Studies on Auxin Protectors. Protection by Protector-I V. On the Mechanism of IAA of the Japanese Morning Glory

The mechanism of auxin protection by auxin protector-I (Pr-I) of the Japanese morning glory was studied in vitro. Four lines of evidence indicate that Pr-I acts as a strong reductant which prevents 'the peroxidase-catalyzed oxidation of IAA: 1) The kinetics of the reaction are best explained on this basis. 2) The Pr-I-induced lag preceding auxin destruction by peroxidase is completely eliminated by a strong oxidant such as H,0., at a concentration which does not appreciably affect the reaction rate. 3) Strong organic reductants mimic the Pr-I-induced lag. And 4) when the reaction rate is altered by varying the concentrations of the reactants, or the temperature, the length of the Pr-I-induced lag varies inversely with the reaction rate. In the Japanese mlorning glory there exist substances wvitlh relatively higlh molecular weights wlhich inhibit the destruction of indoleacetic acid ('IAA) both by commercial horseradish peroxidase and by enzymes present in Japanese mlorning glory stenms (15). Experinments on young plants have indicated that the inhibition of enzymatic destruction of IAA varies inversely with the age of the tissue, i.e., inhibition decreases as one descends along the stem, and the interference with IAA destruction correlates with the rate of internode and leaf elongation. Thus auxin protection is greatest in leaves and internodes elongating nmost rapidly, and dimlinishes in maturing and senescing tissue (21, 22). At least 3 substances (or comp)lexes or polymers of the same substance) appear to be involved: protector-A (Pr-A). protector-T (Pr-I), and protector-II (Pr-I1). Based on gel filtration studies Pr-A has aI molecular weight exceeding 200000 g/mole and has been found to be highily active in seeds. Activity in the slhoot tips of mature plants is also lhigh (22). Pr-I has a nmolecular weiglht of al)l)roxinmatel 8000 g/mole. and Pr-II hals a molecular weiglht of approximatel 2000 g/mole ( 15). Only smiall quantities of Pr-I, and no Pr-II, could be detected at the shoot apexes, whereas these 2 protectors are found in large quantities in the juvenile tissue of the shoot, and appear to play an important role in stem and leaf elongation. Tn old stem or leaf tissue, only snmall quantities of Pr-I and II are found and Pr-A could not be detected (22). Similar substances have been observed in other 1 Permanent address: National Institute of Genetics, Mishima, Japan. plant systems such as tobacco ( 11, 12), sulnflower, cocklebur, coconut water (cocoinut milk), Nicotiana rifstica, N. glauica, and N. affinis (16, and unpublished observations). Clearly the morphogenetic role of these substances should be explored further. One of the questions that needs to be answered is howv do the protectors exert their action? What is the molecular mechanism of auxin protection? This paper addresses itself to this question by investigating the action of Pr-I. As working hypotheses, one might consider 3 possibilities: 1) The protector acts as an enzyme inhibitor. 2) The protector complexes with IAA, thus preventing enzymatic attack. 3) The protector acts in somle other fashion. The first 2 possibilities can be considered improbable on kinetic grounds: Examination of the curves depicting the destruction of IAA by commercial horseradish peroxidase ( 17) shows that the presence of the protector brings about a lag in destruction not a change in rate. If Pr-I were to comlplex with either IAA or peroxidase alone, thereby inhlibiting the destruction of IAA, it would have to do so witlh sonmething approaclhing 100 % efficiency for a limited time (i.e., during the lag whenno destruction occurs) and then would have to dissociate (tthe conmplex) suddenly and act with zero efficiency (i.e., as destruction proceeds at a rate comparable to controls). The evidence presented below indicates that Pr-I exerts its protective action by virtue of the fact that it can act as an antioxidant. This interpretation is consistent with our previous observations (17), that Pr-I is rapidly inactivated by Mn3` even in an atmosphere of nitrogen, wlhile Mn2+ will do so only in the presence of oxygen. 1141 www.plantphysiol.org on July 23, 2018 Published by Downloaded from Copyright © 1968 American Society of Plant Biologists. All rights reserved.