[International Review of Cytology] Volume 128 || The Replication, Differentiation, and Inheritance...

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 128

The Replication, Differentiation, and Inheritance of Plastids with Emahasis on the

Concept of Organelle Nuclei TSUNEYOSHI KUROIWA

Department of Biology, Faculty of Science, Division of Developmental Biology, University of Tokyo, Hongo 113, Japan

I. Introduction

During the past 15 years, there have been remarkable advances in at least three areas related to the chloroplast genome. One of the most active areas involves the molecular biology of the organization of the genes, which has been based on sequencing of the chloroplast genome. Higher- plant chloroplast (cp) DNA can be isolated as a covalently, closed circular molecule with a molecular mass of 85-95 x lo3 kDa (Kolodner and Tewari, 1975). Denaturation mapping (Kolodner and Tewari, 1975) and restriction endonuclease analysis (Bedbrook and Bogorad, 1976) have shown that the majority of the circular molecules in the chloroplasts of a given species are identical in sequence. Since the construction of the physical maps of the cpDNA from Zea mays (Bedbrook et al., 1977) and Chlamydomonas reinhardtii (Rochaix, 1978), the maps of cpDNA from various plants have been reported (see Palmer, 1985). Shinozaki et al. (1986) and Ohyama et al. (1986), respectively, sequenced the entire cp- genome from the chloroplasts of Nicotiana tabacum and Marcantia polymorpha. The cpDNA from N . tabacum and M . polymorpha contains 155,844 and 121,024 bp, respectively. Each contains about 80 genes which encode a complete set of 30 tRNAs, four rRNAs (23 S, 16 S, 5 S, and 4.5 S), 20 ribosomal proteins, and 22 proteins of thylakoid membrane complexes. In addition there are about 30 open reading frames for which the functions remain to be determined. Thus, each of the chloroplasts contains a specific genome that is essential for the semiautonomy of the organelle.

A second area that has seen great progress is related to the biogenesis of the chloroplast membrane system. ATPase and the complexes of photosystems I and 11 are supramolecular complexes of enzymes located in the membrane of chloroplasts. The ATPase consists of a set of different subunits designated a, p, y , 6, and E (Nelson et al., 1980). Three subunits (a, p, and 6) are synthesized within chloroplasts and the remaining two in the cytoplasm (Watanabe and Price, 1982). Close cooperation between the two genetic systems and the two compartments of the cell is necessary for

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2 TSUNEYOSHI KUROlWA

the biosynthesis of the ATPase. The complex molecular and cellular mechanism of formation of the enzyme involves: (1) synthesis of different components in the cytoplasm and in the plastids; (2) transport and integra- tion of the enzyme precursors; (3) organization of precursor molecules in an assembly process; and (4) arrangement of the final supramolecular complex within the membrane layer. Coordination of these individual processes is required for the formation of a functional supramolecular complex, such as the ATPase and photosystems I and 11. It is suggested that the molecular chaperone is related to the assembly or formation of supramolecular complexes of the proteins, such as RuBisCO, ATPase, LHCP 11. etc. in chloroplasts (Lubben et al., 1989).

As described above, research related to the organization of the cp- genome and the biogenesis of plastids, including assembly of subunit proteins, developed as a result of experiments with a population of DNA or proteins isolated from whole tissues or organs of plants. However, it must be remembered that one leaf is composed of a variety of tissues such as spongy parenchyma, palisade parenchyma, epidermis, etc. These tissues contain large numbers of cells. One cell contains many plastids which do not divide synchronously. Accordingly, it should be noted that results obtained from a population do not always reflect events in an individual plastid .

Progress in cell biology related to the distribution, organization, separation, differentiation, and inheritance of plastids has also been consider- able. One remarkable development has added new dimensions to our concepts of the distribution, organization, separation, and inheritance of the pt-genome within the last 10 years. Results of electron microscopy suggested that plastids arise from the division of preexisting organelles and can differentiate into various types of plastid, such as amyloplasts, chromoplasts, chloroplasts, leucoplasts, etc. However, the following questions remain to be answered. Do all of these plastids (pt) contain DNA? How are the ptDNAs organized into organelles? How are pt-chromosomes separated into daughter organelles? How does plastidkinesis occur? How are ptDNAs transmitted to a cell's descendants? How many copies of ptDNA does each plastid contain? What interactions are there among the cell nuclear, mitochondrial (mt), and pt-genomes? The answeres to these basic questions appear to offer a key to the understanding of the timing of expression of genes on the pt-genome and the insertion of newly synthesized proteins for biogenesis occurring during the division cycle and differentiation of plastids.

A DNA-binding fluorochrome, 4'-6-diamidino-2-phenylindole (DAPI), which emits stronger fluorescence than do conventional fluorochromes, was synthesized by Dann et al. (1971) and applied to observations of organelle DNA. The use of DAPI combined with epifluorescence micro-

PLASTIDS AND ORGANELLE NUCLEI 3

scopy makes it possible to visualize extremely low levels of DNA in various organelles to analyze their behavior. The results of such analyses indicate that cp- and mt-DNA are not naked but are organized, with proteins, to form organelle nuclei. Therefore, all of organelle DNA can be observed under DAPI-epifluorescence microscopy.

A description of the molecular organization of the plastid genome and the biogenesis of plastids is omitted here since each has been reviewed exhaustively elsewhere (Rochaix, 1985; Sugiura, 1987; Lubben et al., 1989). However, since only the rough outlines are known of the organization, separation, and inheritance of the pt-genome, as revealed by various cell-biological techniques (including DAPI-epifluorescence microscopy) it seems appropriate to review these issues at this time.

11. Location of the Plastid Nuclei in Plastids of Various Plants

Ris and Plaut (1962) described DNA-like fibers within the chloroplasts of C. reinhardtii. These fine fibrils appeared to become clumped in an electron-transparent area, the “nucleoid,” in the matrix of chloroplasts after conventional fixation. Such DNA-containing regions have also been reported in the chloroplasts of many other plants, such as red alga, brown alga, green alga (Werz, 1966; Yokomura, 1967), and higher plants (Kislev et al., 1965; Gunning, 1965; Yokomura, 1967). In numerous plants, a small number of DNA-like fibrils appear in an electron-transparent spherical area, 0.1-0.5 pm in diameter, within individual chloroplasts. However, even within a single species, such an area (the DNA-like fibers containing the area) is not always visible in all chloroplasts under an electron microscope because a very small amount of DNA is embedded in the semi- electron-dense matrix in chloroplasts under standard physiological conditions and with conventional fixation techniques. Even when the DNA-like fibers are visible in electron-transparent areas, the amount of DNA fibers is much lower than anticipated. Probably, some parts of the DNA-like fibers are embedded in a somewhat electron-dense matrix around the electron-transparent regions.

Although the DNA-containing regions in the electron-transparent area (ETA) of plastids are conventionally called “plastid nucleoids,” they do not contain the entire DNA of the genome. By contrast, after staining with DAPI, the fluorescent spots and intact isolated ptDNA regions show all pt-chromosomes in plastids. Therefore, we consider it preferable to desig- nate these compact isolated structures found in situ as “pt-nuclei” (Kur- oiwa et al., 1981; Kuroiwa, 1982; Nemoto et d., 1988), analogous to “bacterial nuclei’’ (Robinow, 1956) and “mitochondria1 nuclei”.(Kuroiwa et al., 1976; Kuroiwa, 1982). Based on this definition, the nuclei are called “cell nuclei.” As described above, electron microscopic examination is

4 TSUNEYOSHI KUROIWA

not always a suitable technique for monitoring the distribution of all the DNA in chloroplasts.

DAPI was used for the first time for staining mtDNA in yeast by Wiliam- son and Fennel1 (1973, cpDNA in a higher plant by James and Jope (1978), and in green algae by Coleman (1978). The blue-white fluorescence of DAPI is stronger than that of other fluorochromes such as ethidium bromide, Hoechst 33258, acridine orange, etc., which were previously used to stain DNA in organelles. In addition, epifluorescence microscopes have been improved to generate strong fluorescence as a result of strong excita- tion light. Furthermore, DAPI epifluorescence microscopy and an apparatus that combines it with conventional microscopic fluorimetry, or video- intensified photon counting system (VIMPICS), made it easy to observe the distribution or behavior of cpDNA and to estimate extremely low and to estimate extremely low levels of DNA per plastid.

Kuroiwa el al. (1981) examined the distribution and behavior of pt- nuclei during the development of chloroplasts and the cycle of plastid division in many plants by staining with DAPI. The number, size, shape, and distribution of pt-nuclei were found to change during chloroplast development and during the division of plastids, and these parameters differed among various plants. The small proplastids of early embryonic cells of Brassicaljuncea and of cultured cells of N. tabacum (Figs. l a and 2) contain only one small, spherical proplastid (pp) nucleus, 0.2 pm in diameter, whereas the pp-nucleus in the proplastids of dormant embryonic cells is ovoid, 0.5-1 .Opm in diameter and, thus, several times larger than the pp-nucleus of the early embryonic cells. Since the proplastids divide actively, the increase in volume of pt-nuclei seems to be due to endoduplication of ptDNA. When proplastids develop into etioplasts in the dark, the size of the pp-nucleus increases 2- to 4-fold, the pp-nucleus becomes cup shaped, and is often found near starch grains or a prolamellar body. Once etioplasts are illuminated, the pt-nuclei begin dividing into tiny spherical structures and their numbers increase markedly to reach more than 20 cp-nuclei in fully mature chloroplasts, which emit red autofluorescence (Figs. lb and 2). In most land plants and algae examined extensively by Kuroiwa et al. (1981, 1989b) and Coleman (1989, the patterns of distribution of pt-nuclei in mature chloroplasts were peculiar to plant groups, although they changed during the division cycle (Kuroiwa et al., 1981; Zachler and Cepfik, 1987) and the development of plastids.

FIG. 1 . Photomicrographs of cell nuclei (CN), a proplastid nucleus (large arrow in a), mitochondria1 nuclei (small arrows in a and b), and a chloroplast nucleus (large arrow in b) in a Nicoriunu rubacum cultured cell (line BY-2) (a); and a mature leaf cell (line BY-2) (b) after staining with DAPI. Bar = 10 pm. (Photographs courtesy of Dr. Y. Nemoto, Tokyo Agricul- ture and Technology University, Japan.)


Proplastid division Proplastid division Etioplast division Chloroplast division

FIG. 2. A diagram of plastid nuclear events during chloroplast development and senescence. The chloroplasts of the SN, CN, CL, PS, and SP type are differentiated from proplastids throughout several plastid divisions. The term plastid nucleus indicates the cp-nucleaus and the terms plastid genome and chloroplast genome mean the same thing. Pt-M, Pt-S, and CDC show the plastid-division stage, the plastid DNA-synthetic stage, and the chloroplast division cycle, respectively. (From Kuroiwa ef al., 1981; reproduced by permission of the Japanese Society of Plant Physiologists.)

Most eukaryotic plants can be classified into five types according to differences in the shape, size, and distribution of the cp-nuclei in their mature chloroplasts (Fig. 2; Kuroiwa er al., 1981). The first, the SN type (scattered pt-nuclei), is characterized by chloroplasts with small, uni- formly dispersed cp-nuclei in the matrix between thylakoid membranes and/or granas. The land plants and algal groups, such as Chlorophyceae, Prasinophyceae, Chloroohyceae, Euglenophyceae, Cryptophyceae, Eu- stigmatophyceae, and Dinophyceae, are of the SN type (Fig. 2; Kuroiwa er al., 1981; 1989b). The second, the CN type (centrally located pt-nuclei), is characterized by chloroplasts with one or a few cp-nuclei located in the central area surrounded by lamellae. Cyanidiurn caldarium RK-1, which was reported to be a red alga, is a typical example of this type (Fig. 2). Recently, Miyamura and Hori (1989) found an unusual type of chloroplast


in Caulerpa okamurae, the pyrenoid of which contained one large cp- nucleus during a particular phase of the life cycle. Sodmergen et al. (1989) reported that the cells of the coleoptile of Oryza satiua contain mature chloroplasts with 1-3 centrally located cp-nuclei of the CN type while the cells in the first and second leaves contain chloroplasts with dispersed cp-nuclei of the SN type. The third, the CL type (circular pt-nuclei), has chloroplasts with a large, ring-shaped cp-nucleus inside the girdle lamellae (Fig. 2). Such a circular pt-nucleus isolated from the brown alga Ectocar- pus indicus appears to be a chain of small spherical particles which may correspond to the small cp-nuclei of the SN type (Kuroiwa and Suzuki, 1981). Algae, such as the Chrysophyceae, Xanthophyceae, Bacilla- riophyceae, Phaeophyceae, Rhaphidphyceae, and Haptophyceae, are of the CL type. The shape of the circle changes with the shape of the chloroplast in different species. In some diatoms and brown algae, the chloroplasts are disc shaped and the cp-nucleus is a ring; in other diatoms and in C. caldurium M-8, which has irregularly shaped chloroplasts, the pt- nucleus forms an irregular circle that lies along the periphery of the chloroplast (Nagashima et al., 1986; Kuroiwa et al., 1989). The fourth, the PS type (peripherally scattered pt-nuclei), and the fifth type, the SP type (spread pyrenoid pt-nucleus), are modifications of the CL type and the SN type, respectively. The PS type is characterized by chloroplasts with pt-nuclei scattered along their peripheries beneath an inner limiting membrane. The rhodophycean algae, such as Gellkidium amansii and Sym- phyocfadia latiusculu, are of this type. The last SP type has numerous small pt-nuclei which form a shell around a pyrenoid in the chloroplast (Fig. 2). The green alga Bryopsis plumos is of this type. The pt-nuclei tend frequently to be located near the pyrenoid.

It is interesting that the CN type, which can be found among the lower eukaryotes, was also observed in undifferentiated proplastids and chloroplasts, and the CL type, to which chloroplasts in some red algae and almost all brown algae belong, can be observed in etioplasts before the development of chloroplasts in monocots (Sellden and Leech, 1981). Such diversity in the distribution, number, size, and shape of pt-nuclei in various plants must depend basically on the development of the thylakoid membrane during the evolution of membrane systems and the development of chloroplasts (Kuroiwa, 1982; Rose, 1988).

