Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system.

Role of Pseudomonas putida Indoleacetic Acid in Development of the Host Plant Root System

Polyphenol oxidases in plants

We have developed a new assay that differentiates between indoleacetic acid (IAA)-producing and -nonproducing bacteria on a colony plate lift. Medium supplemented with 5 mM L-tryptophan is inoculated with isolates of interest, overlaid with a nitrocellulose membrane, and then incubated until bacterial colonies reach 1 to 2 mm in diameter. The membrane is removed to a filter paper saturated with Salkowski reagent and incubated until distinct red haloes form around the colonies. The colorimetric reaction to IAA is limited to a region immediately surrounding each colony, is specific to isolates producing IAA, occurs within 1 h after the membrane is placed in the reagent, and is sensitive to as little as 50 pmol of IAA in a 2-mm2 spot. We have used this assay for quantifying epiphytic and endophytic populations of IAA-producing isolates of Pseudomonas syringae subsp. savastanoi and for detecting IAA-producing colonies of other pseudomonads and Erwinia herbicola. The assay provides a rapid and convenient method to screen large numbers of bacteria. Images

Rapid In Situ Assay for Indoleacetic Acid Production by Bacteria Immobilized on a Nitrocellulose Membrane

Cytochrome c oxidase is discussed as a possible photoacceptor when cells are irradiated with monochromatic red to near-IR radiation. Four primary action mechanisms are reviewed: changes in the redox properties of the respiratory chain components following photoexcitation of their electronic states, generation of singlet oxygen, localized transient heating of absorbing chromophores, and increased superoxide anion production with subsequent increase in concentration of the product of its dismutation, H2O2. A cascade of reactions connected with alteration in cellular homeostasis parameters (pHi, [Cai], cAMP, Eh, [ATP] and some others) is considered as a photosignal transduction and amplification chain in a cell (secondary mechanisms).

Primary and secondary mechanisms of action of visible to near-IR radiation on cells.

Polyphenol oxidases and peroxidases are among the most studied enzymes in fruits and vegetables. Owing to the deleterious effects of discoloration and off-flavor formation induced by their actions, these enzymes have not ceased to be a matter of concern to food technologists, while their versatility as catalyst and their diversity as protein present a challenge to the biochemist. This article gives an account on the present state of knowledge in this field. The occurrence of polyphenol oxidases and peroxidases in food and food raw materials, and their role and importance in food processing are briefly outlined. Results of biochemical research including catalytic properties, substrate specificity, susceptibility towards pH and temperature, action of inhibitors, isolation, purification, and characteristics of the enzymes are given, with special emphasis on recent achievements based on high resolution separation and isoenzyme techniques. Finally, the behavior of polyphenol oxidase and peroxidase in selected major groups of fruits and vegetables is discussed. Some contradictions found in the literature are pointed out and some questions that have not been given the necessary attention by researchers so far are mentioned.

Polyphenol oxidase and peroxidase in fruits and vegetables.

The wide use of the auxin, indoleacetic acid, in physiological and biochemical experiments has promoted interest in methods for its colorimetrie estimation. Mitchell and Brunstetteb (1) have proposed both the nitrite and the ferric chloride-sulphuric acid tests for the quantitative estimation of indoleacetic acid (IAA) in aqueous solutions, basing their suggested procedures upon a study of optimal reaction conditions for these two reagents. According to them, the nitrite method is sensitive to 10 /tig. IAA/ml. and develops a red color that is stable after two hours. In several attempts to duplicate their nitrite method using solutions of IAA varying from 20 to 45 /tg./ml., we could not obtain a stable red color with IAA at the two hours proposed, or at any other time. A faint pink develops almost immediately which rapidly fades to orange or yellow, depending on IAA concentrations, within i hour. If the concentration of nitrite is reduced, the red color becomes sufficiently persistent to be read. Indole likewise gives a strong, relatively stable, red color in this test (cf. table II)?a reaction which is sometimes used as a qualitative test for indole (Nitroso-Indole reaction). Tang and Bonner (2) have modified the ferric chloride-sulphuric acid method for IAA, combining the iron and sulphuric acid as a single reagent to yield improved sensitivity. However, the color produced is also unstable, rapidly developing and then fading. We have found, as have these workers, that the fading color can be practically dealt with by adopting a standard time between addition of reagent and reading of absorbancy or transmittance. Both of the methods discussed above possess disadvantages, lacking either specificity, sensitivity, or stability of color complex formed. During a study of the inactivation of IAA in aqueous solutions, it was frequently necessary to assay at one time many samples where the IAA concentrations were low, or where the degree of significance of small differences in concentrations between experimental unite required evaluation. Hence, we considered it desirable 'to re-examine the ferric chloride-sulphuric acid procedure. Several alterations have been made which produce a more stable color, of increased specificity, which changes in density more rapidly with variation in IAA concentration. 1. The procedure of Tang and Bonner can be improved somewhat by reading at 15 minutes after addition of reagent (instead of 30 minutes as they suggest), since the transient color reaches a maximum at the former time. Maximum absorption was found to occur at 530 ???.

