The final step of the ethylene biosynthesis pathway in turnip tops (Brassica rapa): molecular characterization of the 1-aminocyclopropane-1-carboxylate oxidase BrACO1 throughout zygotic embryogenesis and germination of heterogeneous seeds y Marı´a del Carmen Rodrı´guez-Gacio a , Carlos Nicola´s b and Angel Jesu´s Matilla a, * a Departamento de Fisiologı´a Vegetal, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur s/n. 15782-Santiago de Compostela, Spain b Departamento de Fisiologı´a Vegetal, Facultad de Farmacia, Centro Hispano-Luso de Investigaciones Agrarias. Universidad de Sala- manca, Plaza de los Doctores de la Reina s/n. 37007-Salamanca, Spain *Corresponding author, e-mail: bvmatilla@usc.es Received 9 May 2003; revised 10 November 2003 In a previous report from the present authors, it was shown that the 1-aminocyclopropane-1-carboxylate (ACC) oxidation may play a crucial role during zygotic embryogenesis of turnip tops seeds. The present study was performed to elucidate the contribution of the silique-wall and seeds in ethylene produc- tion during this developmental process. ACC content in the silique wall is only higher than in seeds during the middle phases of zygotic embryogenesis. The ACC-oxidase (ACO) activity peaks in the silique-wall and seeds during the onset of embryogenesis, declining gradually afterwards, being undetect- able during desiccation period. Using reverse transcriptase- polymerase chain reaction, one cDNA clone coding for an ACO and called BrACO1, was isolated. The deduced protein for BrACO1 has a molecular weight of 36.8 kDa and a high homology with other crucifer ACOs. The heterologous expression of this cDNA confirmed that BrACO1 is an ACO. The expression of this gene was high during the first phases of silique-wall development, low during the middle phases and undetectable during desiccation. By contrast, BrACO1 transcript was accumulated only in the earliest phases of seed embryogenesis and may participate in the high- est ACO activity and ethylene production by seeds at the beginning of embryogenesis. Finally, in this work a correlation between the heterogeneity of Brassica rapa L. cv. Rapa seeds and the ability to oxidize the ACC to ethylene has been demonstrated. Introduction Zygotic embryogenesis is a complex process regulated by the developmental programme directed at the formation of viable seeds able to germinate under appropriate environmental conditions (Torres-Ruiz 1998). This process is often divided into three stages co-ordinated by phytohormones, abscisic acid (ABA) being the most thoroughly studied (Hilhorst 1995). During the first stage, cell-division processes predominate, whereas, in the second stage, growth is due mainly to cell elongation. The cessation of cell elongation marks the beginning of the third stage, which leads to accumulation of storage compounds in the seeds, the development of desiccation tolerance, an increasing susceptibility to dehiscence and final water loss (Meinke 1994, Ferra´ndiz 2002). Many physiological and molecular studies have clearly demonstrated that the synthesis and perception of ethylene PHYSIOLOGIA PLANTARUM 121: 132–140. 2004 Copyright # Physiologia Plantarum 2004 Printed in Denmark – all rights reserved Abbreviations – ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylic acid; ACO, ACC-oxidase; ACS, ACC-synthase; AdoMet, S-adenosyl-methionine; DPA, days post-anthesis; IPTG, b-d-thiogalactopyranoside. y The nucleotide sequence reported in this paper has been submitted to the EMBL and Genebank Nucleotide Sequence Database under accession number AJ309321. 132 Physiol. Plant. 121, 2004