J. Plant Res. 111: 53-58, 1998 Journal of Plant Research by The Botanical Society of Japan 1998 C oskeletal Pattern Changes during Branch Formation in a Centrifuged Adiantum Protonema Masamitsu Wada, Kazunari Nozue and Akeo Kadota Biology.Department, Faculty of Science, Tokyo Metropofitan University, Minami Osawa, Hachioji, Tokyo, 192-03 Japan A protonemal branch was induced on a side wall of a fern filamentous protonema by cell centrifugation and subse- quent polarized-red light irradiation as described in a previ- ous paper (Wada 1995, J. Plant Res. 108: 501-509). Changes in microtubule (MT) and microfUament (MF) pat- terns during the branch development were observed under fluorescence microscopy. A ring-like band of cortical MTs (MT-ring) and MFs similar to a preprophase band or a subapical ring structure (Murata et al. 1987) appeared transiently at the future branching site before cell swelling, the first visible step of branch formation. At this stage, the nucleus was located far from the branching site and the MT-ring appeared to be connected to the nucleus by endoplasmic MFs as well as with endoplasmic MTs. The MT-ring disappeared when cell wall swelling occurred. When the cell wall swelling began, a fan-like pattern of cortical MTs emanating from the new growing tip was established and the MTs reached the opposite flank of the protonema. When a new branch started to elongate and the nucleus moved into the branch, a faint subapical ring of MTs appeared at the subapical part of the new branch, Strands of MTs and MFs emanating from the nuclear front end reached a part of the subapical ring. Key words Adiantum m Branching-- Cytoskeleton m Microfilament m Microtubule-- Protonema (fern) Tip growth of filamentous cells, such as root hairs and pollen tubes, is one of the typical growth patterns in plant cells. Overall growth is another type of growth which occurs in most higher plant cells. These two growth pat- terns look very different from each other. Their basic growth mechanisms may however, be shared at various points, such as the involvement of MTs, rossetts (cellulose synthesizing complexes), turgor pressure, cell wall loosening. In a tip-growing cell, the growing region is very much restricted compared to that of overall growth, suggesting that machinery for cell growth is concentrated in a small region, so that we can focus our attention only on the tip region to study the mechanism of cell growth. In this respect, tip growing cells are a good model system for studying plant cell Abbreviations: MT, microtubule; MF, microfilament; DAPI, 4', 6, diamidino-2-phenylindole growth. The cytoskeleton is thought to play an important role in tip growing cells (Wada and Murata 1991) as well as in overall growing cells (Shibaoka 1994), although the mechanisms are not well understood. In tip growing cells, patterns of MTs and/or MFs have been observed in various organisms by immunofluorescence microscopy, for example, in root hairs (Lloyd 1983), pollen tubes (Joos et al. 1994, Terasaka and Niitsu 1994), fern and moss protonemata as well as their rhizoids (Murata et a/. 1987, Murata and Wada 1989b, Kadota and Wada 1989, Doonan 1991), or by electron microscopy in moss caulonemata (Schmiedel and Schnepf 1980), fern protonemata (Stetler and DeMaggio 1972, Wada and O'Brien 1975, Kiss et al. 1995) and Nicotiana pollen (Lancelle et al. 1987). To study the roles of the cytoskeleton, and espe- cially of MTs, however, their distribution should be observed carefully and categories in which they are included, cortical or endoplasmic, be considered because the function of MTs clearly differ depending upon their localization. In fern protonemata, a band of cortical MTs (subapical MT-ring) which forms at the subapical part of growing protonemata (Murata et al. 1987) may play a role in control- ling the orientation of cellulose microfibrils and consequently, the diameter of the protonemata (Murata and Wada 1989b). However such structure have not been found in rhizoids of the same species (Murata et al. 1987) as in the other tip growing cells so far observed. Similar pattern of MTs have only been found in fertilized Arabidopsis eggs (Webb and Gunning 1991). At the growing tip of these cells including fern protonema and rhizoid cells, cortical MTs either show a random pattern or are not localized there, although in the non-growing zone they run roughly parallel to the cell axis. The reason why, among other tip-growing cells, only fern protonemata have subapical MT-rings is unkown. Endoplasmic MTs, on the other hand, are often observed emanating from one or both ends of a spindle-shaped interphase nucleus in tip growing cells (Wada and O'Brien 1975, Mineyuki and Furuya 1986, Kadota and Wada 1995). They may play a role in nuclear migration or nuclear anchor- age (Schmiedel and Schneph 1979, Vogelmann et al. 1981, Mineyuki and Furuya 1986, Llyod et al. 1987, Kadota and Wada 1995) as shown by experiments using cytoskeletal inhibitors, colchicine and cytochalasin B. Doonan et al. (1986) showed that MTs connect the migrating nucleus to the division site during side branch formation.