PHYSIOLOGIA PLANTARUM 115: 155–165. 2002 Copyright C Physiologia Plantarum 2002 Printed in Denmark – all rights reserved ISSN 0031-9317 Pyrimidine nucleotide and nucleic acid synthesis in embryos and megagametophytes of white spruce (Picea glauca) during germination Claudio Stasolla a,1 , Natalia Loukanina a , Hiroshi Ashihara b , Edward C. Yeung a and Trevor A. Thorpe a, * a Plant Physiology Research Group, Department of Biological Sciences, University of, Calgary, Calgary, Alberta T2N 1N4, Canada b Department of Biology, Faculty of Science, Ochanomizu University, Tokyo 112–8610, Japan 1 Present address: NC State, Forest Biotechnology Group, 2500 Partners II Bldg., Raleigh, NC 27695, USA *Corresponding author, e-mail: tthorpe/ucalgary.ca Received 23 May 2001; revised 8 October 2001; in final form 26 October 2001 Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophyt- es of white spruce by following the metabolic fate of 14 C- labelled orotic acid, uridine, and uracil, as well as by measur- ing the activities of the major enzymes participating in nucle- otide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autora- diographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential util- ization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar Introduction White spruce is an important coniferous species in North America, especially for its use in pulp and lum- ber production (Farrar 1996). In recent years, the es- tablishment of an efficient regeneration system via so- matic embryogenesis (Lu and Thorpe 1987) has consti- tuted a valuable tool for the propagation of this species, as well as a good system which has allowed comparative studies of in vivo and in vitro embryogen- esis. Today, in fact, our studies have increased knowl- edge on the structural, physiological, and biochemical events occurring during zygotic and somatic embryo- genesis (Joy et al. 1991, 1997, Kong and Yeung 1992, 1995, Kong et al. 1997, 1999, Yeung et al. 1998, Sta- solla and Yeung 1999, 2001, Ashihara et al. 2000, 2001a,2001b, Stasolla et al. 2001a,2001b, 2001c). Be- Abbreviations – DTT, dithiothreitol; OPRT, orotate phosphoribosyltransferase; PRPP, 5-phosphoribosyl-1-pyrophosphate; NPT, nucleoside phospho- transferases; TK, thimidine kinase, UPRT, uracil phosphoribosyltransferase; UK, uridine kinase. Physiol. Plant. 115, 2002 155 metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may repre- sent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of 14 C-thymidine for nucleic acid synthesis increased in both dissected embryos and mega- gametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reacti- vation was a later event, and the root meristem became acti- vated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germi- nation. sides its theoretical significance, knowledge on zygotic embryo maturation and germination represents a valu- able tool for improving the somatic embryogenic pro- cess via the design of rational media and improved culture conditions. This is particularly true for post- embryonic growth, as failure to germinate and convert (emergence of root and new leaf primordia) is often observed in somatic embryos. Such a failure has been ascribed to the lack of resumption of mitotic activity in the apical meristems (Kong and Yeung 1992). Thus, comparative studies on nucleic acid metabolism and cell reactivation in germinating zygotic and somatic embryos might provide insights into the causes of the low conversion frequency observed in the latter. Pyrimidine nucleotides have important functions,