The Effects of Exogenous Auxins on Endogenous Indole-3-Acetic Acid Metabolism (The Implications for Carrot Somatic Embryogenesis)

Abstract

(D.M.R., N.I., T.J.C.)l h e effect of auxin application on auxin metabolism was investigated in excised hypocotyl cultures of carrot (Daucus carota). Concentrations of both free and conjugated indole-3-acetic acid (IAA), [2H,]IAA, 2,4-dichlorophenoxyacetic acid, and naphthaleneacetic acid (NAA) were measured by mass spectroscopy using stable-isotope-labeled interna1 standards.[l3C1]NAA was synthesized for this purpose, thus extending the range of auxins that can be assayed by stable-isotope techniques. 2,4-Dichlorophenoxyacetic acid promoted callus proliferation of the excised hypocotyls, accumulated as the free form in large quantities, and had minor effects on endogenous IAA concentrations. NAA promoted callus proliferation and the resulting callus became organogenic, producing both roots and shoots. NAA was found mostly in the conjugated form and had minor effects on endogenous IAA concentrations. [ZH,lIAA had no visible effect on the growth pattern of cultured hypocotyls, possibly because it was rapidly metabolized to form inactive conjugates or possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism. l h e presente of exogenous auxins did not affect tryptophan labeling of either the endogenous tryptophan or IAA pools. This suggested that exogenous auxins did not alter the IAA biosynthetic pathway, but that synthetic auxins did appear to be necessary to induce callus proliferation, which was essential for excised hypocotyls to gain the competence to form somatic embryos.Since the 1940s (Thimann, 1974), exogenous auxins have been used frequently as experimental tools for studying their effects in developing structures (Schiavone and Cooke, 1987); for screening for mutants impaired in the uptake, metabolism, and physiology of endogenous auxins (Timpte et al., 1994); and even to find potential IAA receptors (Jones and Venis, 1989; LoSchiavo et al., 1991). One important use for the application of exogenous auxin is as a growth regulator for maintaining plant-cell-and tissue-

Low-Fluence Red Light Increases the Transport and Biosynthesis of Auxin

“…Because IAA has been found to promote the plasma membrane localization of PIN proteins and therefore increase IAA's own efflux from cells (Paciorek et al, 2005), the increased IAA biosynthesis at the top section may be the driving force for increased PAT triggered by light exposure. Although it was expected that the level of labeled IAA at the top section might be increased by NPA due to disrupted PAT, it was actually unchanged, suggesting a feedback regulation of IAA biosynthesis by IAA itself, which has also been reported in other systems (Ribnicky et al, 1996;Ljung et al, 2001a). The level of labeled IAA in the major portion (H5) of the hypocotyls was not changed by NPA during the 4-h feeding period, suggesting that the enrichment of labeled IAA in this tissue section was primarily due to local biosynthesis of IAA at a rate that was lower than that found in the upper part of the shoot.…”

Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation

“…The pooled eluate containing free purified IAA was methylated by incubation with 1 ml ethereal diazomethane for approximately 5 minutes, evaporated to dryness under N 2 , and resuspended in 25 l ethyl acetate. Quantification was by GC-MS-selected ion monitoring as described by Ribnicky et al (Ribnicky et al, 1996) …”

Developmental Regulation of Indole-3-Acetic Acid Turnover in Scots Pine Seedlings

“…We have recently presented evidence suggesting that these two pathways are operational in tobacco during vegetative growth (Sitbon et al, 2000). Utilization of dual pathways has also been described in carrot cell cultures where different IAA biosynthesis pathways operate in callus cultures compared with developing somatic embryos (Michalczuk et al, 1992;Ribnicky et al, 1996). In the study presented here we have analyzed possible developmental regulation of the two putative pathways.…”