Influence of 2,3,5-Triiodobenzoic Acid and 1-N-Naphthylphthalamic Acid on Indoleacetic Acid Transport in Carnation Cuttings: Relationship with Rooting.

(3)H-IAA transport in excised sections of carnation cuttings was studied by using two receiver systems for recovery of transported radioactivity: agar blocks (A) and wells containing a buffer solution (B). When receivers were periodically renewed, transport continued for up to 8 h and ceased before 24 h. If receivers were not renewed, IAA transport decreased drastically due to immobilization in the base of the sections. TIBA was as effective as NPA in inhibiting the basipetal transport irrespective of the application site (the basal or the apical side of sections). The polarity of IAA transport was determined by measuring the polar ratio (basipetal/acropetal) and the inhibition caused by TIBA or NPA. The polar ratio varied with receiver, whereas the inhibition by TIBA or NPA was similar. Distribution of immobilized radioactivity along the sections after a transport period of 24 h showed that the application of TIBA to the apical side or NPA to the basal side of sections, increased the radioactivity in zones further from the application site, which agrees with a basipetal and acropetal movement of TIBA and NPA, respectively. The existence of a slow acropetal movement of the inhibitor was confirmed by using (3)H-NPA. From the results obtained, a methodological approach is proposed to measure the variations in polar auxin transport. This method was used to investigate whether the variations in rooting observed during the cold storage of cuttings might be related to changes in polar auxin transport. As the storage period increased, a decrease in intensity and polarity of auxin transport occurred, which was accompanied by a delay in the formation and growth of adventitious roots, confirming the involvement of polar auxin transport in supplying the auxin for rooting.

Indole-3-carbinol as a scavenger of free radicals.

The ability of indole-3-carbinol (indole-3-methanol) to trap a metastable synthetic-free radical is presented. Indole-3-carbinol is capable of acting as a scavenger of free radicals in an in vitro system. The presence of indole-3-carbinol determines the disappearance of the free radicals, the reaction being time- and concentration-dependent. The scavenging activity of different indoles is compared. Indole-3-carbinol and indole-3-acetic acid are both able to scavenge free radicals, but indole-3-carbinol is more effective. Other indoles such as indole-3-aldehyde and indole-3-carboxylic acid do not show the ability to trap free radicals. Indole-3-aldehyde appears as a product of indole-3-carbinol reaction with free radicals. The formation of an adduct between the free radical generated in vitro and indole-3-carbinol has also been detected. Stability of indole-3-carbinol in buffered media at different pH values and formation of 3,3'-diindolylmethane from indole-3-carbinol is also studied. The scavenging activity of indole-3-carbinol and its implications on the anti-carcinogenesis process is discussed.

The decrease in auxin polar transport down the lupin hypocotyl could produce the indole-3-acetic Acid distribution responsible for the elongation growth pattern.

The variation of indole-3-acetic acid (IAA) transport along Lupinus albus L. hypocotyls was studied using decapitated seedlings and excised sections. To confirm that the mobile species was IAA and not IAA metabolites, dual isotope-labeled IAAs, [5-(3)H]IAA + [1-(14)C]IAA, were used. After apical application to decapitated seedlings, the longitudinal distribution of both isotopes at different transport periods showed that the velocity of IAA transport was higher in the apical, elongating region than in the basal, non-growing region. This variation in velocity was not a traumatic consequence of decapitation because after application of IAA to the basal region of decapitated seedlings, both the velocity and intensity of IAA transport were lower than in the apical treatment. The variation in IAA transport down the hypocotyl was confirmed when it was measured in excised sections located at different positions along the hypocotyl. The velocity and, to a greater extent, the intensity of IAA transport decreased from the apical to the basal sections. Consequently, if the amount of IAA reaching the apical zones of lupin hypocotyl were higher than the IAA transport capacity in the basal zones, accumulation of mobile IAA might be expected in zones located above the basal region. In fact, an IAA accumulation occurred in the elongating region during the first 4-h period of transport after apical treatment with IAA. It is proposed that the fall in IAA transport along the hypocotyl might be responsible for the IAA distribution and, consequently, for the growth distribution reported in this organ. An indirect proof of this was obtained from experiments that showed that the excision of the slowly transporting basal zones strongly reduced the growth in the remaining part of the organ, whereas excision of the root caused no significant modification in growth during a 20-h period.

Modification by ethylene of the cell growth pattern in different tissues of etiolated lupine hypocotyls.

