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During ripening of avocado (Persea americana), the CYP71A1 mRNA and protein accumulate to relatively high levels. Although the CYP71A1 gene was the first plant P450 to be cloned and sequenced, the functional role of this P450 remains obscure. Substrate studies have shown that CYP71A1 will metabolize various monoterpenes (nerol and geraniol), although these have not been detected in ripening fruit. Using DNA from a conserved domain of the CYP71A1 gene, we have explored the scope of the CYP71 (or related) gene family in avocado using low stringency DNA hybridization. This analysis suggests that there are approximately 10-12 genes in the CYP71 family. An alternative approach using PCR gave essentially identical results.

Mesocarp protoplasts were isolated from mature avocado fruits (Persea americana cv. Hass) at varying stages of propylene-induced ripening. Qualitative changes in the pattern of radiolabel incorporation into polypeptides were observed in cells derived from fruit at the different stages. Many of these differences correlate with those observed during radiolabeling of polypeptides from fresh tissue slices prepared from unripe and ripe fruit. Protoplasts isolated from fruit treated with propylene for one day or more were shown to synthesize cellulase (endo-ß-1,4-glucanase) antigen, similar to the intact propylene-treated fruit. These results suggest that the isolated protoplasts retain at least some biochemical characteristics of the parent tissue. The cells may also be used in transient gene expression assays. Protoplasts isolated from preclimacteric and climacteric fruit were equally competent in expressing a chimeric test gene, composed of the CaMV 35S RNA promoter fused to the bacterial chloramphenicol acetyltransferase gene, which was introduced by electroporation.

Amino acid conjugates of IAA can be chromatographed on polyvinylpolypyrrolidone (PVPP) using either acidic methanol or aqueous buffers as eluents. In the aqueous system, elution of the compounds is pH-dependent, and the pattern obtained suggests that hydrophobic interactions contribute substantially to the chromatographic behavior of IAA peptides on PVPP. Purification of soybean seed extracts by PVPP chromatography produced fractions containing indole-3-acetylaspartate and indole-3-acetylglutamate, based on chromatographic and mass spectral analysis, as well as three other indolic compounds, tentatively identified as N-acyl tryptophan derivatives. PVPP chromatography provides an effective preliminary purification of IAA peptides from plant extracts prior to their separation by other techniques such as high performance liquid chromatography.

Cell-free extracts of cucumber (Cucumis sativus L. cv. National Pickling) seedlings were found to have amine oxidase activity when assayed with tryptamine as a substrate. Studies of the effect of lowered pH on the extract indicated that this activity was heterogeneous, and three amine oxidases could be separated by ion exchange chromatography. The partially purified enzymes were tested for their activities with several substrates and for their sensitivities to various amine oxidase inhibitors. One of the enzymes may be a monoamine oxidase, although it is inhibited by some diamine oxidase inhibitors. The other two enzymes have properties more characteristic of the diamine oxidases. The possible relationship of the amine oxidases to indoleacetic acid biosynthesis in cucumber seedlings is discussed.

The present studies showed that about 80% of the indole-3-acetic acid extractable from Avena kernels by aqueous acetone was esterified to polymers precipitable by ammonium sulfate and ethanol or acetone. The polymers were positively charged, being adsorbed to cation exchange columns at a pH of 3, or below, and eluted at a pH greater than 4. The polymers were heterogeneous with respect to size, about 5,000 to 20,000 daltons, and charge, exhibiting apparent pK(a) values of 4.2 and 4.7. The polymer fractions contained esterified IAA, anthrone-reactive material that liberated glucose upon acid hydrolysis, phenolic compounds, and peptidic material with a high proportion of hydrophobic amino acids. Since the esterified IAA was unstable, establishing polymer purity was not possible, and the designation IAA-glucoprotein fraction was adopted.Dehusked Avena kernels contained 8 mg/kg total IAA of which 5.5% was free and 94.5% esterified. IAA bound through a peptidic linkage was present, but in only trace amounts.

We report the further characterization of indole-3-ethanol oxidase from cucumber seedlings. The effects of various inhibitors suggest that the enzyme may be a flavoprotein with a metal ion and sulfhydryl groups required for full activity. Indole-3-acetaldehyde, a product of the reaction, inhibits the enzyme. This inhibition is overcome by O(2) but not by indole-3-ethanol, indicating that the kinetic mechanism of the enzyme is a ping-pong Bi-Bi. The enzyme undergoes cooperative interactions with indoleethanol, yielding Hill coefficients as high as 2.96. Gibberellins are without effect on the enzyme, but it is inhibited by several acidic indoles possessing growth-promoting activity and by two synthetic auxins, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid. Increasing concentrations of indoleacetic acid (IAA) brought about a slight reduction in the indoleethanol concentration producing halfmaximal velocity. Increasing levels of indoleethanol decreased the concentration of IAA required for half-maximal inhibition. At low concentrations of indoleethanol, low levels of IAA activated rather than inhibited. The effect of IAA was not overcome at higher levels of indoleethanol. These results may be interpreted as showing that IAA is a noncompetitive inhibitor which binds to that conformation of the enzyme which also binds indoleethanol. The significance of these interactions for the regulation of IAA biosynthesis is discussed.