PHYSIOLOGIA PLANTARUM 116: 20–27. 2002 Copyright C Physiologia Plantarum 2002 Printed in Denmark – all rights reserved ISSN 0031-9317 Allelochemical stress produced by the aqueous leachate of Callicarpa acuminata: effects on roots of bean, maize, and tomato Rocı ´o Cruz-Ortega*, Gabriela Ayala-Cordero and Ana Luisa Anaya Departamento de Ecologı ´a Funcional y Aplicada. Instituto de Ecologı ´a, UNAM . Circuito Exterior, Ciudad Universitaria, M e ´xico, D. F. 04510, M e ´xico *Corresponding author, e-mail: rcruz /miranda.ecologia.unam.mx Received 20 March 2002; revised 19 April 2002 The in vitro effects of an aqueous leachate (1%) of Callicarpa acuminata Kunth. (Verbenaceae) on radicle growth, protein expression, catalase activity, free radical production and membrane lipid peroxidation in roots of bean, maize, and tomato were examined. Aqueous extract of C. acuminata in- hibited the radicle growth of tomato by 47%, but had no effect on root growth of maize and beans. 2D-PAGE and densitome- try analysis showed that C. acuminata aqueous leachate modi- fied the expression of various proteins in the roots of all treated plants. In treated bean roots, microsequencing analysis of an 11.3-kDa protein, whose expression was enhanced by leachate treatment, revealed a 99% similarity with subunits Introduction Plants produce diverse secondary metabolites (alleloch- emicals) that are released into the environment. Some of them have a biological activity on other plants and microorganisms, a phenomenon called Allelopathy (for reviews see R ice 1984, Anaya 1999). Consequently, plants must cope with allelochemical stress as well as with other environmental stresses (i.e. drought, tempera- ture, salinity, and pathogens). Like many other stress factors, allelochemicals have several molecular targets, and some of their physiological processes or modes of action have been described (Einhellig 1995, Reigosa et al. 1999). Allelochemical compounds have been shown to affect many different cellular processes in tar- get organisms, including disruption of membrane per- meability (Galindo et al. 1999), ion uptake (Lehman and Blum 1999), inhibition of electron transport in both photosynthesis and the respiratory chain (Calera et al. 1995, Pen ˜uelas et al. 1996, Abrahim et al. 2000), alter- A bbreviations – CHAPS (3[3-cholamidopropyl) dimethylammonio]-1-propanesulphonate); DTT, dithiotreitol; LUMINOL, 5-amino-2,3-dihydro-1,4- phthalazinedione; PVDF, polyvinylidene difluoride. Physiol. Plant. 116, 2002 20 of a-amylase inhibitor of other beans. A 27.5-kDa protein induced in treated tomato showed 69–95% similarity to gluta- thione-S-transferases (GST) of other Solanaceae. Spectro- photometric analysis and native gels revealed that catalase activity was increased by 2.2-fold in tomato roots and 1.4- fold in bean roots. No significant changes were observed in treated maize roots. Luminol chemiluminescence levels, a measure of free radicals, increased 3.8-fold in treated tomato roots and 2.1-fold in treated bean roots. Oxidative membrane damage in treated roots was measured by lipid peroxidation rates. In tomato we observed a 2.4-fold increase in peroxi- dation, however, no effect was observed in maize or beans. ations of some enzymatic activities (Cruz-Ortega et al. 1990, Silva et al. 1996, Politycka 1998), and inhibition of cell division (Cruz-Ortega et al. 1988, Anaya and Pelayo- Benavides 1997). Currently, we are performing a biopro- specting study on the abundant shrub or small tree (1– 6 m height) Callicarpa acuminata Kunth. (Verbenaceae) from the secondary plant communities of the ecological reserve El Eden, Quintana Roo, Mexico. Anaya and del Amo (1999) reported that the aqueous leachates from leaves and stems of this plant exhibit a strong allelopath- ic effect on other plants such as Amaranthus hypochon- driacus and Echinochloa crus-galli. The present study in- vestigates the effects of a 1% (w/v) aqueous leachate of C. acuminata on the radicle growth, protein expression, catalase activity, free radical production and membrane lipid peroxidation in roots of Phaseolus vulgaris L. (Fab- aceae), L ycopersicon esculentum L. (Solanaceae), and Zea mays L. (Poaceae), with the aim of contributing to