III. Organization of the Plastid Nucleus

As described above, almost all of the mature chloroplasts of land plants and algae contain a pt-genome which is located in the specific regions of chloroplasts that develop from tiny proplastids. The proplastids also can


differentiate both directly and indirectly from other plastids, such as etioplasts, chloroplasts, chromoplasts, leucoplasts, amyloplasts, etc., in a tissue-specific manner and/or depending upon such environmental factors as light and temperature. These plastids also contain pt-genomes within a specific area of the plastids.

The functional and structural changes in plastids during leaf development are accompanied by an accumulation of plastid proteins, many of which are encoded by pt-genes (Klein and Mullet, 1986). Pt-genes are located on a circular strand of DNA which is 1.2 X Id to 1.8 x 10’ bp in length (Figs. 4e and 4h). This DNA contains up to 137 genes which encode tRNA, rRNA (16 S, 23 S, 4.5 S, and 5 S), and numerous proteins (Fig. 4h). It has been pointed out that the packing mode, i. e., the dispersion and condensation of the cp-nuclei, is intimately related to the photosynthetic oxygen-evolving activity of the cells in Chfamydornonas (Nakamura et al., 1986). Therefore, the three-dimensional structure of pt-nuclei may be important for understanding the function of ptDNA during the division and differentiation of plastids. To elucidate the organizations of pt-nuclei, the intact pt-nuclei must be isolated from the plastids of various plants. Sev- eral groups have tried to isolate pt-nuclei from chloroplasts or chromoplasts (Kuroiwa and Suzuki, 1981; Briat et af., 1982; Reiss and Link, 1985; Hansmann et al., 1985). It is, however, difficult to purify the isolated pt-nuclei because the thylakoid membrane system in chloroplasts and chromoplasts is highly developed. By contrast, proplastids have poorly developed membrane systems and they contain the smallest numbers of copies of pt-genome. Thus, they are useful for the isolation of intact pt-nuclei.

Nemoto et al. (1988) developed a method for isolating morphologically intact proplastids in large quantities from protoplasts of N. tabacum by a method that involves disruption of cells by forcing them through a layer of nylon mesh (Fig. 3). Isolated proplastids contain one to several pp-nuclei which are similar in appearance to pp-nuclei in viuo (Figs. 3d-3n). After treatment with the detergent Nonidet P-40, the pp-nuclei remain as small, spherical particles which are composed of fine fibrils (Figs. 4a-4e). The isolated pp-nuclei contain a number of polypeptides, only four of which (69, 31, 30, and 14 kDa) are bound to the ppDNA. After treatment with proteinase K or deproteinization, a number of loops of DNA fibrils are also released from the cp-nuclei and the circular DNA is observed (Fig. 4f, g). It is possible to reconstruct the pt-nucleus from the pt-DNA and the relevant polypeptides after dialysis (Nemoto et af., 1989). Therefore, it appears that one to several copies of the ptDNA molecule, which is approximately 53 pm in length, are packed into a pt-nucleus of approximately 0.5 p m in diameter (Fig. 4h). Since the packing ratio of DNA in the metaphase chromosomes in animals is about 140 (DuPraw, 1970), that of the pt-nuclei


may be higher than those of the cell nucleus. This hypothesis is supported by the results of fluorimetry of cellular DNA in the higher plant (Kuroiwa et al., 1990a) and of immunogold electron microscopy in which the number of gold particles per unit area in the cp-nuclei was higher than that in the cell nucleus of the algae (Johnson and Rosenbaum, 1990; Scheer et al., 1987).

From these results, it is concluded that the ppDNAs are not naked in situ but are organized by interactions with some basic proteins to form compact structures called the “pt-nuclei.” This concept should be applicable to the pt-nuclei of other types of differentiated plastid.

IV. Division of Plastids

The concept of organelle nuclei has changed our ideas about the division of organelles themselves. It seems clear that the process of organelle division must be composed of two main events: division of the organelle nucleus and organellekinesis (division of the other components of the mitochondrion or plastid). The latter term has been adopted as an appropriate analogue of cytokinesis.

A. DIVISION OF THE PLASTID NUCLEUS

In general, when the cell volume becomes approximately double during metaphase, the cell divides into daughter cells with an equal division of chromosomes. Then the cell cycle is repeated with a doubling of both DNA and cell volume during the next cell cycle. Similar events may occur in the case of plastids and one might assume that a round of replication of cpDNA occurs during each cycle of chloroplast division in higher plants (Possingham and Rose, 1976; Szmidt et al., 1983; Rose, 1988). However, it

FIG. 3. Isolation of proplastids. (a) Living cells. (b) Protoplasts observed by phase- contrast microscopy (arrowhead indicates a cell nucleus). (c) Fluorescence photomicrograph of a protoplast fixed with glutaraldehyde and stained with DAPI, showing a cell nucleus (CN) and many tiny fluorescent spots from pt-nuclei (large arrow) and mitochondrial nuclei (small arrow) in the cytoplasm (d-i). Part of a protoplast prepared by squashing and observed by DAPI-fluorescence (d, g), DAPI-fluorescence and phase-contrast (e, h), and phase-contrast microscopy (f, i). (d, e, f ) and (g, h, i) are the same fields, respectively. Large and small arrow in (d) indicate a pt-nucleus and a mt-nucleus, respectively. (i-n) Isolated proplastids observed by DAPI-fluorescence (i), fluorescence and phase-contrast (k), phase-contrast ( l ) , and electron microscopy (m, n).(i-l) are the same fields. Large arrows indicate pp-nuclei. a and b, x 330. Bars in c, d, m, and n represent 100,100,l and 0.1 pm, respectively. (From Nemoto er al. with minor modifications, 1988; reproduced by permission of the Japanese Society of Plant Physiologists.)


is difficult to obtain direct evidence to prove this hypothesis because the chloroplast contains amounts of DNA that are too small to examine quanti- tatively by pulse-labeling autoradiography and, furthermore, the chloroplasts do not divide synchronously even within a single cell. Therefore, various phases of the cycle such as plastid GI, plastid S, plastid G2, and plastid M have not been clearly identified.

Division cycles of three different types, namely, CL type, SN type, and CN type, have been studied in detail in plastids. In the CL type, the ring-shaped cp-nucleus segregates into two daughter loops, each of which is transmitted to a daughter chloroplast (Kuroiwa et al., 1981). In the SN type, each of 20-30 small pt-nuclei, which are dispersed throughout the entire chloroplast, divides and the number of pt-nuclei doubles. Subse- quently, equal numbers of pt-nuclei appear to be packaged into daughter chloroplasts (Kuroiwa et al., 1981; Miyamura el al., 1986). In spite of the difference in the location of ptDNA, the pt-chromosomes, which are synthesized during the plastid S phase, must be separated equally into daughter plastids. The CL and SN types of division cycle are observed in chloroplasts of brown algae and in the etioplasts of higher plants, and in chloroplasts of green algae and green land plants, respectively. Since plastid divisions of the CL and SN types have been observed in multiplastidic cells of plants and are often not synchronized, it is difficult to study these division cycles in detail. By contrast, the typical CN type of division cycle is observed in proplastids in the young thalli of algae, in young meristems of leaves (Miyamura et al., 1990), and in chloroplasts of the red alga C. caldarium RK-1.

Since C. caldarium RK-I contains one cell nucleus, one mitochondrion, and one chloroplast (Nagashima and Fukuda, 1981), it is useful for analysis of plastid division cycles. The basic life cycle of C. caldariurn RK-1 is shown in Fig. 5 . If we start with an examination of young cells, we see that each cell contains a cell nucleus of about 0.1 pm3 in volume, a mitochondrion of about 0.06 pm3 in volume, and a chloroplast of about 1 pm3 in volume. Growth of mother cells takes place for up to 50 hours after the

FIG. 4. (a) Pp-nuclei isolated from N. tabacum cultured ceUs (line BY-2) observed by DAPI-fluorescence; (b) DAPI-fluorescence plus phase-contrast; (c) phase-contrast; (d, e) and negative-staining electron microscopy. (f, g) Extracted bauquet of pp-nuclei (f) and circular ptDNA (g) observed by electron microscopy. (h) Circular gene map of the tobacco chloroplast genome and pt-nucleus (arrow). Inverted repeats, I R A and IRB are shown by bold lines. JLA. JKB and J s A and JSB are junctions between a large (LSC) and a small single-copy region (SSC). Genes shown outside the circle are on the A strand and are transcribed counterclockwise. Genes shown inside the circle are on the B strand and are transcribed clockwise. Asterisks indicate split genes. Major open reading frames (ORFs) are included. Bars in a, d, and f represent 10, 1 and 1 pm, respectively. [Gene map (h) (from Shinozaki et al., with minor changes, 1986; reproduced by permission of M. Sugiura.)]


FIG. 5 . Epifluorescence photomicrographs of a cell nucleus (large arrows in a, c), a mt-nucleus (small arrows in a-c), a pt-nucleus (middle arrows in a, c), and a chloroplast (b) in a cell of the red alga Cyunidium cddurium RK-1 cell after staining with DAPI. (a) and (b) are the same field. (c) Diagram of the life cycle of the alga depicting a possible sequence of events based on the observations of cells fixed at various times (hours) after ISC. The chloroplast growth stage: the small, spherical chloroplast increases in volume and becomes a football-like structure. The stage at which formation of the plastid-dividing (PD) ring occurs: the somewhat electron-dense body (SEB) is fragmented into many small somewhat electron-dense granules (SEGs), which are aligned along the equatorial region of the chloroplasts and fine filaments are formed from the SEGs in the equatorial region of the chloroplasts. The fine filaments of the plastid-dividing ring align themselves according to the longest axis of their overall domain. Constriction stage: a bundle of fine filaments begins to contract and generates deep furrow. PD ring conversion stage: after chloroplast division, the remnants of the PD ring are converted into SEGs. Similar events occur during the second cycle of chloroplast division. The divisions of organelle nuclei occurjust before organellekinesis. Cp-S and CN-S indicate the chloroplast DNA-synthetic stage and the cell nuclear DNA synthetic stage, respectively (Mita and Kuroiwa, 1988; reproduced by permission of Springer-Verlag.)


initiation of a synchronous culture (ISC) and these growing cells are mostly four-endospore cells after the second endospore divisions. The divisions of the chloroplast, the cell nucleus, the mitochondrion, and the cell itself occur in that order. The cp-nucleus is located in the central area of the chloroplast; it increases in volume with the growth of the chloroplast and it divides just before chloroplast division. The levels of cpDNA in the chloroplast increase soon after ISC and reach four times the initial value before the first division of the chloroplast. The amount of DNA in the chloroplast decreases stepwise after each endospore division, while the DNA in the cell nucleus is duplicated during each cycle of endospore division (Fig. 5) . Division of the cp-nucleus as well as of the mt-nucleus precedes organellekinesis. The duration of the period of cpDNA synthesis appears to be about 8 hours and the DNA synthesis occurs between 6 and 22 hours after ISC. Similar events have been observed in the chloroplast of another red alga, namely, C. caldarium M-8 of the CL type (Kuroiwa et al., 1989c), in the proplastids in tobacco-cultured cells (Yasuda et al., 1988), and in the leucoplasts in Allium cepa (Nishibayashi and Kuroiwa, 1982). Therefore, we consider that, compared with the division cycle of the cell nucleus, there are two distinguishing features of plastid division: one is an endoduplication of DNA and a greater than 2-fold increase in volume during a single plastid division cycle; the other is the presence of a division cycle without the synthesis of DNA or increase in volume. Stepwise reduction in the volume and DNA content of plastids by division has been observed in the plastids of spermatocytes, during spermatogenesis, in the green alga Bryopsis maxima (Kuroiwa and Hori, 1986), in Charu australis (Sun et ul., 1988), and in the fern Pteris uittata (Figs. 17 and 18; H. Kuroiwa et al., 1988).

In spite of differences in patterns of timing of the synthesis of DNA during the plastid division cycle, pt-chromosomes are basically divided equally among daughter plastids during the division of plastids. However, the molecular mechanism for separation of pt-chromosomes is unclear. An intensive search has been made for the mechanism of segregation of pt-chromosomes since Jacob et al., (1963) first proposed a hypothetical mechanism for the segregation of bacterial chromosomes, namely, that the chromosome is attached to the cell membrane in the region of the replicat- ing fork; indeed, in some species, there is even evidence for additional attachment at the point of origin of replication. The chromosomes may be aided in their separation by the growth of a membrane between these two points of attachment. Rose and Possingham (1976) and Rose (1988) empha- sized the role of the association between DNA and membranes in the segregation of pt-chromosomes. Plastid chromosomes are attached to the thylakoid membrane and, thus, can be separated equally into daughter plastids by an elongation of the binding sites that is accompanied by


growth of the thylakoid membrane system. Rose (1988) proposed an interesting model in which segregation of pt-chromosomes is related to thylakoid membrane systems in dividing chloroplasts, etioplast. and proplastids with various types of cp-nucleus, for example, the SN and PS types. However, there is no direct evidence showing such a specific association for the segregation.

Kawano and Kuroiwa (1985) isolated a membrane-DNA complex by centrifugation of a sheared lysate of isolated mitochondria from Physarum polycephalum. Analyses by Hoechst 33258/CsC1 density-gradient centrifugation and restriction-endonuclease treatment of the complex showed that DNA in the complex was richer in A-T base pairs than the total mtDNA, and contained the specific EcoRI fragment (E-8), which was localized on the right-hand side of the Physarum mt-genome. The sequence of the 1 kbp from the E-8 showed that the region contains an attachment site for topoisomerase, inverted repeats, stem loops, and tan- dem repeats (S. Kawano, personal communication).

