The problem of determining the quantity and quality of peroxidase isoenzymes in plant tissues has persisted over several decades for many reasons important to plant physiologists. First, the phenomenon of isoenzymes itself is perplexing. Why are there so many different forms of peroxidase enzyme all possessing the same catalytic activity? What is the significance of some peroxidase isoenzymes being located in all parts of a cell while other peroxidase isoenzymes are located only in particular organelles, membranes, or walls? Peroxidase is thought to be involved in lignin formation (12). Are all peroxidase isoenzymes associated with this activity, or are just certain isoenzymes involved? Peroxidases from numerous plants are known to exhibit IAA oxidase activity (8). This is an important function in regulating growth in plants; but do all of the peroxidase isoenzymes have this capability to degrade IAA or Jo only a few possess it? Put another way -maybe some peroxidase isoenzymes are much more important in IAA oxidaion than others. Total peroxidase activity generally increases with aging, wounding, pathogen invasion, and exposure to rthylene (6). Does the total complement of peroxidase isoenrymes respond to these factors or do only certain isoenzymes ncrease? More important, does the increased activity come from pre-existing isoenzymes or are new, specific isoenzymes formed de novo?Our ability to answer these questions will parallel our ability to achieve reliable separations of the entire complement of peroxidase isoenzymes and to measure each one's kind and degree of catalytic activity.Shannon's review (10) Continuing work in my laboratory indicated that further improvements in resolution, stability, and linearity of pH gradients, reproducibility, and staining of the isoenzymes could all be achieved. In this paper, I report on the development of an IEF technique in polyacrylamide gel slabs that shows more than twice the number of commercial HRP isoenzymes reported previously. Also, I present evidence that all of the isoenzymes exhibit both peroxidase and IAA oxidase activity to the same degree.

The relationship of indoleacetic acid oxidase activity to peroxidase activity is complicated by numerous multiple forms of this enzyme system. It is not known if all isozymes of this complex system contain both types of activity. Isozyme analysis of commercial horseradish peroxidase and leaf extracts of yellow birch (Betula alleghaniensis) by isoelectric focusing in polyacrylamide gels was used to examine this problem. Horseradish and birch exhibited 20 and 13 peroxidase isozymes, respectively, by staining with benzidine or scopoletin. Guaiacol was less sensitive. Indoleacetic acid oxidase staining (dimethylaminocinnamaldehyde) generally showed fewer bands, and left doubt as to the residence of both types of activity on all isozymes. Elution of the isozymes from the gels and wet assays verified that all peroxidase isozymes contained indoleacetic acid oxidase activity as well. Estimation of oxidase to peroxidase ratios for the major bands indicated small differences in this parameter. A unique isozyme for one or the other type of activity was not found.

Seeds of paper birch (Betula papyrifera Marsh.) were induced to germinate by prechilling at 3 C or by red light. The light requirement was mediated by phytochrome and the action of phytochrome during prechilling was investigated. Red irradiation (R) prior to prechilling markedly enhanced the effectiveness of the prechilling treatment in inducing subsequent germination at 18 C. Reversal of this enhancement by far-red irradiation (FR) was more effective when FR was supplied after a 1-week prechiUl treatment than after a 2-week treatment. The R enhancement effect exhibited a sharp drop as prechilling temperature was increased from 5 to 7 C. This decline is consistent with a membrane phase transition at about 7 C where Pfr action is diminished by a loss in sensitivity of its receptor sites. Although phytochrome action was observed during prechillng treatments, the seeds failed to germinate at prechilling temperatures. Therefore, it was concluded that while potentiation of germination by Pfr occurred during prechiling, some other reaction(s) leading to radicle protrusion requires higher temperatures. In one seed source loss of germination potential was observed with protracted storage at 3 C. This was prevented by R supplied during the prechiling treatment. Taken colectively the data suggest that action of phytochrome during prechilling is accentuated in these seeds by two factors: (a) an increase in the sensitivity (or number) of Pfr receptor sites; and (b) preservation of Pfr by deferment of thermal reversion.

Foliage samples and diameter measurements were taken periodically from yellow birch (Betula alleghaniensis) trees on well-drained Hermon and poorly drained Whitman soils. Foliar analyses were made to determine seasonal changes in composition, and differences between trees and soil series. Leaf weights increased with season and were higher on the Whitman. Leaf moisture trends reflected soil moisture regimes. Nutrient percentages decreased during early June. Thereafter N, P, S, and K percentages fluctuated slightly until September, then decreased until abscission, while Mg fluctuated considerably and Ca increased steadily. Absolute amounts of all six elements increased during June. Nitrogen, P, S, and K varied slightly during summer and decreased prior to leaf yellowing. Total Mg reached peak values in August and then decreased while total Ca increased continually. Total P, K, and S was comparable on both soils. Total N, Ca, and Mg was higher on the Whitman. Greater diameter increments of study trees were also recorded on the Whitman soil. The total content expression provided more meaningful information than nutrient percentages. Specific periods of sampling birch foliage are given.

The isolation of a unique enzyme capable of oxidizing indoleacetic acid, but devoid of peroxidase activity, has been reported for preparations from tobacco roots and commercial horseradish peroxidase. Experiments were made to verify these results using enzyme obtained from Betula leaves and commercial horseradish peroxidase. Both indoleacetic acid oxidase and guaiacol peroxidase activity appeared at 2.5 elution volumes from sulfoethyl-Sephadex. These results were obtained with both sources of enzyme. In no case was a separate peak of indoleacetic acid oxidase activity obtained at 5.4 elution volumes as reported for the tobacco enzyme using the same chromatographic system. Both types of activity, from both sources of enzyme, also eluted together during gel filtration. Successful column chromatography of Betula enzyme was dependent upon previous purification by membrane ultrafiltration. These results indicate indoleacetic acid oxidase activity and guaiacol peroxidase activity are dual catalytic functions of a single enzyme.The molecular "residence" of IAA oxidase activity is not known precisely. And this imprecision in our knowledge about IAA oxidase activity is a hindrance toward further understanding about the regulation of IAA levels in living plants.Up to now, three hypotheses, with varying amounts of substantiating evidence, have been reported in the scientific literature. The first considers that the two types of activity (i.e., IAA oxidase and peroxidase) are present on separable and distinct enzymes; the second considers that the two types of activity are resident on one enzyme (peroxidase) but with two active centers; and the third calls attention to the fact of peroxidase isoenzymes where one member of the family of isoenzymes may be the primary residence of IAA activity. These are examined briefly.The idea of separate enzymes was reported by Sequeira and Mineo (8). They had noted that fresh preparations (tobacco roots) lost IAA oxidase activity after several weeks in storage, whereas peroxidase activity was unchanged. Further, they found that thermal inactivation points and pH optima were different. Attempts to separate the two types of activity on columns of silica gel, carboxymethyl cellulose, diethylaminoethyl cellulose, and diethylaminoethyl Sephadex failed, but with SE'-Sephadex and 0.1 M eluting buffer they reported a Service, United States Department of Agriculture, Durham, major IAA oxidase peak (at 5.4 elution volumes) with little or no peroxidase activity from both tobacco root extracts and commercial HRPO.The belief that both types of activity reside with one enzyme (i.e., peroxidase) is more widely held. Evidence offered in support of this belief is mainly that both types of enzyme activity remain together through various stages of purification (7, 10), and also there is evidence that thermal inactivation is the same for both (7). The work of Siegel and Galston (9)