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Figures 12-2 to 12-7. Thecal plate formation in motile cells of Scrippsiella hexapraecingula in images 2-5. Fluorescence images of thecal plates stained with calcofluor white M2R. Dorsal views of motile cells 5 min (2), 10 min (3), 15 min (4), and 30 min (5) after motile cells began to swim out of pellicles. A bar in 2-5 indicates 10 |m. Image 6: Replica of a mature thecal plate, showing the surface structure of the thecal plate exposed by extraction of amorphous materials. Note microfibrils deposited in the thecal plate with random orientations. Image 7: Microfibrils labeled with CBH-I gold particles, which were isolated from thecal plates.

Figures 12-2 to 12-7. Thecal plate formation in motile cells of Scrippsiella hexapraecingula in images 2-5. Fluorescence images of thecal plates stained with calcofluor white M2R. Dorsal views of motile cells 5 min (2), 10 min (3), 15 min (4), and 30 min (5) after motile cells began to swim out of pellicles. A bar in 2-5 indicates 10 |m. Image 6: Replica of a mature thecal plate, showing the surface structure of the thecal plate exposed by extraction of amorphous materials. Note microfibrils deposited in the thecal plate with random orientations. Image 7: Microfibrils labeled with CBH-I gold particles, which were isolated from thecal plates.

Figures 12-8 to 12-12. Pellicle formation in nonmotile cells of Scrippsiella hexapraecingula. Images 8-10: Cross sections of nonmotile cells. Image 8: Thin pellicle (P) on the plasma membrane (npm) of a nonmotile cell just after ecdysis. Image 9: Pellicle (P) thickening in a nonmotile cell 15 min after ecdysis. Image 10: Thick pellicle (P) in a nonmotile cell 2 h after ecdysis. Image 11: Electron micrograph of freeze-fractured pellicle in a nonmotile cell 5-6 h after ecdysis. Note lateral association of some microfibrils. Image 12: Microfibrils labeled with CBH-I gold particles, which were isolated from thecal plates. Arrowheads show microtubules th, thecal plate.

Figures 12-8 to 12-12. Pellicle formation in nonmotile cells of Scrippsiella hexapraecingula. Images 8-10: Cross sections of nonmotile cells. Image 8: Thin pellicle (P) on the plasma membrane (npm) of a nonmotile cell just after ecdysis. Image 9: Pellicle (P) thickening in a nonmotile cell 15 min after ecdysis. Image 10: Thick pellicle (P) in a nonmotile cell 2 h after ecdysis. Image 11: Electron micrograph of freeze-fractured pellicle in a nonmotile cell 5-6 h after ecdysis. Note lateral association of some microfibrils. Image 12: Microfibrils labeled with CBH-I gold particles, which were isolated from thecal plates. Arrowheads show microtubules th, thecal plate.

In S. hexapraecingula, the pellicle develops at the outside of the plasma membrane of nonmotile cells after ecdysis (Sekida et al. 2001a). Just after ecdysis, the cell produces an electron-dense, thin layer (Figure 12-8). Thirty minutes after ecdysis, an electron-transparent layer is deposited on the electron-dense, thin layer (Figure 12-9) and develops as the pellicle thickens to about 300 nm until 2 h after ecdysis (Figure 12-10). The electron-transparent layer appears homogeneous in thin sections (Figure 12-10), but it actually contains closely packed microfibrils (Figure 12-11). Microfibrils isolated from the pellicle have diameters of 2-14 nm and consist of very fine fibrils (about 2 nm in diameter) (Figure 12-12), which are more slender than those in the thecal plates. The microfibrils are identified as cellulose by labeling with CBH-I gold (Figure 12-12) as well as by their electron diffraction pattern (Figures 12-13 and 12-15).