In an attempt to examine whether or not specific regions of cpDNA are involved in interaction with spinach thylakoids by use of restriction endonucleases (Lindbeck and Rose, 1987), cpDNAs from vesicles and chloroplasts were found to have similar restriction patterns and all cpDNA sequences were represented in the vesicle-bound cpDNA. This result suggests that all cpDNA sequences are represented in the vesicle-bound cpDNA. The simplest explanation for these data is that random DNA sequences are responsible for the DNA-membrane interactions. By contrast, Nemoto et al. (1991) recently obtained evidence that specific sites on the cp-chromosomes in two species of N. tabacum were bound to a complex that consists of thylakoid membrane and proteins. When isolated, intact cp-nuclei were digested by the restriction enzyme, EcoRI. The pattern of the restriction fragments was different from that obtained by direct digestion of the purified DNA: a few fragments containing the specific EcoRI fragment designated E-2 were preferentially deleted (Fig. 6a). The result suggests that, when specific regions were associated to form a complex which was composed of the membrane system and specific proteins, they were not digested by the restriction enzyme. This hypothesis was confirmed by the following experiments. When the complexes were treated with SDS or proteinase K, their restriction patterns were similar to those of total purified DNA. Nemoto et al. (1991) showed that at least four regions (the region between IRF 168 and rpo B in LSC; the region between rps 16 andpsb A in LSC; and the regions between rps 7 and 23 S in inverted repeats) in the circular genome (Figs. 4h and 6b) bind to membranes. The region containing psa A is stronger in binding capacity than the other regions. However, when intact pp-nuclei isolated from tobacco-cultured cells were treated with restriction enzymes, their frag-


FIG. 6. Analysis of DNAs by agarose gel electrophoresis after cpDNA, cp-nuclei, and pp-nuclei were digested with EcoRI. Lane M, Hind111 fragments of lamda phage DNA as molecular-size markers; Lane I , cpDNA; lane 2, cp-nuclei; lane 3, cp-nuclei treated with SDSlproteinase K; lane 4, cp-nuclei treated with SDS; lane 5 , cp-nuclei treated with proteinase K; lane 6, cp-nuclei treated with RNase A; lane 7, cp-nuclei; lane 8, pp-nuclei treated with SDS/proteinase K; lane 9, pp-nuclei; lane 10, pp-nuclei treated with SDS/proteinase K. Cp-nuclei were isolated from N. rabacum (line BY-Z)(lanes 2-6) or from Xanthi Nc (lanes 8,9). Pp-nuclei were isolated from N. tabacum cultured cells (line BY-2). Arrowhead shows the position E-2 of the restriction fragment that appears to bind tightly to protein in cp-nuclei but not in pp-nuclei. (From Nemoto et al., 1990; reproduced by permission of the Japanese Society of Plant Physiologists.)

ment patterns were similar to those of purified DNA (Fig. 6). There is, as yet, no direct evidence for an association between a specific part of the pt-chromosome and a membrane system that is a prerequisite for the separation of pt-chromosomes. If there is such an association, the weak binding at one or all of three binding regions may be related to the separation of pt-chromosomes.


B. PLASTIDKINESIS BY THE PLASTID-DIVIDING RING

The mitochondria of P. polycephalum contain electron-dense, rod- shaped mt-nuclei, and they are therefore particularly suitable for investi- gations of mitochondrial division (Kuroiwa et al., 1977). When a small explant of a plasmodium was incubated in a solution that contained cytochalasin B, a large number of mitochondria did not exhibit the dumbbell shape but developed a large spherical or ovoid configuration (Kuroiwa and Kuroiwa, 1980). It is likely that cytochalasin B disrupted the function of actin such that mitochondria failed to form dumbbells. However, we have been unable, as yet, to discern any fine structures that are intimately related to mitochondriokinesis.

Since plastids are considerably larger than mitochondria and contain more highly developed membrane systems, a bulkier apparatus may be required for plastidkinesis than is required for mitochondriokinesis. In the plastids of land plants, it is difficult to clarify whether or not the dumbbell- shaped plastids in higher plants are dividing plastids, because dumbbell- shaped plastids are not always dividing plastids in living cells (Whatley, 1980). Thus, it is difficult to observe the behavior of the plastid-dividing (PD) ring during the division of plastids in multiplastidic land plants. It has proved advantageous for analyses of the apparatus involved in chloroplast division to use synchronous cultures of the monoplastidic cells of C. caldariurn RK-1. Mita et al., (1986) were the first to identify PD ring located in the cytoplasm of the alga.

Synchronous cultures of C. caldarium RK-1 can be initiated from young cells. The number of cells increases stepwise after ISC and finally reaches a value of about 4 times the initial number. Growth of mother cells takes place up to 50 hours after ISC and these growing cells are mostly four- endospore cells after the second endospore divisions (Fig. 5 ) . In control preparations, when the mother cells are fixed at 36 hours after ISC and excited with UV after staining with DAPI, four spherical cell nuclei and four irregularly shaped ct-nuclei, emitting blue-white fluorescence, can be seen (Fig. 7a). When the same field of such cells is excited with green light, four chloroplasts, emitting red autofluorescence, can clearly be seen in the areas where the ct-nuclei are located (Fig. 7a). When cells are exposed throughout two sequential endospore divisions to cremart, an inhibitor of the assembly of tubulin, each mother cell can be seen to contain one cell nucleus and four chloroplasts (Fig. 7c, d). By contrast, when the cells are exposed throughout two sequential endospore divisions to cytochalasin B, an inhibitor of the polymerization of actin into filaments, each mother cell can be seen to contain four cell nuclei and one large chloroplast (Fig. 7e, f). These results suggest that microtubules are not involved in the division of the chloroplast but are involved in the division of the cell nucleus, while


FIG. 7. Epifluorescence photomicrographs illustrating cell nuclei (CN 1-4) and the chloroplast (CP) in mother cells C. caldarium RK-1 fixed immediately after incubation for 30 hours without any inhibitor (a, b); and after incubation for 30 hours with cremart at 10 pg/ml (c,d). Fluorescence photomicrographs with UV light (a, c, e) and green tight (b, d, f ) were taken in the same field for each treatment. In the control (a,b), one mother cell is composed of four discrete endospore cells, each of which contains a cell nucleus and a chloroplast. When cells are treated with cremart through two sequential endospore divisions, the mother cell contains one cell nucleus and four chloroplasts (c, d). By contrast, when the cells are treated with cytochalasin B, the mother cell contains four cell nuclei and one large chloroplast (e, f). Bar = 1 pm. (Mita and Kuroiwa, 1988; reproduced by permission of Springer-Verlag.)


polymerized actin filaments are not involved in division of the cell nucleus but play an important role in the division of chloroplasts and in cytokinesis.

When cells of C. caldarium are stained with rhodamin-conjugated phalloidin, a fluorescent dye that is specific for actin, a ring that emits orange- colored fluorescence appears faintly in the equatorial region of the dividing chloroplast. However, such a result does not exclude the possibility that the ring may correspond to the cytoplasmic contractile ring involved in cytokinesis.

In synchronized cells of C. caldarium RK-1, it is possible to observe plastidkinetic events and fine structures in detail, from the formation of the PD ring during the early stages to the disapperance of the PD ring during the late stage, as summarized in Fig. 5. When the cell, the cell nucleus, and the chloroplast increase in volume about 3, 2, and 3.5-fold, respectively, the shape of the chloroplast changes from a spherule to a football-like structure and concentric, circular, thylakoid membranes in the chloroplast begin to separate into two parts. At that time, many somewhat electron- dense granules (SEG), each 40-90 nm in diameter, and electron-dense deposits appear in the cytoplasm close to the outer envelope membrane and begin to be distributed at the equatorial region of chloroplast (Mita and Kuroiwa, 1988). The PD ring is made up of SEG. A portion of the PD ring can be seen as a bar, about 60 nm in width, at the edge of the PD ring. The bar consists of fine filaments, each about 5 nm in diameter, which are aligned parallel to the longitudinal direction of the bar. The arrangement indicates that the PD ring is a bundle of fine filaments. By the time the PD ring starts to contract, the small SEGs have completely disappeared. When sequential thin sections are cut through the constricted isthmus of a dividing chloroplast of the alga during the middle phase of division, it appears that the electron-dense deposits at the bridge between daughter chloroplasts are distributed as a close ring or beltlike structure, 60 nm wide and 50 nm thick, lying in close apposition to the outside of the outer envelope of the chloroplast. At the final stage, the width of the deposits that make up the constricted PD ring appears to be somewhat greater than that of the deposits at the early and middle stages of chloroplast division (Fig. 8a,b). However, the width of the PD ring does not deviate very much from the cited value of 60 nm, regardless of the stage of division or the steepness of the walls of the furrow between the daughter chloroplasts, nor does it vary much among chloroplasts of vastly different volumes. At higher magnification, the cross sections of the PD ring clearly reveal that the PD ring is located on the cytoplasmic side of the outer envelope (Fig. 8b,c). Inside the inner envelope, some electron-dense deposits can also be seen, but their width and thickness do not change from the early stage of

FIG. 8. Electron micrographs of sections cut in directions perpendicular to (a, b) and parallel to (c), the plane of division at the constricted isthmus of dividing chloroplast in C. culdurium RK-I. When the chloroplast is progressively pinched, the PD ring becomes more electron dense and is seen to increase in thickness (arrow in a). At higher magnification, the PD rings can be seen on the cytoplasmic side of the outer envelope membrane (arrows in b and c), while the inner electron-dense belt (arrowheads in b and c) does not change from what is observed at the early phase of the division. Bars = 0.1 pm. (Mita and Kuroiwa, 1988; reproduced by permission of Springer-Verlag.)


chloroplast division until it is complete. Similar events can be seen in the second division of the chloroplasts. The PD ring, when visualized in sections cut in a direction parallel to the plane of division, is clearly made up of a circular belt that appears to be composed of tightly packed, fine filaments. When the chloroplast has ceased dividing, a centriolelike plaque with microtubules develops outside the cell nucleus and electron-dense deposits, which consist of actinlike filaments and are related to cytokinesis, appear beneath the cell membrane.

Mita and Kuroiwa (1988) proposed that the main components of the PD ring are actinlike filaments on the basis of the following pieces of evidence: (i) Cytochalasin B inhibits the division of chloroplasts without inhibiting division of the cell nucleus; (ii) the ringlike structure around the chloroplasts can be stained with rhodamin-conjugated phalloidin; and (iii) fine filaments, observed in the PD ring, are very similar in diameter to actin filaments. However, such a proposal is not supported by the results of immunogold-staining experiments with actin-specific antibodies: a few gold particles, which showed the localization of actin, were found on the SEGs and none were found on the PD ring (Mita and Kuroiwa, 1988).

A cytoplasmic PD ring, like that in C . caldarium, was also observed around the plastids of a green alga that was the green alga Trebouxia (Senda and Ueda, IW), in Pyrarninornonas uirginica (T. Hori, personal communication), and in the moss Funaria hygrometrica during the division of plastids, but no inner matrix ring was observed in any of these cases. In the green alga P. uirginica, the division of the chloroplast with a large pyrenoid occurs after the cell nucleus division and immediately before cytokinesis (Fig. 9a, b, d). The PD ring appears to generate the contractile force that is involved in the division of the chloroplast with a pyrenoid (Fig. 9). In the chloronema and caulonema of the moss F. hygrometrica, Tewinkel and Volkmann (1987) observed a distinct filamentous structure similar to the PD ring in the plane of division outside the plastids, but close to the envelope, in three-dimensional reconstructions prepared from electron micrographs. The PD ring was also visible around the narrow isthmus of dividing chloroplasts and amylopiasts during the late phase of plastid division. The cross-sectioned filamentous structures were 10-40 nm in width and 10-15 nm thick and ran parallel to the outer envelope at a distance of about 10 nm.

In higher plants, Suzuki and Ueda (1975) and Luck and Jordan (1980) reported the appearance of electron-dense material, which they considered to be evidence of “buffles” and a “septum,” respectively, at the constricted isthmus between daughter proplastids and daughter amyloplasts in Pisum satiuum and Hyacinthiodes nonscripta. Similar electron- dense deposits have been observed at the narrow neck of dumbbell-shaped


FIG. 9. Electron micrographs of sections cut in directions perpendicular to the plane of division at the constricted isthmus of a dividing chloroplast in the green alga Pyraminomonas virginica (a, b) and in A. satiuum (c). d and e show two models of a single PD ring of the C . caldarium type ( arrow in d) and a PD ring doublet with an outer ring (large arrow) and an inner ring (small arrow) of the A. satiuurn type (e), respectively. Bars = 0.1 p n . [Photo- graphs (a, b) courtesy of Dr. T. Hori, Tsukuba University, Japan; (c) reproduced by permission of H. Hashimoto and Springer-Verlag.]

chloroplasts and have been described as "fuzzy plaques" in Triticum aestivum, Atriplex semibaccata, and Sesamum indicum var. glauca (Leech et al., 1981). Fuzzy plaques of electron-opaque material were frequently, but not always, seen covering or displacing the membranes of the isthmus (Leech et al., 1981). Such early observations were made from individual thin sections. Subsequently, Chaly and Possingham (1981) sur-


veyed the deposits located at the constricted isthmus between daughter chloroplasts in various plants, such as P . sativum, Phaseolus vulgaris, Lycopersicon esculentum, Lactuca sative, Citrullus lanatus, Hordeum vulgare, Z . mays, T. aestivum, and Pisum radiata, using serial sections and they concluded that electron-dense deposits, which were parts of an annulus, were located in the interspace between the outer and inner envelope membranes at the constricted isthmus of dumbbell-shaped proplastids of these plants. To explain the formation of the “fuzzy plaque” or “annulus,” Leech et al. (1981) and Possingham and Lawrence (1983) applied to plastids the theoretical model of cell division proposed by Greenspan (1977), namely, that the internal fluid flow generated by surface changes leads to a concentration of material in the equatorial region and to formation of an annulus. They suggested that the electron-opaque material might be present, but diffuse at earlier stages of division and only become visible when sufficiently concentrated within a narrow constriction. However, in earlier observations of higher plants, there is no definite evidence to indicate that dumbbell-shaped plastids are dividing plastids. By contrast, Hashimoto (1986) observed the presence of an electron-dense double ring structure (PD ring doublet) around the constricted isthmus of dumbbell-shaped plastids of Avena sativum, using a serial thin-sectioning technique. The inner and outer rings of the doublet were reported to coat the inside (stromal side) of the inner envelope membrane and the outside (cytoplasmic side), respectively (Fig. 9c, e).

