastic leaves, lack of lateral roots, reduced ethylene production, and insensitivity to auxin (Ursin and Bradford, 1989). The inability of auxin to induce growth in the dgt mutant apparently results from a defect in auxin-induced cell-wall loosening (Daniel et al, 1989), since the endogenous levels of IAA are the same in the mutant and wild-type parent (Fujino et al, 1988).…”

Auxin Physiology of the Tomato Mutant diageotropica

“…Although endogenous levels of IAA are the same in the shoot tips of dgt and VFN8 (8), dgt mutants are nearly insensitive to exogenously applied auxin with respect to hypocotyl elongation and ethylene production (2,13). The Additional evidence supporting the notion that dgt and its isogenic parent, VFN8, provide a unique system to study auxin action stems from recent work (1 1) utilizing the photoaffinity auxin analog, 3H-5N3-IAA (azido-IAA).…”