A number of cDNAs corresponding to auxin-regulated mRNAs have been isolated from tobacco and found to be encoded by a multigene family consisting of three subfamilies. Homologous proteins have been isolated independently from soybean and potato. Here we report that the encoded proteins show a limited but significant homology to both plant and animal glutathione S-transferases (GST, EC 2.5.1.18). For the protein NT103, encoded by a member of the Nt103 subfamily, we demonstrate an in vitro GST activity. This is the first time a function is attributed to a member of this group of auxin-induced proteins or any of its homologues. The implications of this finding and the possible relationships between auxins and GSTs are discussed.

Using a 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell-suspension culture we analyzed early hormone-induced molecular events preceding cell division. By differential screening of a cDNA library to mRNAs derived from hormone-starved cells treated with 2,4-D for 4 h, seven non-cross hybridizing cDNA clones to 2,4-D-induced mRNAs were obtained. Accumulation of these mRNAs started as early as 15-30 min or less after 2,4-D application. The lowest 2,4-D concentration necessary to induce the mRNAs varied between less than 2.2 × 10(-8) M and 2.2 × 10(-6) M, one mRNA being induced to nearly maximal values at 2.2 × 10(-6) M. Generally, 2,4-D was the most active compound to induce mRNA accumulation, followed by naphthalene-1-acetic acid (NAA). The level of 4 mRNAs increased independently from protein synthesis. Run-off transcription studies showed that the accumulation of some mRNAs was at least partly due to enhanced transcription rates. In different organs of the tobacco plant, the levels of the mRNAs were about as low as in hormone-starved cells. A similar low level of the 2,4-D-induced mRNAs was observed in cells growing in mid-log phase on 2,4-D-containing medium. Only quiescent cells that were triggered to undergo cell division, accumulate these mRNAs transiently.

The effect of exogenous phytohormones on proliferation of the root cortex, and their relation to the division factors from Rhizobium which participate in the initiation of root nodules, were studied using explants of root-cortex tissue from 7-day-old, sterile pea plants. The explants were cultured for 7 days on a synthetic nutrient medium supplemented with auxin, or auxin and cytokinin. With only auxin present in the medium, ca. 10% of the explants showed cell proliferation. With both auxin and cytokinin this percentage was much higher (ca. 80%). The active explants showed proliferation patterns which were similar to or could be derived from a pattern with three predominant meristematic areas in the inner cortex opposite the three xylem radii of the excised central cylinder. These proliferation patterns were similar to the initial proliferative stages in root-nodule formation in seedling intact roots. From this restricted division response of the explants to the hormones, a hypothesis of endogenous division factors is proposed. To test this hypothesis, extractions of root tissue were performed. The addition of a crude alcoholic extract from the central cylinder or the cortex to the medium resulted in cell divisions throughout the cortex. The results are interpreted as evidence for the presence of a transverse gradient system of (an) unknown cell-division factor(s) in the root cortex which may control the induction of cell divisions in nodule initiation brought about by the release of auxin and cytokinin from Rhizobium.

In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5' regions were translationally fused with the beta-D-glucuronidase reporter gene (GUS). The GNT1 5' region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5' regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.