There are discrepancies in the interpretations of the localization of the PD ring at the constricted isthmus, as observed in different species and in the same species of higher plants. Therefore, Kuroiwa (1989a) examined the localization of the PD ring of higher plants using the serial thin- sectioning technique. The localization of the PD ring in proplastids of N . tabacum was found to be similar to that of the PD ring doublet reported by Hashimoto (1986). A similar PD ring doublet was reported in spinach, bean, tobacco, and wheat by Oross and Possingham (1989). T. Hori (personal communication, 1990) observed a PD ring in plastids of the gymnosperm Ginkgo biloba. It is likely that the cytoplasmic PD ring is to be found in plastids of red, brown, and green algae, in mosses and ferns, and in gymnosperm, while the clear PD ring doublet is to be found in all angiosperms if a careful search is made. The PD ring doublet has also been seen in a mutant deficient of the monocot that is deficient in plastid ribosomes (Hashimoto and Possinham, 1989). The observation does not conflict with the hypothesis that the PD ring consists of an actinlike protein, which must be encoded in the cell nuclear chromosomes. The plants in which electron- dense deposits, an annulus, or a PD ring have been observed to date are summarized in Table I.

TABLE 1 OBSERVATIONS OF ELECTRON-DENSE MATERIALS AT CONSTRICTED ISTHMUS OF DUMBBELL-SHAPED PLASTIDS AND PLASTID-DIVIDING RINGS IN

VARIOUS PREPARATIONS OF PLANT MATERIAL ~ ~~ ~~ ~ ~~

Species Tissue Plastid Stage Name or type Distribution Ref.

Red algae Cyanidium caldarium RK-1

Green algae Trebouxia sp.

Pyrarninomonas virginica

Moss Funaria hygrometrica

Higher plats Ginkgo biloba Avena sativa Nicotiana tabacum Spinacia oleracea

Phaseolus vulgaris

Nicotiana tabacum

Triticum aestivum

Single cell

Single cell

Single cell

Protonema

Sperm First leaf Cultured cell Leaf Root Leaf

Leaf

Leaf

Chloroplast Early PDring Cytoplasm Mita ef al. (1986) Middle Mita and Kuroiwa (1988) Late Kuroiwa (1989a)

Chloroplast Middle PDring Cytoplasm Senda and Ueda (1990)

Chloroplast Middle PD ring Cytoplasm T. Hori (1989)" Late

Late

Chloroplast, amyloplast Late PD ring Cytoplasm Tewinkel and Volkmann (1987)

Proplastid Proplastid, chloroplast Proplas tid Chloroplasts Proplastid Chloroplasts

Chloroplasts

Chloroplasts

Late PDring Cytoplasm T. Hori (1990)" Late PD ring doublet Cytoplasm, matrix Hashimoto (1986) Late PD ring doublet Cytoplasm Kuroiwa (1989a) Late PD ring doublet Cytoplasm Oross and Possingham

Late PD ring doublet Cytoplasm Oross and Possingham



( 1989)

( 1989)

(1989)

( 1989)

(continued)

TABLE I Continued

Species Tissue Plastid Stage Name or type Distribution Ref.

Pisum sativum Hyacinthiodes non-scripta Triticum aestivum Sesamum indicum

Pisum sativum

Phaseolus vulgaris

Lycopersicon esculentum

Lactuca saliva

Citrullus lanatus

Hordeum vulgare

Zea mays

Triticum aestivum

Pinus radiata

Root tip Pollen Root tip Root tip

Root tip

Root tip

Root tip

Root tip

Root tip

Root tip

Root tip

Root tip

Root tip

Proplastid amyloplast Plastid (proplastid) Proplastid Proplastid

Proplastid

Proplastid

Proplastid

Proplastid

Proplastid

Proplastid

Proplastid

Proplastid

Proplastid

Late Late Late Late

Late

Late

Late

Late

Late

Late

Late

Late

Late

Septum Septum Fuzzy plaque Annulus

Annulus

Annulus

Annulus

Annulus

Annulus

Annulus

Annulus

Annulus

Annulus

Plastids Plastids Plastids Plastids (interspace)

Plastids

Plastids

Plastids

Plastids

Plastids

Plastids

Plastids

Plastids

Plastids

Suzuki and Ueda (1975) Luck and Jordan (1980) Leech et al. (1981) Chaly and Possingham

Chaly and Possingham




Chdy and Possingham





(1981)

(1981)

(1981)

(1981)

(1981)

(1981)

(1981)

(1981)

(1981)

(1981)

a Personal communication.


When does the cell nucleus generate the information that induces the division of plastids? Kamata et al. (1989) caused matured mesophyll protoplasts of tobacco to fuse with protoplasts from cultured cells by electric fusion. When the fusion products were cultured for 2 days, the division of chloroplasts was observed in the heterokaryocytes, while such division of chloroplasts was not observed when mesophyll protoplasts alone were cultured under the same conditions. Since the matured mesophyll cells never divide in the leaf (whereas the cultured cells multiply regularly), these results suggest that proliferating cells may synthesize an unknown substance that induces the division of chloroplasts.

Many cytologists accept the hypothesis that plastidkinesis occurs by partition of the inner limiting membrane (Modrusan and Wrischer, 1990). However, more data must be gathered before this mode of division is confirmed. (i) Each step of plastid division has not yet been studied by the serial thin-sectioning technique. (ii) The complete step-by-step division of a plastid, accompanied by division of pt-nuclei by partition, has never been observed. (iii) A quantitative analysis of the number of plastids before and after the division of plastids by partition has not yet been made in detail. In cells of algae and higher plants, the majority of plastids divide by constriction, but about 1% or even fewer of the total dumbbell-shaped plastids examined appear to have been partitioned at the center by the inner limiting membrane. However, three-dimensional reconstructions created from serial sections have indicated that, in alI the partitioned plastids examined, the two daughter progeny were actually still connected to each other by a channel of plastid matrix. Then the invagination of the inner limiting membrane was cut in a direction perpendicular to the invagination, an image of the partition was seen at the equatorial region of the dumbbell-shaped plastids. As the invagination of inner limiting membrane often occurs in a direction perpendicular to a cross section of the stack of grma and thylakoid membranes in the chloroplast, the direction of invagination can be judged easily by reference to the morphological characteristics of the grana.

V, Differentiation of Plastids

‘The electron microscopic approach to differentiation of plastids was initiated in the late 1950s. Miihlethaler and Frey-Wyssling (1959) suggested that proplastids, etioplasts, and chloroplasts arise from smaller organelles termed “plastid initials” (0.002-0.05 pm in diameter) which are present in leaf buds and leaf meristems of a number of higher plants. With new and improved methods for fixation for electron microscopy, it is now possible


to distinguish between most of the organelles in meristematic tissue and there have been no recent observations of “proplastid initials” (Pos- singham and Lawrence, 1983). With the advances in DAPI staining and epifluorescence microscopy, the number of copies of ptDNA per plastid is easily counted. Such studies have shown that there are variations among proplastids, which can be conveniently classified into at least two types (Fig. 2; Kuroiwa et ul., 1981). One type of proplastid is characterized by the presence of one to two copies of the plastid genome per plastid of about 1 p m in diameter, and by the presence of a single pt-nucleus per plastid. The other type is characterized by several copies of the pt-genome per plastid of about 2-3 p m in diameter and about 2-5 pt-nuclei per plastid. Herein, the latter will be referred to as “proplastids” (pp) to distinguish them from the morphologically less complex and smaller “pp-precursors” characteristic of the former type. Often these two types of proplastids were mixed in one cell as seen in tobacco culture cells (Yasuda et a/., 1988).

Typical morphological changes in pt-nuclei during the development of chloroplasts of monocots and dicots are shown in Figs. I and 2. The pp-precursor in higher plants contains only one small, spherical pt-nucleus and can divide according to the pp-division cycle 1 (Fig. 2). When the pp-precursors develop into proplastids, and then into etioplasts, if growth takes place in the dark the pt-nucleus becomes cup shaped with concomitant endoduplication of ptDNA, and is often found to be associated with starch grains or the prolamellar body (Kuroiwa et al., 1981). Since the association between the pt-nucleus and starch grains occurs commonly in various plastids, such as etioplasts, chloroplasts, and amyloplasts, it must play an important role in an as yet unknown way. The proplastids and etioplasts can also divide according to the proplastid division cycle 2 and the etioplast division cycle, respectively. In etioplasts of monocots, the pt-nucleus becomes a ring-shaped structure (Sellden and Leech, 1981; Hashimoto, 1985; Miyamura et al., 1986). Once etioplasts have been illuminated, the pt-nuclei begin dividing into 20-30 small, spherical pt- nuclei, which are distributed individually into mature chloroplasts. During greening of pea leaves, the synthesis of proteins in the plastids may be a prerequisite for the dispersion of pt-nuclei into an entire plastid since such dispersion does not occur after addition of the inhibitors chloramphenicol and lincomycin (Fig. 13; Sasaki and Kuroiwa, 1989).

The changes in numbers of copies of ptDNA have been examined by biochemical techniques and cytochemical fluorimetry. In Brussica, Kur- oiwa et af. (1981) showed qualitatively, by staining with DAPI, that the DNA content per plastid increased markedly during the division cycle of proplastids and during the development from proplastids to etioplasts, but


the DNA content increased only slightly after illumination, even though the cp-nucleus divided into small, scattered cp-nuclei. Miyamura et al. (19:36) examined the fluorescence intensity of each cp-nucleus by use of VIlvlPICS and showed clearly that the number of copies of ptDNA increased approximately 8-fold during the division cycle of plastids and during the differentiation of the proplastid to the etioplast; the number of copies reached a plateau when the chloroplasts began to engage in photosynthesis. As a result of the division of young chloroplasts according to the chloroplast division cycle, a 10-fold increase in the number of chloroplasts per cell occurs in spinach leaves and a 2- to 3-fold increase occurs in wheat leaves. This process is, therefore, important in determining the photosynthetic potential of the mature leaf(Leech, 1976). Plastids of both epidermal and palisade cells of P. vulgaris also divide at all stages of plastid development, but division ceases soon after the plastids become mature (Whatley, 19110). The matured chlorophyll-containing chloroplasts of red, brown, and green algae and the green leaves of land plants exist in a specialized form for photosynthesis and the fixation of carbon, using the energy of the sun, and synthesize amino acids as precursors of various proteins. However, when &he disks of young green leaves of spinach are dissected and grown in sterile culture, the division of chloroplasts continues and the number of chloroplasts per cell increases 5- to 10-fold over a 7-day culture period in the light. There is virtually no cell division during this time (Rose er a/ . , 1 974).

Since the synthesis of cpDNA occurs in mutants that are deficient in chloroplast ribosomes (Hermann et al., 1974; Scott etal., 1982), in chloroplasts without substantial portions of the cp-genome (Day and Ellis, 1984) as well as in plastids that have ceased to synthesize proteins in the presence of inhibitors (Sasaki and Kuroiwa, 1989), the synthesis of cpDNA may not be related to the genome of chloroplasts themselves. By contrast, the synthesis of cpRNA is dependent on the genome of chloroplasts (Cozens and Walker, 1986; Shinozaki et al., 1986; Zaitlin et al., 19139; Hu and Bogorad, 1990). The mature chloroplasts of the CL, PS, and SP type can also develop from proplastids that contain only one pp- nucleus throughout several divisions (Fig. 2).

[n addition to differentiation from proplastids to chloroplasts (Figs. 1 an'd 2), proplastids are also able to differentiate into functional plastids of various forms, such as amyloplasts, elaioplasts, and chromoplasts, according to the differentiation of tissues. Small amyloplasts occupied by starch grains can be seen during the differentiation from proplastids to etioplasts in leaves of angiosperms, during embroygenesis, and during the formation of pollen grains, egg cells, central cells, and root caps. Spe- cialized large amyloplasts are filled with large starch grains and can be


observed in the cells of storage tissues, such as the cotyledons of dicots, endosperms of monocots, and tubers of potato. Their function is to accu- mulate starch as a reserve material. Elaioplasts, which are found in the epidermal cells of some monocots, are plastids that are largely filled with oil. Chromoplasts are carotenoid-containing plastids responsible for the colors of fruits and flower petals. As seen in the fruits of tomato and pepper, the chloroplasts are transformed into the chromoplasts during maturation of fruits. In general, the numbers of pt-nuclei and the DNA content per plastid are smaller in amyloplasts, chromoplasts, and elaioplasts than in chloroplasts.

In addition to the cytological studies, some researchers have tried to elucidate the molecular mechanism of plastid differentiation. The proteins encoded by ptDNA are involved in transcription, translation, and photosynthesis. Therefore, the differentiation of chloroplasts requires selective activation of pt-gene expression and selective translation of pt-genes. The modes of chromoplast gene expression during the development of fruit and of gene expression in amyloplasts have been examined in comparisons with those operating in chloroplasts (Bathgate et a / . , 1986; Piechulla et al., 1986; Ngernprasirtsiri et al., 1988a, b). For example, Ngernprasirtsiri et al. ( 1988a) proposed that the amyloplast genome is mostly inactive with the exception or the gene for 16 S rRNA and psb A, which are presumably regulated at the transcriptional level. Several workers (Sasaki and Kur- oiwa, 1989; Sasaki et d., 1990) have also shown that the photogenespsb A, psb B, and rbc L are active in green tissues, such as leaves and stems, but are inactive in noncolored root tissue, while the housekeeping genes are active in many tissues including noncolored roots (Fig. 10a). Sasaki et al. (1990) showed that variations in the relative levels of transcripts of the photogenes in different organs were similar to the variations in gene dosage, but those in levels of the transcripts of ribosomal protein L2 were not. They proposed that gene dosage affects the organ-specific expression of photogenes. Baumgartner et al. (1989) showed that plastid transcriptional activity and numbers of copies of ptDNA increase early in chloroplast development, and they suggested that transcriptional activity per DNA template varied up to 5-fold during the biogenesis of barley leaf.

