Two Elongation Responses to Auxin Respond Differently to Protein Synthesis Inhibition

Abstract

The first and second responses to auxin react differently to the inhibition of protein synthesis by cycloheximide. It was determined that the protein with the shortest half-life, among the several necessary for the first response, is different from its counterpart among the several necessary for the second response. Specifically, the protein half-lives are 28 minutes and 11 minutes for the first and second responses, respectively. "Growth-limiting proteins" have been frequently discussed in connection with auxin activity (e.g., refs. 3 and 19 and refs. therein). There is no doubt that such growth-limiting proteins exist in all living systems. For example, in growing higher plant cells which respond to auxin there are protein components in the cell wall, and there are enzymes involved in wall assembly. It is possible that without these proteins the cells will not grow; therefore, they are potentially growth-limiting proteins. If consideration is limited to only those processes directly involved in wall formation (e.g. in a direct line between amino acids and sugars, and the cell wall) and loosening, there are likely dozens of potentially growth-limiting proteins. This, although somewhat oversimplified, is why experiments which use protein synthesis inhibitors have not been very enlightening in determining the mode of action of auxin; even though a protein is required for growth, it may have nothing to do with auxin action (10,12,22). This is not to say that these general inhibitors have been without use in the study of plant hormone action. For example, the RNA inhibitor 5-fluorouracil was essential to the determination by Key and co-workers (9, 11) that ribosomal RNA synthesis was not essential to auxin activity. Similarly, the use of actinomycin D and cycloheximide was vital to experiments which have shown abscisic acid to be active at the level of translation in cotton seed germination (6).Analogously, the study of growth-limiting proteins in auxin action can provide useful information. Even though there may be many essential (potentially limiting) proteins in auxin-induced growth, experiments which use a protein synthesis inhibitor, such as cycloheximide, will measure only the half-life of one protein-that protein, among the several essential to auxin-induced growth, which has the shortest half-life.These experiments were designed to compare the half-lives of those proteins for the two responses to auxin (23,25 for the first and second responses, respectively). This result indicates that separable responses to auxin are the result of different cellular activities. MATERIALS AND METHODSSoybean seedlings (Glycine max L. Merr. var. Wayne) were germinated in the dark, and the elongating segment of the hypocotyl was excised as described (24) except that all procedures were performed under green light (460-590 nm) at 30 C.Hypocotyl extension was continuously measured with a linear transducer. The apparatus was modified after that reported by Green and Cummins (7).To facilitate clamping of the segment in t...

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).…”

“…When IAA and cycloheximide are added simultaneously, IAA-induced growth lasts for approximately 60 min. Vanderhoef et al (1976) found that the half-life of the growth-limiting protein for the first IAA-induced growth response is longer than that of the protein limiting the second response. Similar findings have been made in monocots.…”

“…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.…”

Rapid Auxin-Induced Cell Expansion and Gene Expression: A Four-Decade-Old Question Revisited

“…By the 1960s, the central question in the auxin field was whether the rapid auxin-induced change in instantaneous growth strictly requires the immediate synthesis of growth-limiting proteins through auxininduced gene expression (Cleland, 1971) or rather is triggered by a direct stimulation of growth-limiting processes (Hager et al, 1971). It has been proposed that sustained growth involves both gene expression and stimulation of growth-limiting processes (Vanderhoef et al, 1976). This long-standing question remains unanswered.…”

Auxin-regulated Wall Loosening and Sustained Growth in Elongation

“…We suggested, and others have confirmed (8), that auxin-induced elongation could be separated into two phases, the early burst of growth (simulated by lowering the pH from 6 to 4) and a later phase associated with long-term, steady-state growth. Subsequent experiments showed that elongation during the first phase was biochemically distinct from elongation during the second phase (15,16,21,23 Figure 1. The figure is based on the data that prove the existence of separable responses, on the data which support the wall-acidification and gene expression hypotheses and on the consideration of what very likely happens to an excised rapidly elongating segment which is preincubated and then treated with acid or auxin.…”