Defined Nuclear Changes Accompany the Reprogramming of the Microspore to Embryogenesis P. S. Testillano,* M. J. Coronado,* J. M. Seguı ´,* J. Domenech,* P. Gonza ´ lez-Melendi,* I. Ras ˇka,† , ‡ and M. C. Risuen ˜ o* ,1 *Laboratory of Nuclear Organization during Plant Development, Centro de Investigaciones Biolo ´gicas, CSIC, Vela ´ zquez 144, 28006 Madrid, Spain; †Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic; and ‡Laboratory of Gene Expression, 3rd Medical Faculty, Charles University, Prague, Czech Republic Received December 17, 1999, and in revised form February 14, 2000 The switch of the gametophytic developmental program toward pollen embryogenesis to form a haploid plant represents an important alternative for plant breeding. In the present study, the switch of the gametophytic developmental program to- ward a sporophytic pathway, ‘‘embryogenesis,’’ has been studied in three different plant species, Bras- sica, tobacco, and pepper. The switch has been induced by stress (heat shock) at the very respon- sive stage of the microspore, which is the vacuolate period. As a result, the cell nucleus undergoes strik- ing structural changes with regard to late gameto- phytic development, including alterations of biosyn- thetic activities and proliferative activity. An enrichment in HSP70 heat-shock protein and in the presence of Ntf6-MAP kinase was observed after induc- tive treatment in the nuclei during early embryogen- esis. This apparently reflected the possible roles of these proteins, specifically the protective role of HSP70 for the nuclear machinery, and signal transduction of Ntf6-MAPK for the entry of cells into proliferation. Importantly, the observed nuclear changes were simi- lar in the three species investigated and represented convenient markers for early monitoring of embryogen- esis and selection purposes for obtaining double- haploid plants in plant breeding.  2000 Academic Press Key Words: plant nucleus; microspore embryogen- esis; stress response; heat-shock proteins; MAP ki- nases. INTRODUCTION Pollen embryogenesis represents an important tool in plant breeding for obtaining isogenic lines and new varieties through double-haploid plants (Clem- ent et al., 1999). This process involves the switch of the gametophytic developmental program toward a sporophytic pathway. It can be induced in vitro at specific stages during pollen development such as the vacuolate microspore (Gonza ´ lez-Melendi et al., 1995; Touraev et al., 1996, 1997; Chupeau et al., 1998). Various approaches (hormones, vitamins, su- crose, starvation, cold treatment, or heat shock) have been used to induce pollen embryogenesis, but the efficiency of the induction to produce embryos was low (Raghavan, 1986; Chupeau et al., 1998). Bio- chemical and molecular biology approaches have been extensively implemented in the past few years to achieve a deeper understanding of the mecha- nisms controlling this change in the developmental program (Chupeau et al., 1998), but the induction process remains largely unknown. While cell biology studies on nuclear changes accompanying pollen gametophytic development are well documented (Medina et al., 1983; Cresti and Tiezzi, 1992; Risuen ˜ o and Medina, 1986; Risuen ˜o et al., 1988; Testillano and Risuen ˜o, 1988; Testillano et al., 1993, 1995a) those describing pollen embryogen- esis are rare (Hause and Hause, 1996; Gonza ´ lez- Melendi et al., 1996; Straatman, 1999). To gain deeper insight into pollen embryogenesis, we docu- ment in the present study nuclear changes that accompany heat-shock-induced pollen embryogen- esis in two model species, Brassica napus and Nicoti- ana tabacum, and in an agronomically interesting crop, Capsicum annuum (pepper). The light and electron microscopy approach was complemented by immunocytochemistry of several markers reflecting nuclear metabolic activity, including a heat-shock protein and a MAP kinase. MATERIALS AND METHODS Plant material and microspore embryogenesis in in vitro culture. The plant species used were C. an- nuum L., American variety; N. tabacum L.cv Petit 1 To whom correspondence should be addressed. Fax: 34-91-562 75 18. E-mail: risueno@cib.csic.es. Journal of Structural Biology 129, 223–232 (2000) doi:10.1006/jsbi.2000.4249, available online at http://www.idealibrary.com on 223 1047-8477/00 $35.00 Copyright  2000 by Academic Press All rights of reproduction in any form reserved.