Since the physical restriction map of cpDNA is basically similar to those of other differentiated plastids such as amyloplasts (Macherel et ai., 1985; Scott et al., 1984; Ngernprasirtsiri et al., 1988a), chromoplasts (Hansmann et al., 1985), and proplastids (Nemoto et al., 1988; Fig. lob), it is difficult to explain the mechanism of plastid differentiation at the structural levels of DNA molecules. The transcriptional and posttranscriptional regulations are considered to control protein synthesis in plastids (Gruissem et al., 1988). If there are at least two regulatory systems, the system based on


FIG. 10. (a) Northern analyses of the total RNA in various tissues from P. satiuum plants. Three-microgram samples of total RNA were loaded on gels from which each autoradiogram was prepared. Sp, specific activity of the probe (10' cpm/pg); Ex, exposure time for autoradiography (hours); Se, seeds; L, leaves; S, stems; P, petals. 16 S rDNA, gene for 16 S ribosomal RNA; psb A, gene for the p700 apoprotein Al of photosystem I; psb B, gene for p700 apoprotein A2 of photosystem I; rbc L, gene for the large subunit of RuBisCO; psb E, gene for the 8-kDa subunit of cytochrome b 559; atp A, gene for a subunit of H'-ATPase subunit; rpl2, gene for the 50 ribosome subunit CL 12; pet A, gene for cytochromefin the cyiochrome blfcomplex; rbc S, gene for the small subunit of RuBisCO; Cab, gene for LHCP 11. (From Sasaki et al., 1990; reproduced by permission of the Japanese Society of Plant Physiologists); (b) Patterns after electrophoresis on agarose gels of DNAs from cp-nuclei (lanes C) and pp-nuclei (lanes P) isolated from N. tabacum BY-2 digested by three restriction enzymes, namely, EcoRI, HindIII, and BamH I. (From Nemoto et al., 1990; reproduced by permission of Japanese Society of Plant Physiologists.)


transcriptional regulation must be more active than that based on posttranscriptional regulation during the early and middle period of chloroplast development, while the posttranscriptional regulation may be more important after maturation of chloroplasts, since cpDNAs are digested soon after maturation (Sodmergen et al., 1989; 1990).

Two approaches have been developed to elucidate the molecular mechanisms of regulation at the level of transcription, as related to plastid differentiation. One is based on the physical changes in ptDNA itself and the other on changes in proteins that bind to ptDNA.

A. METHYLATION OF PLASTID DNA Ngernprasirtsiri et al. (1988b) analyzed methylation of ptDNA from

fully ripened red fruits, green mature fruits, and green leaves of tomato. They found from Southen blots that no methylation could be detected of DNA fragments that contained certain genes that are actively expressed in chloroplasts, whereas DNA fragments of genes that are barely transcribed in chromoplasts were methylated. In addition Ngernprasirtsiri et al. ( 1988a) also examined gene expression of amyloplast DNA in the hetero- trophically grown white cells of sycamore as compared with expression of the cpDNAs isolated from the green mutant cells. They demonstrated that the cp-genes in which methylation was not detectable were active as templates for transcription in uitro by RNA polymerase from Escherichia coli, but the methylated amyloplast genes were apparently inactive. They proposed that methylation of DNA is a likely mechanism for the regulation of expression of amyloplast DNA in sycamore cells. By contrast, it has been reported that both amyloplast and chloroplast genes in P. satiuum are methylated and chromoplast DNA during tomato fruit ripening (Marano and Carriilo, 1991) is not methylated (N. Ohta et al., unpublished data). We cannot yet explain the discrepancy between methylation of cytosines in ptDNA and gene expression in plastid.

B. PLASTID DNA-BINDING PROTEINS In the early 1980s some workers began to consider the role of proteins

that bind to cpDNA, and the concept of “organelle nuclei” (Kuroiwa, 1982) increased in importance.

Based on an analogy with mt-nuclei (Kuroiwa, 1973; 1974; 1982) and the isolation of pt-nuclei, the possibility that the ptDNA is not naked but is organized in situ by some proteins to form a compact structure has been claimed by many investigators (Kuroiwa et al., 1981; Kuroiwa and Suzuki, 1981; Briat et al., 1982; Reiss and Link, 1985; Hansmann et al., 1985). If this is indeed the case, then intact pt-nuclei, and not ptDNA, must be


isolated and their components must be characterized in terms of both structure and function. Hallick et al. (1976) and Briat et al. (1979) isolated a DNA-protein complex from chloroplasts of Euglena and spinach, respectively, and showed that they retained their transcriptional activity. Yoshida et al. (1978) isolated a looped and folded structure of cpDNA from spinach which remained even after a vigorous deproteinization. These results conflict with the later observation that the cp-nuclei isolated from several plants become swollen and are easily dispersed by deproteinization. Kuroiwa et al. (1981) found by DAPI-fluorescence microscopy that the cp-nuclei of Nitella become dispersed in situ upon treatment with proteinase K, a result which suggests that the cpDNAs are organized into cp-nuclei by some proteinaceous components. Furthermore, in an attempt to avoid contamination from spherical fragments of celI nuclei and mt- nuclei, Kuroiwa and Suzuki (1981) succeeded in isolated ring-shaped cp- nuclei, with their structures intact, from the brown alga E. indicus. Results of enzymatic treatment again indicated that the ring-shaped structure of the cp-nuclei was maintained by some proteins. However, none of these earlier studies included a biochemical analysis of any proteinaceous components associated with the ptDNAs.

Briat et al. (1982) examined the transcriptionally active DNA-protein coniplexes from spinach chloroplasts by electron microscopy and also analyzed the acid-soluble polypeptides contained in the complexes. They showed that some basic and low-molecular-weight proteins were present in the preparation. Reiss and Link (1985) compared similar DNA-protein coniplexes from etioplasts and chloroplasts of Sinapis alba and showed that they contained common as well as unique polypeptides. Meanwhile, Hatismann et al. (1985) succeeded in isolating cp- and chromoplast nuclei in a more condensed form from leaves and coronae, respectively, of Narcissus pseudonarcissus. They demonstrated characteristic differences between the polypeptide patterns of the two types of pt-nucleus as well as several polypeptides common to both. However, these studies did not reveal which polypeptides actually bind to ptDNA. It is likely that isolation of pt-nuclei (with their compact structures intact) by using a mild detergent might solve the problem of contamination by the membrane frac:tion which is not solubilized under such conditions. Therefore, methods must be developed to distinguish the proteins that are directly involved in the organization of ptDNA from membrane proteins that are unrelated to the pt-nuclei.

Proplastids are very suitable for the isolation of pt-nuclei from organelles. Nemoto et al. (1988) succeeded in identifying four polypeptides (69, 31,30, and 14 kDa) that were bound to ptDNA in the pt-nuclei by combin- ing DAPI-fluorescence microscopy with biochemical techniques. For


studies of the interactions between the ppDNA and the DNA-binding proteins in more detail, neutral salts seem to be the most useful among various agents, since they can disrupt the DNA-protein interaction so gently that their effects can be reversed by removal of salt by dialysis. The organization of cell nuclear chromatin has been studied intensively by reconstituting the structure of nucleosomes by dialysis of purified DNA with purified histones, which had previously been dissolved in 2 M NaCI, against decreasing concentrations of neutral salt (McGhee and Felsenfeld, 1980; Igo-Kemenes et al., 1982). Nemoto et a f . (1989) examined the effects of various concentrations of NaCl on the compact structure of pt-nuclei using DAPI-fluorescence microscopy and, at the same time, they investi- gated the behavior of the four proplastid DNA-binding proteins by SDS- PAGE after centrifugal fractionation. They found that the pp-nuclei, after dispersion by 2 M NaCl, could be organized once again into compact structures similar to those of the original pp-nuclei by a reduction in the salt concentration. Furthermore, the four DNA-binding proteins from proplastids, even though they had been dissociated from ppDNA by the treatment with 2 M NaC1, were found to have reassociated with ppDNA in the reassembled pp-nuclei. These results strongly suggest that the DNA- binding proteins in proplastids play a critical role in the organization of the compact pp-nuclei. Recently, Nemoto et al. (1989, 1990) isolated cp-nuclei from the chloroplasts in N . tabacum (Fig. 11) and compared their DNA- binding proteins with those of proplastids (Fig. 12). Major polypeptides, which were present in pp-nuclei, were not found in cp-nuclei, a result that suggests that the population of DNA-binding proteins changes during the differentiation of plastids (Figs. 12 and 13).

It seems feasible that certain special regions of the mt- and pt- chromosomes are involved in interactions with the respective membrane systems. There must be more than three roles for membrane-bound ptDNA.

In addition to a role in DNA replication, a role in the separation of daughter pt-chromosomes, and a role in RNA transcription, there must be a role for such interactions in the dispersion of the clumped pt- chromosomes during greening (Figs. 2 and 13). It has been shown in chloroplasts of N . tabacum that binding of two types occurs: there are weak and strong associations between ptDNA and the membrane system (Figs. 6 and 13). The weak association between the regions that contain psb A or between genes for rRNA and the thylakoid system may be a prerequisite for the DNA synthesis and for the separation of pt- chromosomes into daughter plastids. The strong association between the region that contains the psa A, psa B, psb C, and psb D genes of pt- chromosomes and the membranous systems does not seem to occur in

FIG. 11. Three types of nuclei in mesophyll protoplasts of N. tabacum cv. Bright Yellow-2. (a) Living protoplasts and (b) fluorescence photomicrograph of a protoplast fixed with glutaraldehyde and stained with DAPI, showing a cell nucleus and many tiny fluorescent spots from cp-nuclei (large arrow) and mt-nuclei (small mow) in the cytoplasm; (c,d, e) Part of a protoplast, observed by DAPI-fluorescence (c), DAPI-fluorescence and phase-contrast (d), and phase-contrast (e). (c, d, e) are the same field. (f, g, h) Isolated chloroplasts containing cp-nuclei (arrow) observed by DAPI-fluorescence (f), DAFT-fluorescence and phase-contrast (g), and phase-contrast (h); (i-k) Isolated cp-nuclei (arrow) observed by DAPI-fluorescence (i), DAPI-fluorescence and phase-contrast (i), and phase-contrast (k). (i, :#, k) are the same fields. Bars = 10 wm. (From Nemoto et al., 1990; reproduced by permission of the Japanese Society of Plant Physiologists.)

34 TSUNEYOSHI KUROI WA

FIG. 12. SDS-PAGE patterns of polypeptides from the pallet (a) and supernatant (b) of isolated pp-nuclei (lane PN) and isolated cpnuclei (lane CN) treated with 2 M NaC1. Polypeptides for which molelcular masses are shown are DNA-binding proteins. The DNA- binding proteins (35.28. and 26 kDa) of cpDNA differ markedly in molecular mass and in their binding sites from those of ppDNA (69, 31, 30, and 14 m a ) . (From Nemoto et al., 1990; reproduced by permission of the Japanese Society of Plant Physiologists.)

proplastids, but occurs during the development of chloroplasts (Nemoto et al., 1991). Since the pt-nucleus is divided and distributed into many tiny spherical cp-nuclei throughout the entire chloroplast during greening, and since the transcriptional activity of the region which contains the photogenes related to the photosynthesis is preferentially higher in the chloroplasts than in other areas, the membranous complex must play an important role in the control of both the dispersion of the clumps of copies and pt-chromosomes and the expression of pt-genes (Fig. 13).

It has proved possible to isolate pt-nuclei from a variety of plastids, such as proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts, from various plants. Since the physical structure of ptDNA in proplastids does not differ from that of the DNA in the other differentiated plastids, the DNA-binding proteins found in the various types of differentiated


\ Cell nucleus

/ Chloroplast

Dispersion of

chromosome

Greening

+ proplastid ,/ Etioplast

plastid

___)

; I 1 1 I '

', Etioplast I I I I

I I I I

I I I I

I I I I I I

t I I I

-tL iPlastid chromosome

1 1 1 , , I

DNA-binding proteins

'Weak binding 'Strong binding@

Membrane system

FIG. 13. Schematic representation of the changes in the molecular architecture of plastid nuclei during the differentiation of plastids from proplastids to chloroplasts. Signals from the cell nucleus are prerequisite for the endoduplication and dispersion of pt-chromosomes.

plastid must be important contributors to the regulation of the transcription that is essential for the differentiation of plastids.

c. PREFERENTIAL D~GESTION OF PLASTlD NUCLEI PRIOR TO LEAF SENESCENCE

In the mature chloroplasts of higher plants, small spherical cp-nuclei are dispersed throughout the entire chloroplast and the ptDNAs appear to be actively transcribed. It has been shown by biochemical analysis (Scott and Possingham, 1980) and conventional fluorimetry (Lawrence and Poss- ingham, 1986) that the DNA content of chloroplasts in spinach increases markedly during the development of the chloroplasts but is reduced in mature leaves. A similar decrease in the number of copies of ptDNA during the latter phase of chloroplast development has been observed in several other plants (Boffy and Leech, 1982), barley (Baumgartner et al., 1989), and pea (Lamppa and Bendich, 1979; Lamppa et al., 1980). Poss- ingham and Lawrence (1983) suggested that the decrease in number of copies of DNA per mature chloroplast was due to the division of chloro-


plasts that was not accompanied by cpDNA synthesis. This suggestion does not conflict with the hypothesis that the DNA content of the chloroplasts of pea leaves (Lamppa er al., 1980) is inversely related to the number of chloroplasts per cell.

As green leaves begin to turn yellow in Prunus persica, the cell nuclear DNA and the ptDNA in palisade cells are degraded completely (Nii et al., 1988). The levels of protein, DNA, and RNA per cell all decrease signifi- cantly during aging of the leaf. During the decrease in levels of DNA in senescing peach leaves, a nuclease that requires Zn2+ for full activation is gradually activated, while the activity of a nuclease that requires Ca2+ does not change during leaf senescence (Nii et al., 1988). The mechanism for the conversion from an inactive state of the Zn2+ -dependent nuclease to an active state at the time when digestion of cpDNA starts is of consider- able interest. Sodmergen et al. (1989) found in the coleoptile of 0. sariue that when the grana and thylakoid membrane system had developed sufficiently in the mature chloroplast, the digestion of each ptDNA had already begun but the cell nucleus remained intact. Since the number of plastids per cell remained constant throughout the subsequent growth and aging of the coleoptile, the preferential reduction in the amount of cpDNA per plastid was not due to the division of plastids but might, perhaps, be associated directly with the aging of the cells of the coleoptile, which precedes senescence of the coleoptiles. Similar degradation of cpDNA occurred before the first and the second leaves of 0. sariua turned yellow (Sodmergen et al., 1990). It is likely that such degradation occurs in the third leaves and in the other leaves. Since the nuclease that requires Zn2+ is activated in the chloroplasts with concomitant degradation of cpDNA, the nuclease may be involved in the digestion of cpDNA in the coleoptile, and in the first and second leaves (Fig. 14).