In S. hexapraecingula, amphiesmal vesicle membranes in the motile cells and the plasma membrane in the nonmotile cells were examined by freeze-fracture electron microscopy to determine whether they contained cellulose-synthesizing TCs (Sekida et al. 2004). According to Sekida et al. (2004), no microfibril impression was found in the protoplasmic face (PF) and the extracellular face (EF) of either the inner or outer amphiesmal vesicle membranes in the motile cells. No particle

Figures 12-13 to 12-15. Electron diffraction patterns of cellulose microfibrils isolated from the tunicate Halocynthia as a standard cellulose sample (13), microfibrils isolated from thecal plates (14), and pellicles (15) of Scrippsiella hexapraecingula. Note the typical equatorial (110, 110, 200) and meridional (004) reflections of cellulose I.

Figures 12-13 to 12-15. Electron diffraction patterns of cellulose microfibrils isolated from the tunicate Halocynthia as a standard cellulose sample (13), microfibrils isolated from thecal plates (14), and pellicles (15) of Scrippsiella hexapraecingula. Note the typical equatorial (110, 110, 200) and meridional (004) reflections of cellulose I.

aggregates that could be TCs were observed on the fracture faces in any of 200 specimens examined. However, Sekida et al. (2004) found that TCs are present in the nonmotile cells 0.5-3 h after ecdysis but they are not observed more than 4 h after ecdysis. In the nonmotile cells of S.hexapraecingula, microfibrils of the pellicle and their impressions on the EF of the plasma membrane are arranged with random orientations (Figure 12-17). Some microfibrils are associated laterally with each other to form a band that is often curved. Particle arrays are found only in the PF of the plasma membrane (Figure 12-16). Since these particle arrays are associated with the ends of individual microfibril impressions, they are regarded as TCs (Sekida et al. 2004). This was the first to report the presence of TCs in dinoflagellates. The TCs consist of two rows of particles (Figures 12-16, 12-18 to 12-21). The particles have diameters of 5-15 nm (an average of 9.1 nm) and are not necessarily arranged at regular intervals. The number of particles ranges from 5 to 40 and averages 19. The TCs have a length of 62.5-290 nm (139.4 nm average) and a width of 15-31 nm (21.5 nm average). The TCs of S. hexapraecingula form two rows, and thus are of the linear multiple row type. However, the multiple rows of TCs of S. hexapraecingula differ from those of other species in two ways. S. hexapraecingula has two rows of TCs, while glauco-phycean, chlorophycean, and ulvophycean green algae have three rows (Willison and Brown, Jr. 1978; Brown, Jr. and Montezinos 1976; Itoh 1990). The TCs of S. hexapraecingula are also irregularly spaced, while in some red algae, which have from 2 to 4 rows of TCs (Tsekos 1999), the TCs are almost regularly spaced. Thus, S. hexapraecingula has a new linear type of TC that has not been found in other organisms examined so far. In S. hexapraecingula, several TCs up to 7 are often associated laterally with each other and formed a cluster (Figures 12-16, 12-18-12-21). These clusters may synthesize a band of microfibrils and consolidate to function as a single TC, because they were followed by parallel impressions of microfibrils (below broken lines in Figure 12-16). This is consistent with

Figures 12-16 to 12-21. Electron micrographs of freeze-fractured plasma membranes of nonmotile cells in Scrippsiella hexapraecingula. (16) TCs (arrows) in the PF of the plasma membrane. Parallel microfibril impressions seen below broken lines. (17) Microfibril impressions in the EF of the plasma membrane. Note lateral association of some microfibrils (arrowheads). (18-21) Consolidation of TCs. Two (18), three (19), four (20) or seven (21) TCs consolidating as a cluster. A bar in 18-21 indicates 100 nm.

Figures 12-16 to 12-21. Electron micrographs of freeze-fractured plasma membranes of nonmotile cells in Scrippsiella hexapraecingula. (16) TCs (arrows) in the PF of the plasma membrane. Parallel microfibril impressions seen below broken lines. (17) Microfibril impressions in the EF of the plasma membrane. Note lateral association of some microfibrils (arrowheads). (18-21) Consolidation of TCs. Two (18), three (19), four (20) or seven (21) TCs consolidating as a cluster. A bar in 18-21 indicates 100 nm.

the case where individual rosette TCs form hexagonal arrays during secondary wall formation in zygnematalean green algae belonging to the Charophyceae (Giddings et al. 1980), and with the formation of multiple linear TCs in a slime mold (Grimson et al. 1996). The consolidation of TCs might be the result of parallel evolution in distinct phylogenetic groups.