The influence of ethylene on growth in etiolated lupine (Lupinus albus L.) hypocotyls was studied in ethephon-treated plants. Ethephon reduced the length and increased the diameter of hypocotyls. At the end of the hypocotyl growth period (14 days), the fresh weight was reduced by 53%, and the dry weight was reduced by 16%. Thus, ethylene reduced water uptake in the tissues to a greater extent than the incorporation of new materials. Light microscopic measurements showed that the thickness of tissues was stimulated by ethylene, the vascular cylinder and cortex exhibiting greater increases (55 and 45%, respectively) than pith (26%) or epidermis (12%). Ethephon modified the cell growth pattern, stimulating lateral cell expansion and cell wall thickness, while reducing cell elongation. The response to ethylene varied in the different tissues and was higher in cortex and pith cells than in the epidermis cells. The ethylene-induced cell expansion in the cortex varied according to the localization of cells in the tissue: the central and subepidermal layers showed little change, whereas the innermost layers exhibited the greatest increase. Electron microscopy revealed that ethylene increased both the rough endoplasmic reticulum and dictyosomes, suggesting that ethylene stimulated the secretion of cell wall materials. In untreated seedlings, the pattern of cell growth was similar in cells from the epidermis, cortex, and pith. The final cell size varied along the hypocotyl, the cells becoming shorter and broader the closer to the basal zones of the organ.

Lateral diffusion of polarly transported indoleacetic acid and its role in the growth of Lupinus albus L. hypocotyls.

The transport and metabolism of indole-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L, cv. Multolupa) hypocotyls, following application of dual-isotope-labelled indole-3-acetic acid, [5-(3)H]IAA plus [1-(14)C]IAA, to decapitated plants. To study the radial distribution of the transported and metabolized IAA, experiments were carried out with plants in which the stele was separated from the cortex by a glass capillary. After local application of labelled IAA to the cortex, radioactivity remained immobilized in the cortex, near the application point, showing that polar transport cannot occur in the outer tissues. However, following application of IAA to the stele, radioactivity appeared in the cortex in those hypocotyl sections below the first 1 cm (in which the capillary was inserted), and the basipetal IAA movement was similar to that observed after application of IAA to the complete cut surface. In both assays, longitudinal distribution of (14)C and (3)H in the stele outside the first 1 cm was positively correlated with that of cortex, indicating that there was a lateral migration of IAA from the transport pathway (in the stele) to the outer tissues and that this migration depended on the amount of IAA in the stele. Both tissues (stele and cortex) exhibited intensive IAA metabolism, decarboxylation being higher in the stele than in the cortex while IAA conjugation was the opposite. Decapitation of the seedlings caused a drastic reduction of hypocotyl growth in the 24 h following decapitation, unless the hypocotyls were treated apically with IAA. Thus, exogenous IAA, polarly transported, was able to substitute the endogenous source of auxin (cotyledons plus meristem) to permit hypocotyl growth. It is proposed that IAA escapes from the transporting cells (in the stele) to the outer tissues in order to reach the growth-responsive cells. The IAA metabolism in the outer tissues could generate the IAA gradient necessary for the maintenance of its lateral flow, and consequently the auxin-induced cell elongation.

Oxygen consumption during the indole-3-acetic acid oxidation catalyzed by peroxidase. Effect of the enzyme/substrate ratio.

Oxygen consumption during the oxidation of indole-3-acetic acid (IAA) by peroxidase has been studied, in conditions of no limitation by oxygen. For different enzyme/substrate ratios the quotient O2 consumed/IAA oxidized shows rather small differences and it is always less than unity. During the reaction, however, the quotient undergoes important variations because oxygen uptake ceases long before the decarboxylation of IAA. The results are discussed in the light of previous proposed mechanisms.

Effects of enzyme/substrate ratio and of cofactors on the oxidation products of indole-3-acetic acid catalyzed by peroxidase.

During the oxidation of indole-3-acetic acid catalyzed by peroxidase, the relative amounts of the products closely depends on the enzyme/substrate ratio. In the absence of cofactors, high enzyme/substrate ratio induces a rise in the level of indole-3-aldehyde and indole-3-methanol, and a drop in that of oxindoles. 2,4-dichlorophenol, although a very efficient cofactor, promotes inhibition of the oxidation after a few minutes, presumably through the formation of a phenol-derivative inhibitor. 2-4-dichlorophenol also inhibits the production of oxindoles at all stages. Both inhibitory effects are abolished by a low concentration of enzyme. Mn2+, itself a weak inhibitor, synergizes the catalytic effect of 2,4-dichlorophenol, perhaps by preventing the formation of the inhibitor. The results are discussed against more widely accepted mechanisms of indole-3-acetic acid oxidation.