VI. Cytoplasmic Inheritance of Plastids

Uniparental and biparental patterns of transmission of cp-genes were found in studies of the non-Mendelian inheritance of leaf color in angiosperm plants by Correns (1909) and Baur (1909), respectively. Correns (1909) showed that the green variegated, and white patterns in leaves of Mirabilis jalapa could be inherited in a uniparental fashion while, at the same time, Baur (1909) found that the green and variegated patterns of leaves of Pefargoniurn tonale were inherited in a biparental fashion. The literature on transmission of plastids has been summarized in a number of reviews (Connett, 1987; Gillham, 1978; Gillham et al., 1985; Hagemann, 1976, 1983; Kirk and Tilney-Bassett, 1978; Sears, 1980; Smith, 1988;


FIG. 14. Electrophoretic patterns ofcoiE1 plasmid DNA digested by the Ca2+- (a), Mg2+- (b) , Zn2+- (c), and Mn*+- (d) dependent nucleases in crude extracts of chloroplast preparations. The chloroplasts were isolated from leaf blades %hours (lane 2) and 144 hours (lane 3) after imbibition. Lane 1 shows the pattern of colEl plasmid DNA incubated without any chloroplast extract. Lane 4 shows the pattern of colEl plasmid DNA incubated with a crude extract of chloroplasts isolated from leaf blades 144 hours after imbibition but without any added metal ions. All the incubations were carried out at 37°C for 80 minutes. 0, open- circular; L, linear; and C, closed circular form of colEl plasmid DNA. (From Sodmergen et a/ . 1991; reproduced by permission of Springer-Verlag.)

Tilney-Bassett, 1975; Whatley, 1982; Boffey and Lloyd, 1988) and, most recently, in “Transmission of Plastid Genes’’ by Gillham et af . (1990). These reviews summarized biochemical, genetic, and electron microscopic approaches to this problem and their results. Mechanisms of non- Mendelian inheritance of ptDNA in algae and higher plants have been explained in terms of the specific behavior of plastids, such as the exclu- sion of plastids from one parent in the gamete, destruction of plastids in one parent in the zygote, and plastid fusion in the zygote followed by destruction of ptDNA from one parent (Gillham et al., 1990).

Eukaryotic organisms have been classified by differences in the sizes of female and male gametes into at least three different groups: isogamous, arusogamous, and oogamous species. When gametes are alike in appearance they are called isogametes, as in the case of Chfamydomonas, Ufoth- rix, and Ectocarpus, and the plants exhibit isogamy. Gametes having only a slight difference in size are said to be anisogametes and such species exhibit anisogamy. Bryopsis maxima is classified as a number of this group. When gametes differ in size and activity, as they do in algae such as Vdsluox, Fucus, and Polysiphonia, in mosses, ferns, and higher plants, the plants are said to exhibit oogamy. In higher plants, genetic analyses of mechanisms of maternal inheritance have proved possible, but biochemical analyses have been dimcult since double fertilization must be per- formed in cells embedded in various tissues. Many workers prefer to use


microorganisms with a short life cycle in which genetic and biochemical analyses are relatively easy. Sager (1954) showed for the first time that uniparental inheritance of cytoplasmic genes for resistance to streptomy- cin occurred in C. reinhardtii. Since then, many studies on mechanisms of plastid inheritance at the genetic and biochemical level have been per- formed using C. reinhardtii (Gillham, 1974).

A. ISOGAMOUS ALGAE The findings of uniparental inheritance and DNA in chloroplasts (Sager

and Ishida, 1963) in the isogamous alga C. reinhardtii led to the important hypothesis that the mechanisms of maternal inheritance were based on enzymatic reactions, and maternal transmission of cp-genes was connected to the behavior of organelle DNA. Sager and Lane (1972) showed that cpDNA of female origin remained in zygotes for at least 6 hours after mating, during which time cpDNA from the male parent disappeared. This result suggested the existence of a mechanism of maternal inheritance based on the preferential degradation of male cpDNA in young zygotes.

To explain the preferential degradation of male cpDNA, it has been proposed that the maternal transmission of cpDNA is governed by a methylation-restriction system analogous to that found in bacteria: after gametic fusion, the male cpDNA is degraded by a restriction enzyme while the modified female cpDNA remains intact. Modification is assumed to occur as a result of methylation. Studies using CsCl density gradients (Burton et af., 1979), high-pressure liquid chromatography (Royer and Sager, 1979), and restriction endonucleases (Sano et al., 1981) have shown that female cpDNAs are methylated but male cpDNAs are not. Further- more, the isolation of DNA methyltransferases from C. reinhardtii with molecular weights of 60,OOO and 200,000 has been reported.

Methylation of cpDNA from both parents has been reported in a mutant of C. reinhardtii in which the cpDNAs of both mating types are heavily methylated but maternal inheritance occurs as in the wild-type strain (Bolen et af., 1980). It was confirmed subsequently that the methylation of female cpDNA is at its lowest level during the vegetative stage and that this basal level of methylation of cpDNA increases more than 20-fold after gametogenesis. A large number of methyl groups are incorporated into cpDNA at the 7-h-zygote stage in C. reinhardtii. However, results of experiments using inhibitors of the methylation of DNA suggest that extensive methylation of female gametic cpDNA during gametogenesis is not required for the maternal inheritance of cp-genes (Feng and Chiang, 1984). The methylation at the 7-h-zygote stage is probably not related to the maternal inheritance of cpDNA because, in most of the zygotes, preferen-


tial degradation of male cpDNA occurs within 60 minutes after mating (Kuroiwa et al., 1982). The methylation-restriction hypothesis is associated with some problems which must be solved.

Cp-nuclei can be easily observed in DAPI-stained cells. Therefore, if male cpDNAs in a zygote of C. reinhardtii are preferentially degraded within 6 hours after mating, as suggested by the results of biochemical experiments (Sager and Lane, 1972), the disappearance of cp-nuclei of male origin should be observable by epifluorescence microscopy during formation of zygotes. The accurate timing and morphological pattern of the preferential destruction of cp-nuclei in each zygote must also be identified in relation to biochemical reactions, such as methylation of cpDNA and activation of as yet unidentified nucleases.

Ten minutes after the mating of gametes of two wild-type strains, the newly formed zygotes are spherical, have four flagella, and contain two cell nuclei and two discrete chloroplasts. Each chloroplast contains 8-10 dispersed cp nuclei. About 40 minutes after mating, the cp-nuclei in the chloroplast from one parent disappear completely, prior to cell nuclear fusion (Fig. 15). Various crosses were arranged between morphologically different gametes by using cells that differed in size, in the size of ct-nuclei, in the length of the flagellum, and in chlorophyll content (Kuroiwa et al., 1982; Tsubo and Matsuda, 1984; Kuroiwa, 1985) and in BUdR label (Mun- aul. et al., 1990). The cp-nuclei that disappeared were confirmed to be of male origin. Since similar uniparental degradation of cp-nuclei has been observed in C. moewusii (Coleman and Maguire, 1983) and in multicellular green algae such as Monostroma nitidum, Dictiosphaeria calvernosa, and Acetabularia calyculus (Kuroiwa et al., 1985a), preferential degradation of organelle DNA of uniparental origin may occur in many isogamous algae, such as Caulerpa brachypus and C. okamurai (Kuroiwa and Hori, 1990). On the other hand, in a few green algae, such as Ulva arasaki and Entero- morpha intestinalis, the preferential digestion of organelle DNA was not observed 10 hours after mating (Kuroiwa and Hori, 1990). In each case, therefore, it is essential to examine whether or not the uniparental transmission of cpDNA occurs.

I3oynton et al. (1990) and Hams (1989) pointed out the possibility that the: disappearance of male cp-nuclei may be due to preferential dispersion of cp-nuclei throughout the entire chloroplast and, thus, cpDNA of male origin may still be present. However, this possibility has been completely excluded. The total population of cp-nuclei contained 80-100 copies of cpDNA and occupied more than 10% of the matrix, exluding starch grains anld thylakoid membranes. Therefore, if the total cp-nuclei had been dispersed throughout the entire matrix of chloroplast of male origin, the chloroplasts that contained the dispersed DNAs would be visualized as


Time (h ) af te r mating 0 1 2

1

Control

mt

. Chloropla8t nuclei

a

FIG. 1.5. Diagrammatic summary of representative events of cell nuclei(N)and chloroplast nuclei in young zygotes during the first 2 hours (a) and epifluorescence photomicrographs (b, c ) of zygotes 10 (b) and 40 minutes (c) after mixing of mt' (male) and mt+ (females) gametes of C. reinhardtii. cn and p show ceU nucleus and pt-nucleus. respectively. Bar = 1 pm.

bright areas by high-resolution epifluorescence microscopy and the fluorescence intensity would be easily measurable by VIMPICS. VIMPICS has the ability to identify one gene in uifro and one molecule of ptDNA. In fact, the fluorescence intensity in the matrix of chloroplasts of female origin did not change either before or after the disappearance of cp-nuclei of male origin (Kuroiwa and Nakamura, 1986). Immunofluorescence patterns obtained with DNA-specific antibodies corresponded to the pattern of staining by DAPI (Sun et al., 1988). Addition of inhibitors of nucleases soon after mating completely inhibited the preferential destruction of cp- nuclei of male origin. These results strongly suggest that the disappearance of cp-nuclei is due to nucleolytic reactions. Therefore, DAPI staining


appears to be a very simple and rapid method for making direct estimation of the extent of degradation of cpDNA in young zygotes.

‘To identify the factors involved in the preferential destruction of cp- nuclei of male origin, young zygotes of C. reinhardtii were treated with specific inhibitors of translation in chloroplasts (chloramphenicol, erythromycin) and in the cytoplasm (cyclohexirnide), with inhibitors of nucleases (anrintricarboxylic acid, ethidium bromide), with inhibitors of transcription in chloroplasts (rifampicin) and in the cytoplasm (actinornycin D and a-amanitin), at various temperatures, with chelating agents (EDTA, EGTA), and with UV light (Kuroiwa et al., 1983a,b). The degradation of cp-nuclei was then examined by staining with DAPI and epifluorescence microscopy. The nucleolytic reaction in uiuo displays the following characteristics: (i) digestion of about eight cp-nuclei of male origin occurs synchronously within a single chloroplast; (ii) each cp-nucleus deflates and dissolves from its periphery toward its center; and (iii) the cp-nuclei are completely digested within 20 minutes after the initiation of degradation. These characteristics suggest that DNases similar to DNase I and micro- coccal nuclease may be involved in the digestion of the cp-nuclei. If cpDNAs are digested only by a restriction endonuclease, as described in the restriction-modification hypothesis, the cp-nuclei should swell slightly and would not be expected to disappear rapidly. Attempts to isolate nucleases from young zygotes of C. reinhardtii revealed that extracted nucleases required the presence of Ca2+ for full activation. Thus this class of nucleases is called nuclease C (Ogawaand Kuroiwa, 1985). The nuclease is pcllymorphic and includes six molecules (16, 18, 20, 22, 25, and 26 kDa). Nucleases with similar properties have been found in all plants examined showing uniparental transmission of ptDNA, such as A. calyculus, Nitella axillformis, M . polymorpha, Neurospora crassa, N . tabacum, and P . ptrsica (Nakamura and Kuroiwa, 1987; Nii et al., 1988).

Since preferential destruction of cp-nuclei of male origin is inhibited by EGTA, nuclease C may be responsible for the preferential digestion of cpDNA in uiuo. However, nuclease C is found in zygotes and in female and male vegetative cells and gametes (Ogawa and Kuroiwa, 1985a). When cpDNAs isolated from female and male vegetative cells and gametes were infabated in a solution of nuclease C, cpDNAs of both male and female origin were completely digested. Therefore, the preferential destruction of cp-nuclei of male origin is probably not controlled at the DNA level. The presence of nuclease C in both female and male gametes led to the hypothesis that nuclease C is presynthesized prior to mating, exists in an inactive form in pregametic cells, and is activated by enzymatic systems in young zygotes.

Preferential destruction of cp-nuclei of male origin can be completely inhibited by addition of cycloheximide soon after mating but not by addi-

42 TS UNEYOSHI KUROl WA

tion of chloramphenicol and erythromycin. This result suggested that proteins which were synthesized de nouo in the cytoplasm after mating were required for the preferential destruction (Kuroiwa ef al., 1983a, b). An examination was made of the polypeptides that are synthesized in the cytoplasm and related to the preferential destruction of cpDNA of male origin by incorporation of [S35]methionine and two-dimensional gel electrophoresis. About 200 polypetides were synthesized within 30 minutes after mating and six of these polypetides [94 (a), 94 (PI, 94 (y ) , 52,50, and 38 kDa] appeared to be essential for the preferential destruction of cp- nuclei of male origin (Nakamura and Kuroiwa, 1989; Nakamura et al., 1988). These polypeptides are probably involved in the activation of nuclease C, perhaps via alterations in biochemical or biophysical parameters such as the concentration of intracellular Ca2', the permeability of chloroplast membrane, the structure of lysosomes, etc. In Monosfroma, the preferential degradation of cpDNA from one parent occurs within 30 minutes. Just before this degradation the fluorescence intensity of the chlorophyll in the chloroplast in question decreases while that of the chlorophyll in the other chloroplast does not change (Kuroiwa and Hori, 1990). Under the electron microscope, the entire chloroplast can be seen to become electron dense while the other chloroplast does not change, suggesting that some parameter, probably the permeability of the membrane systems of the chloroplast from one parent, changes markedly, promoting the action of nuclease C.