3 OCCURRENCE OF DISTINCT TCs IN THE HETEROKONTOPHYTA

Heterokontophytes are a large phylogenetic group including at least eleven taxo-nomic classes (Kawachi et al. 2002) and exhibit a great diversity in thallus organizations, growth patterns, habitats and life histories, which may be equivalent to that in chlorophytes. Although a relatively small number of investigations on cellulose and TCs in heterokontophytes have been carried out, at least three distinct TCs have been reported so far in the four classes, the Phaeophyceae, the Xantho-phyceae, the Phaeothamniophyceae, and the Eustigmatophyceae. The presence of cellulose was shown in several species of the Phaeophyceae (Cronshaw et al. 1958) and the Xanthophyceae (Parker et al. 1963) by x-ray diffraction and chemical analyses. In the zygote of the phaeophycean alga Pelvetia, Peng and Jaffe (1976) found single linear particle rows associated with the tip of microfibril imprints in the freeze-fractured plasma membrane first. Peng and Jaffe (1976) regarded the linear particle rows as elements for orienting microfibrils. Later, in other eight species of the Phaeophyceae, similar linear particle rows have been confirmed to occur at the ends of microfibril imprints in the PF of the plasma membrane and then assumed to be TCs (Katsaros et al. 1996; Reiss et al. 1996; Tamura et al. 1996; SchuBler et al. 2003). Each of TCs in these phaeophycean algae consists of a single liner row of particles 6-7 nm in diameter (Figure 12-22) and synthesizes a thin, ribbonlike cellulose microfibril with a uniform thickness (Figure 12-23). Individual particles constituting TCs may be composed of two closely packed subunits (Reiss et al. 1996). The number of TC particles varies between 10-100, concomitant with microfibrils with a variable width in the range of 2.6-30 nm in Sphacelaria (Tamura et al. 1996). A higher density of TCs has been shown in the apical area of tip growing cells in Syringoderma (SchuBler et al. 2003). The occurrence of short linear particle rows in Golgi vesicles suggests that TC precursors may be transported to the plasma membrane via these vesicles (Reiss et al. 1996).

Mizuta et al. (1989) found a TC on the PF of the plasma membrane in the xanthophycean alga Vaucheria hamata first, which is quite distinctive from TCs in phaeophycean algae. After Mizuta et al. (1989), TCs have been found in other two xanthophycean species Botrydium stoloniferum (Sekida et al. 2001b) and Botrydiopsis intercedens (Okuda et al. 2004). These xanthophycean algae have TCs composed of diagonal rows of particles (Figure 12-24) and assemble a thin, ribbon-like microfibril (Figure 12-25). The number of particles in an individual diagonal row and the number of diagonal rows constituting a TC vary (Table 12-1). As shown in Table 12-1, there are slight differences in TC and microfibril structures among the xanthophycean species so far examined. The length of TCs in B. intercedens and V. hamata is longer than that in B. stoloniferum. However, TCs in B. intercedens consist of a smaller number of diagonal rows than those in V. hamata, since spacings between neighboring diagonal rows in B. intercedens are larger than those in V. hamata. In the TC of B. stoloniferum, the number of particles in diagonal rows at the both ends of the TC is about half as much as that in the other diagonal rows, whereas the length of diagonal rows among a TC is almost the same in B. intercedens and V. hamata. Furthermore, cellulose microfibrils synthesized by TCs in B. intercedens and B. stoloniferum are thicker than those in V. hamata. According to Mizuta et al. (1989) and Mizuta and Brown, Jr. (1992a), Vaucheria TCs are separated into two types: TCs associated with microfibril impressions that are presumably active in microfibril formation; TCs unassociated with microfibril impressions that are presumably inactive in microfibril formation. The average number of diagonal rows in the former TCs is about 14, while in the latter TCs it is about 8. Such two types of TC also have been found in B. intercedens, but TCs unassociated with microfibril impressions usually appear in group (Okuda et al. 2004). In Vaucheria, TC assembly occurs directly on the plasma membrane from particulate precursors (globules) that are supplied by Golgi vesicles to the plasma membrane (Mizuta and Brown, Jr. 1992a)

