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Francis H. Witham, Charles W. Heuser, and Jun Chen

Ethidium bromide (EB), at 10-5 to 10-4 M, progressively inhibits NAA-induced rooting of mung bean cuttings. Cycloheximide (CH), 6-methylpurine (6-MP) and kinetin (KIN) also inhibited rooting at the same concentrations, although CH and 6-MP were more effective.

At 70 and up to 130 hours of incubation, after cuttings received a 1-ml pulse of NAA (10-4 M), they exhibited a progressive increase in the number of observed adventitious roots. The addition of one of the inhibitors, 6-MP, EB or KIN to cuttings, pulsed 48 hours earlier with NAA, showed an initial slight inhibition with increased inhibition over time. CH, however, inhibited rooting immediately after addition. From these and other similar kinetic studies, it appears that 6-MP, EB and KIN operate at the transcriptional level and that CH inhibits translation.

Lineweaver-Burk plot analysis of NAA-induced rooting inhibition showed that EB may act as a competitive inhibitor of NAA. Since EB is a known intercalating agent and competitively inhibits NAA-induced rooting, NAA may influence gene expression by ultimately binding to DNA. Studies with space-filling and computer-generated models show that both NAA and EB can bind to certain dinucleotides by an intercalation mechanism.

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Jun Chen, Dengru Wu, Francis H. Witham, Charles W. Heuser, and Richard N. Arteca

Adventitious root formation (rooting) in `Berken' mungbean [Vigna radiata (L.) Rwiclz.] cuttings is stimulated by indole-3-acetic acid (IAA). To understand the molecular events that occur during IAA-induced adventitious root initiation, a λgt11 cDNA library was made from mungbean hypocotyls treated with 500 μm IAA for 3 hours and differentially screened. Two cDNAs MII-3 and MII-4 were isolated. Southern analysis revealed that both cDNAs are encoded by different genes. Expression studies showed different patterns for both genes. Both MII-3 and MII-4 were highly expressed in IAA treated hypocotyls, whereas MII-4 was also induced in IAA treated epicotyls. There was no expression of either MII-3 or MII-4 in control or IAA treated leaves. With increasing concentrations of IAA from 100 to 1000 μm there was an increase in the average root number per cutting as well as a stimulation in MII-3 and MII-4. Both MII-3 and MII-4 showed a stimulation in expression 4 hours following treatment with 500 μm IAA reaching a maximum from 4 to 8 hours followed by a decline thereafter. Basal expression of MII-3 was evident between 2 and 8 hours, whereas, a high degree of basal expression was found with MII-4 from 1 to 8 hours followed by a sharp decline. Cycloheximide (50 μm) dramatically reduced rooting and MII-3 expression, whereas MII-4 was only slightly affected.