The six polypeptides may be encoded by mRNAs synthesized just after mating since the protein synthesis, RNA synthesis, and the preferential destruction of cpnuclei are inhibited completely by treatment with actino- mycin Dafter mating (Kuroiwa etal., 1983b; 1985b). The question, then, is whether cell nuclei of male or female origin contribute to the synthesis of relevant mRNAs. When female gametes of C . reinhardtii which have been irradiated with UV light mate with unirradiated gametes, the preferential destruction of cp-nuclei is inhibited, but when male gametes are irradiated with UV light, destruction of cp-nuclei occurs normally. The effects of UV irradiation suggest that the target of the irradiation is the DNA molecule. The important mRNAs are probably synthesized in the cell nucleus from the female parent just after mating. 5-Fluorodeoxyuridine (5-FdUrd) selec- tively decreases the amount of cpDNA in plastids by inhibiting the synthesis of organelle DNA but it does not affect the amount of the cell nuclear DNA, the rate of proliferation of cells, or the cell density at stationary phase (Wurtz ef al., 1977). When the female cells are incubated in 5- FdUrd, the cpDNAs in some chloroplasts of female gametes are completely lost (Nakamura and Kuroiwa, 1989). When female gametes without cpDNA are crossed with normal male gametes, the preferential digestion of cp-nuclei of male origin occurs and, as a result, the male zygotes,


containing chloroplasts without their cpDNAs, are generated. When the preferential digestion of cpDNA of male origin occurs after mating of male gametes treated with 5-FdUrd and normal female gamete, the six polypeptides mentioned above are synthesized. These results indicate that mRNA thiit encodes the information of the preferential digestion of cp-nuclei of male origin is synthesized in the cell nucleus of female origin. These results do not conflict with the finding by Sager and Ramanis (1973) that irradiation of female gametes with UV light just before mating induces the biparental inheritance of nonchromosomal genes. However, Wurtz e f al. (1!)77) reported that when the mt' gamete is treated with 5-FdUrd, the decrease in levels of cpDNA appears to perturb the normal maternal transmission of cp-genes, with a dramatic increase in the proportion of exceptional zygotes. The discrepancy between these cytological and genetic experiments can be explained if 5-FdUrd strongly inhibits synthesis of organelle DNA but only weakly inhibits the expression of the cell nuclear gene(s) essential for the preferential digestion of cpDNA of male origin in young zygotes.

Protection of female cpDNA from degradation can be clearly explained by methylation in Sager's model but not in the present nuclease C model. Hlowever, Ogawa and Kuroiwa (1985b) obtained interesting data using cell msodels prepared by treating vegetative and gametic cells of C. reinhardtii wicth a medium that included EDTA, 2-mercaptoethanol, and spermin. When the model cells were incubated with the nuclease C fraction, cp- nuclei in female and male vegetative cells and male gametes disappeared completely, but cp-nuclei in female gametes did not. In each case, cell nuclei were not markedly affected. Numbers of female gametes with cp- nuclei that were not digested by nuclease C increased as gametogenesis advanced. These results suggest that there is an as yet unidentified mechanism that protects cpDNA of female origin from nuclease C in domains such as the chloroplast matrix and the membrane that surrounds female cpDNAs. Such a hypothesis is presented schematically in Fig. 16 (Kur- oiwa, 1985).

For further elucidation of the molecular mechanisms of maternal inheritance, we need to identify the substances that protect female cpDNA and to characterize the proteins that activate nuclease C, which may be encoded by mRNA synthesized de nouo in young zygotes just after mating. Detailed analyses of mutants that do not display preferential destruction should provide important clues.

B. ANISOGAMOUS AND OOGAMOUS ALGAE AND FERN The preferential degradation of male cp-nuclei occurs in young zygotes

after mating in several isogamous algae and it may be responsible for


maternal inheritance of cp-genes. However, little information is currently available on the behavior of ptDNA during gametogenesis and zygote formation in anisogamous algae. In B . maxima, 12 hours before the release of gametes, male and female green lateral branches of thalli became orange-brown and dark green in color, respectively. Large chloroplasts in the male gametangia divide to form about 32 or more small daughter chloroplasts which contain a few cp-nuclei, and the large chloroplasts in the female divide to form eight or more daughter chloroplasts that contain about 20 ct-nuclei. At about 10 hours before the release of gametes, when cleavage of cytoplasm into gametes begins, cp-nuclei and mt-nuclei disap-


pear completely from most male gametangia, though about 10% of the gametes still contain one to three cp-nuclei and one or two mt-nuclei. By contrast, cp-nuclei and mt-nuclei in organelles of female gametangia per- sist. The disappearance of cpDNA in male gametes has been confirmed by CsCl density-gradient centrifugation (Kuroiwa et al., 1991). After the onset of the light period, male and female gametes are markedly anisogamous and contain one spherical cell nucleus, one chloroplast, and one mitochondrion. The chloroplasts in both male and female gametes are spherical and cup-shaped, respectively, and occupy a major part of the posterior hemisphere of the gametes; the mitochondrion lies at the base of the flagella. Only a small percentage of the total population of gametes contains cp- and mt-nuclei, but female gametes contain about 20 cp-nuclei per chloroplast and 3-6 mt-nuclei per mitochondrion. Two minutes after mating of female and male gametes, the anisogamous gametes form an irregular heart-shaped union, in which cp-nuclei and mt-nuclei that origi- nated in the male gamete completely disappear, while the cp-nuclei and mt-nuclei of female origin remain. Twenty-five minutes after mating, both cell nuclei fuse in the young zygote, but the chloroplasts of male and female origin are never seen to fuse. After mating, the Golgi vesicles and ly sosomes develop markedly. When the lysosome is associated preferentially with only the small chloroplast without DNA, the small chloroplast begins to break down and finally is fragmented to small membranes. By contrast, the large chloroplast of female origin remains and increases in volume. The small chloroplast of male origin is digested within 12 hours after mating (T. Hori and T. Kuroiwa, unpublished data). The preferential degradation of ptDNA in male gametes before mating occurs in B. plurnosa (Ogawa, 1988; Saito et al., 1989).

In many oogamous algal species and in some anisogamous species, the preferential degradation of cpDNA during spermatogenesis must be responsible for the disintegration of the plastids that are contributed by the male parent to the egg cell after gametic fusion. Elimination of plastids before mating has been reported for the spermatozoids of a few oogamous Xanthophytes and brown algae (Whatley, 1982).

In the Characeae, the behavior of cpDNA has been examined after double-staining with DAPI and FITC subsequent to treatment with DNA- specific antibodies. The cp-nuclei, which were present in internode cells and cells at the early spermatid stage, disappeared during spermatogenesis (Sun et al., 1988). Similar events also occurred during spermatogenesis in the fern P. uittata, as summarized in Figs. 17 and 18 (H. Kuroiwa et al., 1'988). Spores of P. uittata were grown on a solid medium that contained antheridiogen and an antheridium initial formed on each protonema cell. The antheridium initial divided and produced 16 spermatocytes and three surrounding cells. The chloroplast in the spermatocytes decreased in vol-


FIG. 17. Phase-contrast (a, d, g) and epifluorescence images (b, c, e, f, h) showing cell nuclei (N), plastids (arrows in a, d, and g), pt-nuclei (arrows in c and f), and flagella (F in g) in 16-cell spermatocytes (a-c), the transformed cells derived from 16-cell spermatocytes (d-f ), and sperm (g, h) in the fern P. virrora. In the I k e l l spermatocyctes that are being transformed into sperm, the chlorophyll of the plastids has disappeared completely (arrows in b, e) but plastids (arrows in a , d) and pt-nuclei (DNA) (arrows in c, f ) remain. When the sperm have matured, pt-nuclei also disappear (h). Thus, the plastids in sperm do not contain DNA. Bar = 10 pm. (From H. Kuroiwa er ol., 1988; reproduced by permission of Springer-Verlag.)


ChlOrOPlESt Cp-nuclei

Spore 1

8 ca @ 0

ume as cell division occurred and this process was repeated until the final volume of each chloroplast was 1/15 that of the primary chloroplast, The DNA content of the chloroplasts was also reduced to 1/5 of the original value. When the sperm matured and the shape of its cell nucleus began to transform into a spiral, the ptDNA disappeared. The plastids without DNA remained visible until the final stage of development of the sperm (Figs. 17 and 18).

These results indicate that, in anisogomous algae and fern, the preferential reduction in the amount of cpDNA per chloroplast occurs by division of chloroplasts without DNA synthesis, as the first steps, and then the preferential digestion of cpDNA in the chloroplast or plastid occurs during the transformation of the cell nucleus.

Thus, the fertilization of an egg that contains a large amount of ptDNA by a sperm that contains a very small number of small plastids without


DNA seems to occur in the anisogamous algae, in mosses, and in ferns. It was reported that, in motile spermatozoids of lower plants, a cytoplasmic vesicle which includes the plastids is discarded before the male gamete reaches the egg (Whatley, 1982).

C. HIGHER PLANTS Correns (1909) and Baur (1909) identified two basic patterns of maternal

and biparental transmission of cytoplasmic genes in plants. In angiosperms the pattern of plastid transmission has been examined genetically in crosses of more than 50 genera of dicotyledons and monocotyledons (Smith, 1988), and the pattern of transmission of ptDNA has been observed cytologically in more than 100 genera (Miyamura et al., 1987; Corriveau and Coleman, 1988; Kuroiwa and Hori, 1990; Kuroiwa, T. et al., 1990b). Such studies have shown that plastid transmission is most often maternal, but biparental inheritance is not uncommon and has been demonstrated in about 20 genera (Sager, 1972; Corriveau and Coleman, 1988; Gillham et al., 1990; Sears, 1980; Smith, 1988; Tilney-Bassett, 1975). The progeny of certain species regularly contain at least some paternally derived plastids (Smith, 1988; Gillham er al., 1990).

The mechanism of maternal inheritance of plastids in angiosperms has been explained in terms of elimination of plastids from the pollen parent during the time between the formation of pollen grains and fertilization of the egg by one of the two sperm cells. Therefore, before a description is provided of the mechanism of cytoplasmic inheritance in angiosperms, the process of formation of sperm cells must be summarized (Fig. 20). The haploid microspore cells, which are formed after meiosis, can develop into four pollen grains. During the formation of pollen grains, each microspore divides mitotically and unequally to yield a large vegetative cell that contains large amounts of cytoplasm and a small generative cell that contains a small amount of cytoplasm. The generative cell moves to the internal region of the vegetative cell and, thus, is surrounded by the vegetative cell. The generative cell divides a second time to form two daughter sperm cells. The division of the generative cell can occur either in pollen grains or in pollen tubes. In the pollen tube, the two sperm cells are surrounded by the cytoplasm of the vegetative cell. At fertilization, one of the sperm cells fuses with the egg cell, forming a diploid zygote from which the embryo will develop, while the other fuses with the polar cells to yield a triploid cell from which the endosperm of the seed develops (Fig. 20).

From observations by both light and electron microscopy, Hagemann (1983) recognized four patterns of plastid transmission in angiosperms. In the Lycopersicon type, which is represented by the tomato Lycopersicon


esculentum, all microspore plastids segregate to the vegetative cell, and only chloroplasts from the maternal parent are transmitted to the progeny. In the Solanum (potato) type, the first partitioning of the cytoplasm during pollen mitosis is more equal, with the generative cell as well as the vegetative: cell receiving some plastids. However, in the course of further devel- oprnent of pollen, the plastids of the generative cell are lost, so the sperm cells do not contain plastids. In the Triticum type, which so far appears to be restricted to grasses, plastids are found in the generative and sperm cells. However, when the sperm cell enters the egg cell, enucleated cytoplasmic bodies containing mitochondria and plastids are left outside (Mogensen, 1988; Mogensen and Rusche, 1985). In the Pelargonium pat- ten1 of plastid transmission, found in geraniums, the distribution of plastids to the generative and vegetative cells during the first pollen mitosis results in biparental transmission of plastids. Patterns of plastid transmission can vary within a single taxon such as the Liliaceae (Schroder, 1984; Schroder and Hagemann, 1985; Vaughn et al., 1980).

As described above, mechanisms of maternal inheritance in angiosperms have been explained by the absence of plastids (Sears, 1980) and mitochondria in the generative cells. However, in Lillium longifioem, in which organelle DNA shows maternal inheritance, a few plastids and many small mitochondria are present in sperm cells in pollen tubes (Miki- Hiroshige and Nakamura, 1977). However, when the organelles in the vegetative cells are stained with DAPI, fluorescent spots are never seen, suggesting that the organelles do not themselves contain DNA (Miyamura et d. , 1987). Since the generative cell contains organelle DNA at the early stages of the formation of pollen grains, the preferential elimination of organelle DNA from the organelles in the generative cell occurs during forination of sperm cells and may be responsible for the maternal inheritance, as described in the case of the algae and the fern. Miyamura et al. (1987) selected 18 representative plants in which the two different types of inheritance occurred and examined cytologically whether or not the preferential elimination of the ptDNA and the mtDNA in the generative cellls occurred during formation of pollen grains and in pollen tubes. They cornpared their cytological results with the results of classical genetic studies and found that the plants could be classified into three types according to the behavior of the organelle DNA in the generative cells during the formation of pollen grains. The first type, exemplified by N . tabucum and Lycoris radiata, is characterized by the digestion of organelle DNA in the generative cells immediately after the first mitosis in pollen grains (Figs. 19a and 20). The second type, exemplified by T. aestiuum, is Characterized by the digestion of organelle DNA between the second pollen mitosis and the initiation of the transformation of sperm nuclei (Fig.


FIG. 19. Fluorescence photomicrographs of vegetative nuclei (VN), generative nuclei (GN), a sperm nucleus (SN), pt-nuclei (large arrows) and mt-nuclei (small arrows) in pollen grains of four angiosperms such as Lycoris radiata (a), Opuntiaficus-indica (b), Rhododen- dron indicurn (c), and Pelargonium zonale (d) after staining with DAPI. Bar = 1 Fm.