Colorimetric estimation of indoleacetic acid.

Observations on the Quantitative Determination of Indoleacetic Acid

Plant preparations that convert tryptophan to indoleacetic acid (IAA) characteristically have low reaction yields. Since such preparations sometimes also contain IAA-oxidase activity, inhibition of the oxidase might raise the yields of IAA. Accordingly, polyphenolic inhibitors (1, 11, 19, 33, 40) were added to incubation mixtures containing tryptophan and a seedling enzyme preparation that was able to convert tryptophan to IAA. More than tenfold increases in auxin yield resulted. However, the presence of auxin in enzymeless controls, plus experiments on the recovery of added IAA, indicated that the enhancements observed might be caused by a polyphenolic oxidation of tryptophan to IAA. We shall present evidence that the sequence

Formation of auxin from tryptophan through action of polyphenols

The magnitude of acceleration required to induce growth responses in Avena seedlings grown in the absence of tropic response to earth gravity has been investigated. For this purpose, a clinostat was developed that imposes accelerations from about 10−9 g to 3 g upon the seedling; simultaneously, it nullifies, or compensates for, response to the directional component of the gravitational-force vector by rotating the seedling on a horizontal axis. When accelerations less than 10−3 g are applied in either the acropetal or the basipetal direction, the growth in length and weight of the various organs is not materially different from that of compensated seedlings to which no longitudinal force is applied. At accelerations between 10−3 and 10−2 g, differences in growth become highly significant. When the centrifugal forces are transverse to the seedling during compensation, the threshold acceleration range for geoperception, as manifest by shoot reorientation, is again between 10−3 and 10−2 g. Geotropic reorientation of the root becomes apparent after exposures between 10−4 and 10−3 g.

Thresholds for georesponse to acceleration in gravity-compensated Avena seedlings.

The rate of oxidative phosphorylation by isolated mitochondria was found to be affected by red (650 m mu ) and far-red (725 m mu ) light. Esterification of adenosine diphosphate to adenosine tripliosphate by rat liver mitochondria is decreased by concomitant or prior irradiation with far-red and increased by red light. The effect of far-red is reversible by red in an alternating sequence. An analogous sensitivity was not found when isolated mitochondria of the Avena seedling were irradiated. However, irradiation of the intact seedling significantly affected the phosphorylative capacity of the mitochondria subsequently isolated. Whether enhancement or inhibition of activity took place depended on the time at which the seedlings were irradiated in the post germinative period. There is apparently no direct correlation between radiationinduced changes in phosphorylative rate and either mitochondrial protein or volume. Impairment of ATP generation is suggested as the biochemical basis for the far-red potentiation of x-ray-induced chromatid aberrations. The photosensitivity of this phosphorylation also suggests its consideration as a metabolic determinart of the photomorphogeneses controlled by red and far-red light. (auth)

Solon A. Gordon

Papers

Colorimetric estimation of indoleacetic acid.

Observations on the Quantitative Determination of Indoleacetic Acid

Formation of auxin from tryptophan through action of polyphenols

Thresholds for georesponse to acceleration in gravity-compensated Avena seedlings.

Red and far-red action on oxidative phosphorylation