and may be inhibited by Tinopal LPW (Mizuta and Brown, Jr. 1992b). Unlike in Vaucheria, in B. intercedens diagonal rows of particles also appear in the PF of cytoplasmic vesicles different from Golgi vesicles (Okuda et al. 2004). The diagonal rows of particles are assumed to be TC precursors that may be loaded into the plasma membrane through the fusion of the cytoplasmic vesicles (Okuda et al. 2004).

Bailey et al. (1998) established the class Phaeothamniophyceae in hetero-kontophytes. Recently, a new type of TC was found on the PF of the plasma membrane in three species belonging to the Phaeothamniophyceae (Okuda et al. 2004). The TC generally consists of three linear rows of particles in these species (Figure 12-26). However, Phaeothamnion confervicola and Stichogloea doederlei-nii have also TCs composed of two linear rows of particles. In P. confervicola, asymmetric TCs sometimes occurred, where one row was shorter than the other two. Microfibrils in these three species are characteristic of a thin, ribbon-like structure (Figure 12-27). TCs consisting of 3 linear rows of particles also occur in glaucophycean (Willison and Brown, Jr. 1978), chlorophycean (Brown, Jr. and Montezinos 1976) and ulvophycean (Itoh 1990) green algae. However, the TCs of the phaeothamniophycean species differ from those of these other species in three ways. The TCs of the phaeothamniophycean species are observed only on the PF of the plasma membrane, while TCs are found only on the EF in Glauco-cystis (Willison and Brown, Jr. 1978) and Oocystis (Brown, Jr. and Montezinos 1976) and on both the PF and the EF in ulvophycean algae (Itoh 1990). The TCs of the phaeothamniophycean species synthesize thin, ribbon-like microfibrils with 1-4 nm in thickness and 2-20 nm in width (Okuda et al. 2004), but in the ulvophycean algae much larger microfibrils are synthesized, for example, Valonia microfibrils being 20 nm wide and 17 nm thick in average (Kuga and Brown, Jr. 1989). Finally, the length of the TCs of phaeothamniophycean species is relatively short, ranging from 25-100 nm (Okuda et al. 2004). The TCs of Oocystis are about 500 nm long (Brown, Jr. and Montezinos 1976), and in Valonia the length of the TCs ranges 150-600 nm (350 nm average) during the primary wall formation and 255-799 nm (558 nm average) during the secondary wall formation (Itoh 1990). The dinoflagellate species Scrippsiella hexapraecingula has a TC only composed of two linear rows of particles (Sekida et al. 2004).

Okuda et al. (2004) reported the presence of TCs in the Eustigmatophy-ceae first. The TC of the eustigmatophycean alga Pseudocharaciopsis minuta consists of a single linear row of particles on the PF of the plasma membrane (Figure 12-28). Microfibrils isolated from the cells of P. minuta may be flat, ribbon-like structures, but most of microfibrils are observed to consist of several fine fibrils 2-4 nm in diameter (Figure 12-29). This type of TC found in P. minuta corresponds to that in phaeophycean algae in particle arrangement and formation of thin, ribbon-like microfibrils. Linear rows constituting TCs are occasionally missing particles or consist of some shorter rows with a gap in both eustigmatophycean (Okuda et al. 2004) and phaeophycean (Reiss et al. 1996) algae.

Figures 12-22 to 12-29. Structures of TCs and cellulose microfibrils in heterokontophycean algae. TC (22) and microfibrils (23) in the phaeophycean alga Sphacelaria rigidula. TC (24) associated with a microfibril impression (25) at the upper side of a broken line in the xanthophycean alga Botrydium stoloniferum. TC (26) associated with a microfibril impression (27) at the upper side of a broken line in the phaeothamniophycean alga Phaeothamnion confervicola. TC (28) and microfibrils (29) in eustigmatophycean alga Pseudocharaciopsis minuta. Arrowheads showing thin microfibrils twisted. Each bar indicates 100 nm.