Maternal inheritance Biparental inheritance

Qermination of pollen grain

(0 a@ 4

Trlcellular pollen

2nd pollen grain mitorir

Blcellular pollen

grain

Migration of generative cell

Y mltorlr

Mlrabi i i r t y p e Pe1argonium.type

FIG. 20. Schematic representation explaining mechanisms of maternal inheritance and biparental inheritance of organelle DNA in angiosperms on the basis of whether or not organelle DNAs are present. Maternal inheritance of plastids and mitochondria of Mirabilis type may result from three steps, as follows: a reduction in the number of organelles just after the first pollen mitosis (three arrowheads); the preferential digestion of organelle nuclei during formation of sperm cells; and the digestion of organelles that do not contain DNA during fertilization. Biparental inheritance of organelles of Pelargonium type may be the results of the protection of organelle DNA from nucleases or the absence of nucleases and the separation of organelles from both parents during formation of the embryo. GN, generative nucleus; VN, vegetative nucleus; PN, plastid nuclei; MN, mitochondrial nuclei; EN, egg nucleus; EPN, egg plastid nucleus; EMN, egg mitochondrial nucleus.


20). In general, those plants that are known for the maternal inheritance of ptDNAs belong to the first and second types. The third type is characterized by the presence of organelle DNA in sperm cells in pollen tubes. Plants such as Opuntia Ficus-indica (Fig. 19b), Rhododendron indicum (Fig. 19c), and P . zonale (Fig. 19d), which show biparental transmission, can be classified as the third type. The cytological observations are in basic agreement with the classical genetic data and the results have been confirmed by the extensive observations of Comveau and Coleman (1988) and Kuroiwa and Hori (1990; Table II), and by the technique of molecular biology (Comveau, et al., 1990). Kuroiwa and Hori (1990) also identified a number of additional species in Aozpaceae, Cactaceae, Campanulaceae, Ericaceae, Fabaceae, etc. in which ptDNA can be found in generative or sperm cells of pollen (Kuroiwa and Hori, lw), suggesting that these have the potential for biparental inheritance. These results indicate that the preferential digestion of organelle DNA during formation of sperm cells must be responsible for the maternal inheritance in higher plants as well as in anisogamous algae and ferns.

How does the preferential digestion of the organelle DNA in the generative cells occur in higher plants? In many species an examination has been made of whether or not the volume of cytoplasm in the generative cell, formed by the first pollen mitosis, affects the preferential destruction of organelle DNA. In general, in plants such as the Geraniaceae, in which the ratio of the volume of cytoplasm to that of the cell nucleus is high, the organelles remain and the preferential destruction of the organelle DNA does not occur. By contrast, in plants such as the Compositae, in which the ratio is low, the preferential destruction of the organelle DNA does indeed occur. However, in some cases, such as the Liliaceae, in spite of the ratio not being low, the preferential destruction of organelle DNA does occur. Accordingly, it is not always the case that the decrease in the volume of the generative cell by the first pollen mitosis or unequal division is directly responsible for the destruction of the organelle DNA in the generative cells.

It is commonly held that the elimination of ptDNA from sperm cells (sperms) in anisogamous algae, ferns, and angiosperms involves the elimination of the organelle DNA in two steps, as follows. The number of organelles in the generative cells decrease markedly as a result of unequal division at the first step and then the organelle DNA is digested by Caz+ -dependent or Z2+ -dependent nucleases while the degradation of organelles without organelle DNA may be induced by lysosomes (Fig. 20). The activation of such nucleases may not operate in plants such as Pelar- gonium zonale and Schlumbergera russellienum that show biparental transmission of cpDNA (Fig. 20; Sodomergen and Kuroiwa, unpublished


TABLE I1

IN VARIOUS SPECIES OF ANGIOSPERM RELATIONSHIP BETWEEN THE BEHAVIOR OF Pt-NUCLEI AND CYTOPLASMIC INHERITANCE

Genetic evidence for maternal inheritance (M) or

Taxon of pt-nuclei biparental inheritance (B)" Absence (-) or presence (+)

Helianthus annuus Impatiens capenicis Arabidopsis thaliana Arabis albida

M' Brassica campestris -

M' Bem vulgaris -

M' Chenopodium album -

M' Glycine max -

M' Trijblium pratense -

M' Hytirophyllum virginianum -

MI Go::sypium hirsutum -

M' Pleantago major - M' M' Corx lacryma-jobi -

M ' Hordeum vulgare -

MI Oryza sativa -

MI Sorghum vulgare -

MI Zeci mays -

M' Capsicum annuum -

MI Lycopersicon esculentum -

M' Petunia hybrida -

Solanum tuberosum - MIB' M2 Viola tricolor -

Hy,vericum perforathum + B' Rhododendron maximum + B' Rhododendron indicum + B2 Medicago sativa + B' Geranium maculata + B' Peragonium hortorum + B' Ch forophytum elatum + B'

B' Phmeoius vulgaris - Pisum sativum + M' Ipomoea nil + MI

Mirabilis jalapa - M1.2

Avena sativa -

Triiicum aestivum - M1.2

Nicotiana tabacum - M1.2

Oenothera biennis + B1.2

- a (1) Based on Comveau and Coleman (1988); (2) based on Miyamura er 01. (1987).


data). The transmission of paternal plastids at low frequency has been documented in both Nicotiana (Medgyesy et al., 1986) and Petunia. Such cases may be due to some accidents of the control system that induces digestion of ptDNA during the formation of pollen and of the separation system of plastids during embryogenesis.

The amount of cpDNA in the sperm cells of the biparental type, in which cpDNA remains until the formation of pollen tubes, has been examined by VIMPICS. In the plants tested, the number of copies of cpDNA per cell varied from 24 to 550 (Table HI). It was surprising to us that the number of copies of cpDNA in P . tonale was the highest among all plants examined because Baur (1909) seemed to have already chosen Pelargonium as the most suitable material for research into non-Mendelian and biparental inheritance of leaf color. Triform repens contained a few copies of ptDNA in sperm cells (Miyamura et al., 1987). It is likely that in T. repens, maternal transmission of cpDNA occurs at lower frequency, as shown by Corriveau and Coleman (1988). Among biparental progeny of Oenothera, maternally derived plastids predominate, whereas in the alfalfa, Medicago satiua, paternally derived plastids predominate. These phenomena also can be explained by the number of copies of cpDNA in sperm cells. Both Oenotheru and M . satiua show biparental transmission of ptDNA and their sperm cells contain ptDNA. Since the DNA content is lower than that in other plants, such as Pelargonium and Rhododendron, the frequency of maternal transmission of ptDNA is high. Of course, there is the possibility that additional mechanisms influence the inheritance of plastids (Lee et al., 1988; Smith, 1989). Spatial distribution of the plastids in the zygote

TABLE 111 LEVELS OF ptDNA CONTENT PER SPERM CELL I N VARIOUS PLANTS WITH PI-NUCLEI

Number of Number of Number of copies of

Taxon plastids/cell pt-nuclei/plastid T value/pt-nuclei" ptDNA

Trifolium repens Oenorhera sp. Pelargonium zonale Pelargonium inquinans Geranium yesoense Geranium roberrianum Rhododendron indicum Triticum aestivum

1 1 6

55 51 19 21 12 14

4 5

10 9 8 9

10 2

44 30

550 459 152 189 120 24

" Fluorescence intensity of T4 phage, fured and stained with DAPI in the same way as plant cells, was used as a standard for measurements (7 = 1).


prior to its asymmetric division into a suspensor and a terminal cell may play a critical role, with paternal plastids in alfalfa being more favorably situated for entry into the terminal cell (Tilney-Bassett and Almouslem, 1989).

The asymmetric dispersion of plastids during embryogenesis provides a useful explanation of the biparental pattern of inheritance in the case of Pelargonium, which yields green, white, and variegated plants (Fig. 20). Since the plants showing biparental inheritance of ptDNA are composed of cells that contain organelles from both parents, they will probably be important materials for “organelle technology.”

Paternal inheritance and transmission of cpDNA have been shown by genetic experiments (Ohba et al., 1971) and through the use of restriction fragment polymorphisms (RFLPs) in a number of conifers (Neale et al., 1986; Neale and Sederoff, 1989; Wagner et al., 1987; Szmidt et al., 1983, 1987). Paternal transmission in conifers can be explained by the observation that paternal plastids enter the egg cell and maternal plastids are often degraded (Whatley, 1982).

Apparent cpDNA recombinants have been observed in pine, and Pinus banksiana and P. contorta are known to hybridize naturally (Govindaraju et al., 1988). In the green algaEuglena, Ehara et al. (1984) reported that, at a certain stage of the cell cycle, several spherical chloroplasts conjoin to form a single giant body that contains a threadlike cp-nuclei, which may be formed by the fusion of small, spherical cp-nuclei. Recently, Kawano et al. (1991) found in Physarum polycephalum that mitochondrial fusion, the mt-nuclear fusion, and the recombination of mtDNA were controlled by a plasmid in the mitochondria. Plasmids associated with the fusion of chloroplasts and recombination of ptDNA may be present in many organisms.

w. summary

Our present understanding of the replication, differentiation, and inheritance of plastids can be summarized as follows:

1. Most plants can be classified as being one of five types: the SN, CN, CL, PS, or SP type, based on differences in the shape, size, and distribution of the cp-nuclei in their mature chloroplasts. The differences in patterns of chloroplast nuclei can be explained by the pattern of distribution of the replicated chloroplast genomes in mature chloroplasts. They may be distributed randomly throughout the chloroplast (SN type); gathered around the pyrenoid in the chloroplast (SP type); distributed along the periphery of the chloroplast (PS type; fused to form spherules in the


central area of the chloroplast (CN type); or fused to form a circle along the periphery of the chloroplast (CL type).

2. Proplastids contain one to a few copies of their own unique circular genome, of which the size varies from 12 X lo5 bp to 18 X lo5 bp. The genome is organized to form a pp-nucleus approximately 0.5 p m in diameter in the central area of a proplastid by interactions with specific proteins. The packing ratio is higher than that of cell nuclei. Proplastids can divide into daughter proplastids by binary fission with concomitant separation of pp-genomes after endoduplication of ppDN A.

3. The concept of organelle nuclei, as described above, has changed our ideas about the division of organelles. Thus, the process of organelle division must be composed of two main events: division of the organelle nucleus and organellekinesis (analogue of cytokinesis). The pt-nuclear division occurs after endoduplication of ptDNA or in the absence of prior DNA synthesis. The association between pt-DNA and membrane systems has not been clearly proven in proplastids. The strong binding between specific sites of ptDNA and membrane systems is observed in chloroplasts and may be related to the dispersion of pt-chromosomes and the activation of photogenes after greening. The plastidkinesis is mediated by the plastid- dividing (PD) ring in lower eukaryotes, in moss, and in gymnosperm which is located on the cytoplasm outside plastids. In angiosperm, the plastid- dividing ring is a doublet which is composed of an outer ring and inner ring. The PD ring is composed of a bundle of actinlike filaments. Plastidkinesis occurs by contraction of this bundle.

4. Proplastids can differentiate with a concomitant increase in DNA content and volume of more than 10-fold, and with dispersion of pt-nuclei into etioplasts, chloroplasts, amyloplasts, and chromoplasts, which are very specific organelles in cells in differentiated tissues. Proplastids and all differentiated plastids are called plastids as a general term and all plastids contain a genome (DNA) with basically identical physical characteristics. The genome forms a pt-nucleus by association with different proteins, and it can divide by constriction rather than partition. There are two hypothe- ses to explain the mechanism of plastid development: one is that the development of plastids is induced by demethylation of ptDNA; the other is that the development is controlled by the proteins that bind to the ptDNA.

5. In higher plants, the DNA content per chloroplast is reduced in mature leaves. Some workers have explained the decrease in number of copies of DNA per mature chloroplast as being due to the division of chloroplasts that was not accompanied by synthesis of ptDNA. As green leaves begin to turn yellow in P. persica, the cell nuclear DNA and ptDNA


in palisade cells are degraded completely. During the decrease in levels of DNA in senescing peach leaves, the nucleolytic activity that requires Zn2+ for full activation develops gradually. In 0. sariua, when the grana and thylakoid membrane system are adequately developed in the mature ch1,oroplasts in the coleoptile, the first leaf, and the second leaf, the digestion of each ptDNA has already begun but the cell nucleus remains intact. The nuclease that requires Zn2+ is activated with concomitant degradation of cpDNA, and, thus, is involved in relation to the digestion of cpDNA in the coleoptile, the first, and the second to the other leaves.

6. The preferential digestion of cpDNA of male origin occurs in the young zygotes of isogamous algae and seems to be responsible for the maternal inheritance of cpDNA. In a hypothetical mechanism for the regulation of maternal inheritance of cpDNA, female gametes have the ability to protect their cpDNA against Ca2' -dependent nuclease C during gametogenesis by changing the domains that surround female cpDNA. Soon after mating of male and female gametes, specific mRNAs are synthesized in a cell nucleus of female origin in the newly formed zygote. Then the proteins encoded by the mRNAs are synthesized de nouo in the cytoplasm and directly or indirectly activate nuclease C. After a selective change in the permeability of the membrane systems of chloroplasts of male origin, nuclease C preferentially enters the chloroplasts of male origin and digests the cpDNA. Since cpDNA of female origin remains an'd is transmitted into the progeny, maternal inheritance occurs. In the case of maternal inheritance in anisogamous algae, ferns, and higher plants, the preferential digestion of cpDNA occurs during spermatogenesis (formation of sperm cells) and is responsible for maternal inheritance. The degradation of plastids of male origin occurs in at least three steps: the reduction of the number of plastids per sperm is reduced by unequal division; the ptDNA in sperms is preferentially digested during spermatogenesis; and the degradation of the plastids without DNA occurs during or after fertilization. In plants which show biparental transmission of ptDNA, the preferential degradation of ptDNA does not occur.

ACKNOWLEDGMENTS

1 wish to thank Drs. S. Kawano and H. Duroiwa for their valuable advice and encourage- ment and Drs. T. Hori, Y. Sasaki, S. Nakamura, H. Hashimoto, Sodomergan, Y. Nemoto, and S. Miyamura for providing photographs, and Miss K. Mori for providing samples. This work was supported by grant nos. 63440003 and 02242206 from the Japanese Ministry of Education, Science, and Culture and a grant pioneering research project in biotechnology from Ministry of Agriculture, Forestry, and Fisheries of Japan.


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[International Review of Cytology] Volume 128 || The Replication, Differentiation, and Inheritance...

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