Properties of auxin binding sites in different subcellular fractions from maize coleoptiles

In-vitro binding of labeled auxins to sedimentable particles was tested in subcellular fractions from homogenates of maize (Zea mays L.) coleoptiles. The material was fractionated by differential centrifugation or on sucrose density gradients. It was confirmed that the major saturable binding activity (site I) for 1-naphthyl[1-¹⁴C]acetic acid is associated with vesicles derived from the endoplasmatic reticulum. A second type of specific auxin binding (site II) could be distinguished by several criteria, e.g. by the low affinity towards phenylacetic acid. The particles carrying site II could be clearly separated from markers of the endoplasmatic reticulum, the plasmalemma, the mitochondria and the nuclei, while their density as well as sedimentation velocity correlated with particle-bound acid phosphatase, indicating a localization at the tonoplast. In contrast to site I, binding at site II was hardly affected by a supernatant factor and by sulfhydryl groups. However, the specificity pattern of site II towards auxins and auxin analogs was very similar to that of site I tested in the presence of supernatant factor. The existence of a third auxin receptor localized in plasma membrane-rich gradient fractions was indicated by a preferential in-vitro binding of 2,4-dichlorophenoxyacetic acid.Abbreviations 1-NAA 1-naphthyl acetic acid - 2-NAA 2-naphthyl acetic acid - IAA 3-indolyl acetic acid - PAA phenyl acetic acid - 2,4-D 2,4-D-dichlorophenoxy acetic acid - D-2,4-DP dichlorophenoxy isopropionic acid - NPA 1-N-naphthyl phthalamic acid - ER endoplasmatic reticulum - SF supernatant factor     

Levels of indole-3-acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay

A specific solid-phase enzyme immunoassay for the detection of as little as 3–4 pg of indole-3-acetic acid (IAA) is described. The assay involves minimal procedural efforts and requires only standard laboratory equipment. Up to 50 samples in triplicate, processed simultaneously, can be assayed and evaluated in 2.5 h. As little as 1 mg oat coleoptile tissue is sufficient for a quantitative IAA analysis and little or no extract purification is necessary. Using this assay, levels of IAA have been determined in coleoptiles of maize and oat. The distribution of IAA within single coleoptiles was quantitated and the production of IAA during the regeneration of the physiological tip in Avena coleoptiles was investigated. The changes in levels of IAA and other major phytohormones were quantitated during the growth of oat coleoptiles.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - BSA bovine serum albumin - IAA indole-3-acetic acid - TBS Trishydroxymethylaminomethane buffered saline Part 21 in the series Use of Immunoassay in Plant Science     

Changes in cell wall polysaccharides in developing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) coleoptiles

Cell wall polysaccharides in developing barley coleoptiles were examined using acetic acid–nitric acid extraction, alditol acetate and methylation analyses and enzymatic digestion. The coleoptile cell wall from imbibed grain was rich in pectic polysaccharides (30 mol%), arabinoxylan (25 mol%), cellulose (25 mol%) and xyloglucan (6 mol%), but contained only low levels of (13,14)--D-glucan (1 mol%). During 5 days of coleoptile growth, pectic polysaccharides decreased steadily to about 9 mol%, while (13,14)--D-glucan increased to 10 mol%. Following the cessation of growth of the coleoptiles at about 5 days, (13,14)--D-glucan content rapidly decreased to 1 mol%. The cellulose content of the walls remained at about 35–40 mol% throughout coleoptile growth. Similarly, arabinoxylan content remained essentially constant at 25–30 mol% during growth, although the ratio of substituted to unsubstituted 4-linked xylosyl units decreased from about 4:1 to 1:1. Xyloglucan content ranged from 6 mol% to 10 mol% and the oligosaccharide profile determined using a xyloglucan-specific endoglucanase and MALDI-TOF mass spectrometry indicated that the oligosaccharides XXGG and XXGGG were the principal components, with one and two acetyl groups, respectively, Thus, dramatic changes in wall composition were detected during the growth of barley coleoptiles, both with respect to the relative abundance of individual wall constituents and to the fine structure of the arabinoxylans.     

