Bioassay auxin

The LJT bioassay was developed for an auxin bioassay by Dr. Maeda of Nagoya University in 1965(. As mentioned above, the LJT is extremely sensitive and specific to BS. We established a... 

Yopp JH, Mandava NB, Sasse JM (1981) Brassinolide, a growth-promoting steroidal lactone. I. Activity in selected auxin bioassay. Physiol Plant 53 445-452... 

Auxin inhibitor herbicides include the so-called wild oat herbicides shown in Figure 5.22. Their classification as auxin inhibitors is based on their inhibition of auxin-induced responses in auxin bioassays and their antagonism of auxin herbicides. " This anti-auxin activity of diclofop-methyl is almost certainly secondary in importance to its inhibition of acetyl-CoA carboxylase (see Chapter 3). 

Abscisin II is a plant hormone which accelerates (in interaction with other factors) the abscission of young fruit of cotton. It can accelerate leaf senescence and abscission, inhibit flowering, and induce dormancy. It has no activity as an auxin or a gibberellin but counteracts the action of these hormones. Abscisin II was isolated from the acid fraction of an acetone extract by chromatographic procedures guided by an abscission bioassay. Its structure was determined from elemental analysis, mass spectrum, and infrared, ultraviolet, and nuclear magnetic resonance spectra. Comparisons of these with relevant spectra of isophorone and sorbic acid derivatives confirmed that abscisin II is 3-methyl-5-(1-hydroxy-4-oxo-2, 6, 6-trimethyl-2-cyclohexen-l-yl)-c s, trans-2, 4-pen-tadienoic acid. This carbon skeleton is shown to be unique among the known sesquiterpenes. 

The most promising leads have come from studying the hormonal requirements of the system. By themselves, we can find no indication whatsoever of any growth promotion by any of the alkyl lipides. When gibberellic acid is also applied, response remains small. If indoleacetic acid, or another auxin, is added, the sections show the classic bioassay response—and this is further stimulated by the active lipides. In the presence of both auxin and gibberellic acid, however, the sections show their greatest capacity to elongate still further in the presence of the alkyl lipides (I). Remarkably, the lipides are most effective at concentrations corn-... 

Hoffinger ert al. (177) showed that the naturally occurring betaines such as hypaphorine and oxyneurine induce growth promotion at low concentrations in the lentil root tip bioassay which is an auxin test system. The same compounds turned out to be inhibitors at higher concentrations. 

Auxins are defined as organic substances that promote cell elongation when applied in low concentrations to plant tissue segments in a bioassay. By this definition, there are several other native auxins that have been reported to occur in plants in addition to the most often studied auxin, IAA. These include the halogen-substituted 4-C1-IAA,23 as well as phenylacetic acid and indole-3-butyric acid.24 All native auxins are found in planta as both free acids and conjugated forms through ester or amide linkages. IAA, the auxin most extensively studied, will be the focus of this chapter. 

Figure 1. Effects of CaCl2 on bioassays for auxin, gibberellin, and cytokinin. A effects of CaCl2 on elongation of oat coleoptile sections in the presence and absence of indoleactic acid B effects on elongation of lettuce hypocotyls in the presence and absence of gibberellic add and C effects on enlargement of Xanthium cotyledon pieces in the presence and absence of benzyladenine (13).

An in vitro bioassay was then performed to measure the auxin-like properties of these compounds, based on a procedure from Cleon Ross(14)(Fig. I). This assay is based in principle on the growth response to auxins of stem segments of Pisum sativum. Measurement of segment weight and transectional area was compared to the untreated control.In this manner, a response curve was obtained for each compound. Indoleacetic acid (IAA) gave a typical response curve, as shown AC 78,299 gave an auxin-like response while AC 78,167 was inactive(Fig. 2). 

J.H. Yopp, Southern Illinois University, Carbondale, became interested in the brassins project in its early stages (36) and he conducted cooperative brassins studies with both Mitchell and Mandava. Yopp et al. (37, 38) evaluated brassins responses in a number of auxin, gibberellin, and cytokinin bioassays and showed that brassins responded similarly to some of the known hormones in certain systems but not in others. These studies led to more cooperative investigation of brassinolide and other brassinosteroids (39, 40). 

The First Internode Bioassay (15) is based on the curvature of the first internode section after insertion of a paper disc containing auxin to one side of the morphologically basal part of the internode. When it is used for the testing of BRst, the paper discs containing tested BRst are applied to the internode 1 h prior to auxin application. BRst stimulate the lag-phase of auxin action and its activity is expressed as a difference in internode curvature between BRst + IAA tested sections and controls treated with auxin alone. 

High sensitivity to auxin was closely related to the elevated sensitivity to 24-epiBR (I). As shown in Figure 2, the dependence of curvature of internode sections on the amount of 24-epiBR applied is expressed by a two peak curve. When evaluated on the first peak basis, inversion of sections caused an increase in the sensitivity of bioassay by factor 100. The unusual two-peak-response is probably a result of interactions of the two growth regulators applied successively to the internode sections, that is, 24-epiBR and IAA. The first peak was always recorded when equimolar quantities of the two regulators were used. 

Specificity. Using the first member of the family, brassinolide (BR), an extensive survey of its effects in 17 bioassays, which varied in their responses to gibberellins, auxins and cytokinins, showed that BR did not behave exclusively as any one of those hormones. In some supposedly specific bioassays BR was as effective, or more so, as the hormone the assay was supposed to detect (9,10). This also applies to the rice lamina inclination assay (11), which is now frequently used. 

Brassinolide was tested on 17 bioassays for growth substance. The results led to claims that brassinolide possesses a broad spectrum of biological activity, including gibberellin-, auxin- and cytokinin-like activity (32,34). These claims must be treated with some caution however, since the claimed "specificity of some of the bioassays selected is questionable. At present three bioassay techniques (35,36,37) are used routinely for the detection of brassinolide activity. All three assays are sensitive to auxin, which is a prerequisite for the detection of brassinolide-like compounds. This is not to say that brassinolide has auxin-like activity, but rather there seems to be an interaction of cooperative action between auxin and BR. 

Bean First Internode Bioassay. This assay was originally designed for the detection of auxin activity (37). It takes advantage of the fact that unilaterally applied auxin causes bending of the treated bean internode, which reaches a maximum rate about 20 minutes after auxin application. The sections will respond to 10 pmol of lAA and BR increase the sensitivity of the sections to auxin by one order of magnitude. 

The problem of selectivity of detection has been a major aspect of plant hormone analysis since the early days of bioassay, as shown by the plethora of studies on naturally occurring auxin synergists and auxin antagonists . Two complementary approaches have been taken to improve the selectivity of detection. The first approach has been the application of high resolution techniques of separation science in order to purify the phytohormone away from interfering compounds. The second approach has been the use of selective detectors following chromatographic purification. The quality of an analytical method is dependent on the interplay of these two factors, resolution and detector selectivity. 

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