Growth-limiting Proteins in Relation to Auxin-induced Elongation in Lupin Hypocotyls

Abstract

The role of protein synthesis in auxin-induced cell elongation in lupin hypocotyl segments was studied using cycloheximide. Cycloheximide inhibited protein synthesis by 9 minutes. Experiments adding cycloheximide at various times before and after indolyl-3-acetic acid are reported. Estimates of the relative amounts of growth-limiting protein(s), and a first order rate constant for the apparent turnover of the growthlimiting protein(s) were made. It was shown that there is a sizeable growth promotion by auxin after protein synthesis has essentially ceased. It is concluded that the initial phases of auxin action do not require protein synthesis but that its action depends on the existing pool of growth-limiting proteins which is rapidly depleted, and protein synthesis is then required for continued elongation.It has been shown that inhibitors of protein synthesis have a rapid effect on IAA-induced growth (1,5,9,13,16,19). This has led to the suggestion that an unstable protein, which has been called growth-limiting protein (5), is necessary for auxin-induced cell expansion. It is not possible, however, to determine from these studies with inhibitors whether auxin acts on the synthesis of GLP' or whether the requirement for protein synthesis is a secondary (albeit rapid) effect of auxin action.Other evidence indicates that auxin acts on pre-existing macromolecules and that protein synthesis is not necessary for the initial action of auxin. This is the rapid change of growth rate by: (a) the methyl ester of indolyl-3-acetic acid (21, 22); (b) high temperature at high auxin concentration (14); and (c) the initial decline in growth rate before the auxin-induced stimulation (22). Although we have tried, without success, to obtain these results with lupin hypocotyl segments, they do illustrate the point for coleoptiles at least.In this paper the quantitative study of the effect of cycloheximide leads to a similar conclusion that protein synthesis is not required for the initial action of auxin on cell elongation in lupin hypocotyl. The measurements of elongation were made every minuLte in the apparatus described by Penny (17). Briefly, a segment was held in a chamber which was held on the stage of a microscope. The desired solution flowed through the chamber, and the flow rate of solution was maintained by a Watson Marlow model MHRE flow inducer. Tris-maleate buffer, pH 6.1, 20 mm, was used throughout; the concentration of IAA (when used) was 30 pM and the concentration of CH, when used, was 10 jug/ml. The solutions were aerated and the light energy at the surface of the segments was 3 wm-2 supplied by an incandescent lamp. Temperatures, except where otherwise stated, were maintained at 28 C. There was a tendency for the temperature to increase over the course of a long experiment. but the total difference in temperature during the experiment was never more than 0.5 C. MATERIALS AND METHODS SeedlingsFor measurement of protein synthesis, groups of 120 segments were pretreated in buffer for 2 hr and then blotted a...

Evidence for Two Indoleacetic Acid-Induced Growth Responses in the Avena Straight-Growth Indoleacetic Acid Assay

“…According to Cleland (1987), the initial growth response is caused by IAA-mediated proton secretion, and it is dependent on protein synthesis. However, this view contradicts the evidence that IAA can promote hypocotyl and coleoptile growth in the presence of cycloheximide (Penny, 1971;Pope and Black, 1972;Vanderhoef et al, 1976) and in the absence of proton secretion (Pope, 1983).…”

“…Kinetin inhibited the second response but not the first. Protein synthesis is not required for lupin (Penny, 1971) or soybean hypocotyl segments (Vanderhoef et al, 1976) to show a growth response to IAA. When IAA and cycloheximide are added simultaneously, IAA-induced growth lasts for approximately 60 min.…”

A gene encoding a protein modified by the phytohormone indoleacetic acid

“…IAA-modified proteins, however, appear to be present in vegetative as well as seed tissues, suggesting other possible roles. Prosthetic groups are known to alter protein stability (36), and a recurrent theme in auxin signal transduction has been the involvement of protein metabolism in the process (37)(38)(39). The linkage between auxin activity and protein metabolism seems to be reinforced by a diverse array of experimental approaches, and the presence of specific proteins to which IAA is attached may have significance beyond the important regulatory roles already assigned to conjugates for modulation of IAA levels.…”

Photocontrol of the Germination of Onoclea Spores

“…nous storage products, uptake of ions or inorganic molecules does not seem to be an important consideration in the germination process, unless ion exchange between compartments within the spore plays a role in germination. Furthermore, there is no direct evidence in the literature that inhibition of ion uptake in vivo is not due to inhibtion of protein synthesis rather than to interference with energy transfer (10). The claims that CH interferes with RNA and DNA synthesis and pyrimidine nucleotide metabolism have not been investigated in our system.…”

“…They were then treated with 5 ml of 0.1% (w/v) HgCl2 for 2 min and washed 4 times with sterile distilled H20. Aftre the final washing, 10 ml of sterile distilled H20 were added to the Petri dishes. To check for bacterial and fungal contamination, samples of spores were plated onto bacto-nutrient agar after surface sterilization.…”