Auxin assay

Sevin. 1-Naphthalenol methylcarbanate [63-25-2] (Sevin) (44) was developed as an insecticide. However, the conception of the molecule, in the mid-1950s, was as a possible herbicide. The compound ultimately was useless as a herbicide, but in routine testing it was discovered to be an excellent insecticide. Sevin was active in the oat mesocotyl assay and demonstrated weak auxin-like activity. During the development of Sevin, it caused massive apple drop in the western United States in an orchard being treated for insects. It is used (ca 1993) as an abscising agent to thin apples. 

Scheme 1. Molecular structure of the plant growth hormone auxin (indoleacetic acid, IAA). Extremely small amounts (nanomolar) can be detected by the auxin standard test 5 mm long segments of pea shoots elongate faster in the presence of exogenous auxin, which can be taken as a sensitive assay...

Peas are a low-fat plant, and it is likely, therefore, that our results should not be freely extended to all other auxin- and gibberellin-induced systems. In fact, the equally classic oat coleoptile section hormone assay does not seem to respond to the fatty substances. Even the pea section fails to respond, unless the seedling has received some red light during the early part of its development. 

Figure 3. Development of BS and BI cellulase activity in apices of pea seedlings. Intact seedlings were sprayed with the auxin analogue 2,4-D and decapitated seedlings were painted with the natural auxin IAA with or without an inhibitor of DNA synthesis, FUdR. All treatments resulted in massive swelling at the pea apex because of cell expansion cell divisions also occurred, but not in the presence of FUdR (6). Cellulases were extracted as described in Figure 1 and assayed in unpurified form.

We stress these assays because, while leading to the discovery of IAA, they imposed structure-activity requirements precluding study of the IAA conjugates--and possibly the discovery of other auxins. For a substance to be active in the assays required that they 1) permeate membranes in a cut tissue surface, 2) be transported to the growing zone, and 3) promote growth in that zone. Hopefully these three requirements may someday be studied independently. 

A series of benzylnitramines were prepared by either nitration of the carbamates or N-alkylation of nitrourethane, followed by ammonolysis. These represent one-phenylnitramines, known broadleaf herbicides. Nastic responses and growth inhibition observed for this class suggested similarity to the auxin type herbicides. To determine if die preference for the (R) -configuration extended to nitramines, l-(2 6 -dichlorophenethyl)nitramine was resolved and the absolute configuration determined by asymmetric synthesis. A comparison of the (+) and (-) isomers in herbicidal and in vitro assays was performed and the results are discussed. 

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). 

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. 

Isolated nicotonic acetylcholine as well as plant receptors have been employed in receptrodes for the assay of acetylcholine and its antagonists, and auxin and toxin, respectively (Rechnitz, 1987 Thompson et al., 1986). 

The majority of the papers cited above under the individual hormones contain experimental details of how to set up and use immunoassays to quantify these hormones in extracts of plant tissue. Additional procedures can be found elsewhere for auxins [102-105], cytokinins [106-108], abscisic acid [109-115] and gibberellins [116-120]. The sensitivity of immunoassays for most of the plant hormones is generally in the pmole range but occasionally, when high affinity antibodies (K3=10 lmol ) are available, analysis is possible at the fmol level. Using amplified ELISA assays sensitivity down to 200 amol (200 x 10 mol) has been claimed for abscisic acid [113]. 

Phytotropins are synthetic molecules which affect the tropic responses, inhibit auxin transport and bind to the receptor for 1-N-naphthylphthalamic acid (NPA) (10-1) [91-93]. For a recent review see [94]. A simple assay to detect and compare activities is measurement of antigravitropic activity on cress seedlings [95]. There are two phytotropin recognition sites on the NPA receptor, or two receptors, which recognize phytotropins... 

A combination of promoter deletion analysis, linker scanning, site-directed mutagenesis, gain-of-function analysis, gel mobility shift assays, DNA methylation interference and DNase 1 footprinting have been used to search for AuxREs within auxin-responsive promoters. Two types of AuxREs have been identified using these experimental approaches. One of these is the ocs or as-1 element and the other is theTGTCNC element (discussed in Section 4.3.). 

---