Figures 12-22 to 12-29. Structures of TCs and cellulose microfibrils in heterokontophycean algae. TC (22) and microfibrils (23) in the phaeophycean alga Sphacelaria rigidula. TC (24) associated with a microfibril impression (25) at the upper side of a broken line in the xanthophycean alga Botrydium stoloniferum. TC (26) associated with a microfibril impression (27) at the upper side of a broken line in the phaeothamniophycean alga Phaeothamnion confervicola. TC (28) and microfibrils (29) in eustigmatophycean alga Pseudocharaciopsis minuta. Arrowheads showing thin microfibrils twisted. Each bar indicates 100 nm.

The fact that all heterokontophycean species so far examined synthesize thin, ribbon-like cellulose microfibrils is consistent with the postulation that hetero-kontophytes constitute a monophyletic algal lineage evolved by the secondary endosymbiosis between a heterokontic protozoan and a chlorophyll c-contain-ing eukaryotic alga (Kowallik 1993; McFadden 2001). This contrasts with the case of the Chlorophyta, because in the Chlorophyta chlorophycean and ulvophycean algae synthesize much larger cellulose microfibrils than those synthesized by charophycean algae (see below). In heterokontophytes, each of

Table 12-1. Structural characteristics of TCs and cellulose microfibrils in xanthophycean algae Botrydiopsis interceden, Botrydium stolonifelum and Vaucheria hamata

Botrydiopsis Botrydium

Characters intercedens stolonifelum*' Vaucheria hamata*2

TC structure

TC length (nm) TC width (nm) Number of diagonal rows Number of particles in each diagonal row Spacing between neighboring diagonal rows (nm) Particle diameter (nm)

Microfibril structure

Microfibril width (nm) Microfibril thickness (nm)

Parentheses showing means values. *'Data adopted from Sekida et al. (2001b).

*2Data adopted from Mizuta et al. (1989) and Mizuta and Brown, Jr. (1992a).

three distinct TCs known at present assembles thin, ribbon-like microfibrils. Therefore, a whole of heterokontophytes may be a good example to give evidence for that extant TCs had modified or evolved from a common original TC phylogenetically. The heterokontophyte algae consist of eleven major taxonomic groups: eustigmatophytes, dictyochophytes, pelagophytes, bacillariophytes, synurophytes, chrysophytes, raphidophytes, pinguiophytes, xanthophytes, pha-eophytes, phaeothamniophytes (Kawachi et al. 2002). Among them, phaeophytes, xanthophytes and phaeothamniophytes include multicellular forms with cell walls, and phaeophytes are thought to be the most evolved group in the Hetero-kontophyta (Clayton 1989). According to recent gene sequence analyses on rbcL genes, the Eustigmatophyceae form the basal clade in the rbcL tree, and the Phaeophyceae, the Xanthophyceae and the Phaeothamniophyceae are included in the same clade (Bailey et al. 1998; Kawachi et al. 2002). Further the Phaeo-thamniophyceae are sister taxa to the Phaeophyceae and Xanthophyceae clades according to Kawachi et al. (2002). Based on the rbcL tree, the Eustigmatophy-ceae diverged earliest from the origin of chromophytes. This suggests that the origin had the same TC type of a single linear row as extant eustigmatophycean

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algae. If this would be true, the Phaeophyceae would have inherited the basic organization of original TCs since extant phaeophycean algae have the TC type of a single linear row. The Phaeothamniophyceae have distinct TCs from the TCs of the Phaeophyceae and the Xanthophyceae. This suggests that the following events happened: When the Phaeophyceae, the Xanthophyceae and the Phaeothamniophyceae diverged from their common origin, the diagonal row TC type of the Xanthophyceae would have evolved independently from that of a single linear row. In the Phaeothamniophyceae, the 2-3 linear row TC type would have been acquired from a single linear row TC type.

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