Lateral electrical potential following asymmetric auxin application to maize coleoptiles

Following asymmetric application of indoleacetic acid to maize (Zea mays L.) coleoptiles the early time course of changes in lateral electrical potential was externally monitored with static-drop electrodes. First, an early negative potential change of ca.-1 mV was measured at the surface on the side of a strong auxin application. This negative auxin effect ended after ca. 15 min and was followed by a strong and lasting auxin stimulation of a positive lateral potential up to +12 mV at the auxin-treated side. The initial auxin effect appeared to depend on the size of the step-up in auxin concentration.     

Immunohistochemical observation of indole-3-acetic acid at the IAA synthetic maize coleoptile tips

To investigate the distribution of IAA (indole-3-acetic acid) and the IAA synthetic cells in maize coleoptiles, we established immunohistochemistry of IAA using an anti-IAA-C-monoclonal antibody. We first confirmed the specificity of the antibody by comparing the amounts of endogenous free and conjugated IAA to the IAA signal obtained from the IAA antibody. Depletion of endogenous IAA showed a corresponding decrease in immuno-signal intensity and negligible cross-reactivity against IAA-related compounds, including tryptophan, indole-3-acetamide, and conjugated-IAA was observed. Immunolocalization showed that the IAA signal was intense in the approximately 1 mm region and the outer epidermis at the approximately 0.5 mm region from the top of coleoptiles treated with 1-N-naphthylphthalamic acid. By contrast, the IAA immuno-signal in the outer epidermis almost disappeared after 5-methyl-tryptophan treatment. Immunogold labeling of IAA with an anti-IAA-N-polyclonal antibody in the outer-epidermal cells showed cytoplasmic localization of free-IAA, but none in cell walls or vacuoles. These findings indicated that IAA is synthesized in the 0–2.0 mm region of maize coleoptile tips from Trp, in which the outer-epidermal cells of the 0.5 mm tip are the most active IAA synthetic cells.     

Continuous measurement of initial curvature of maize coleoptiles induced by lateral auxin application

To analyze early effects of auxin application, an apparatus was developed which continuously and simultaneously registered the curvature of 10 individual maize (Zea mays L.) coleoptiles. Resolution was less than 5 m over a range of ±0.5 mm. The data were evaluated and plotted via paper tape and Hewlett-Packard-computer. Unilateral application of 3×10^-5 M indoleacetic acid (IAA) resulted in a transient inhibition of growth on the side of application for ca. 10 min (Phase I), followed by a strong stimulation (Phase II). The phytotoxin fusicoccin (FC) caused an immediate stimulation of elongation. The initial negative reaction of Phase I is auxin-specific. Only active auxins such as IAA and 1-naphtaleneacetic acid produced this initial inhibition; chemical analogs-inhibitory or neutral in long-term growth tests, e.g. phenylacetic acid-did not show any significant effects on Phase I. When the coleoptiles were symmetrically preloaded with different levels of auxin, only a large step-up of subsequent unilateral auxin application resulted in a negative phase I; a small step-up led to an immediate positive reaction. The results are discussed in context with the parallel kinetics for various other auxin-induced reactions of coleoptile cells which have already been published.Abbreviations FC fusicoccin - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - PAA phenylacetic acid     

The interpretation of intracellular measurements of membrane potential,resistance, and coupling in cells of higher plants

Results of microelectrode impalements of parenchymal cells of coleoptiles made in several different laboratories differ widely. The highest membrane potentials correlate with lower input resistance and the presence of intercellular coupling, whereas high input resistance seems to be associated with an absence of measurable coupling and possibly lower membrane potentials. In this paper we demonstrate that these results are consistent with (1) a tonoplast resistance several times greater than the input resistance of the cytoplasmic compartment, and (2) the presence of variable amounts of shunting introduced by insertion of the microelectrode through the cell membranes. The general consequences of this hypothesis are developed quantitatively. If the ideas are applicable to other tissues of higher plants-and on this point the evidence is still insufficient to judgeboth the design of experiments and the interpretation of measurements made with microelectrodes will have to be reevaluated.