10.1.1 Generating cells and organelles: control of division processes

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Figure 10.1 Cell reproduction then cell growth contribute to the final length of normal dark-grown wheat coleoptiles, but if cell division is disrupted by gamma irradiation of seed, the increase of coleoptile length is entirely due to cell elongation which continues much as before. (a) Coleoptile length; (b) cell number; cell length. Normal plants (○); gamma plants treated with 500 krad of γ-radiation (●).

(Based on Rose and Adamon 1969)

Much of the cell division in plants occurs in meristems, which provide a continual supply of new cells for increase in root or stem length (see Section 7.1) or for lateral growth. This is not the case for all plant organs, for example dicotyledonous leaves, cereal coleoptiles and fruits, where there is a phase of cell division throughout most of the young organ followed by cell enlargement, leading to a final organ size dictated by a com-bination of genotype and environment.

The growth pattern of parenchyma cells of dark-grown wheat coleoptiles (Figure 10.1) illustrates the interrelationship between cell enlargement and cell division. These coleoptiles reach a final height of 60 mm. Between 24 and 48 h after germination, cell numbers double and cell length increases slightly. After 72 h, cell division ceases and cell elongation takes over. Coleoptiles at 72 h are therefore a favoured system for studying auxin-induced cell enlargement without the complications of simultaneous cell division. Gamma irradiation inhibits cell division by damaging chromosome structure, but growth by cell enlargement can still occur. Final coleoptile height, however, is reduced proportionately less than the reduction in total cell number, because greater than normal final cell dimensions allow some compensation. Similarly, colchicine stops cell division but roots can still initiate short lateral branches called primordiomorphs, which are entirely the result of cell expansion (Foard et al. 1965).

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Figure 10.2 Four mitotic stages are visible in this squash preparation of a rapidly dividing root apical meristem of Vicia faba: prophase (P), metaphase (M), anaphase (A) and telophase (T).

(Magnification x 1200).

Plant cell division, besides providing a source of new cells, requires replication of the genome, and equal distribution of this coded information in the form of paired chromosomes to daughter cells. The anaphase stage of mitosis is where chromosomes are separated as the microtubule spindle contracts (Figure 10.2), guaranteeing identical gene complements for every somatic cell. Plant cells are encapsulated by a cell wall, so during cytokinesis (i.e. cell division, as opposed to mitosis, which is chromosome separation) a ‘cell plate’ forms to divide the parent cell into two daughters, usually of similar size. The cell plate subsequently matures to become a new primary cell wall. However, replication and division of the nuclear genome is not sufficient to maintain normal cell functioning, because in plants there are two additional genomes, residing in the plastids and mitochondria. These, too, must be multiplied.

(a) The cell cycle

The cell cycle encompasses the growth of a cell during the cell division cycle, the replication of its genomes and the formation of two daughter cells containing all the necessary information to repeat the cycle. For convenience, we normally deal with the cell cycle as a series of discrete segments, based on alternate phases of nuclear DNA replication (the synthesis or S phase) and mitosis (the M phase) where newly replicated chromosomes segregate into daughter cells (Figure 10.3). There are gaps between these phases: G1 (Gap 1) between cytokinesis at the end of mitosis and the S phase, and G2 (Gap 2) between the S phase and mitosis. During G1, crucial events occur that determine the timing of initiation of DNA replication, in other words the entry into the S phase. Once the S phase commences, it needs to follow a highly ordered process starting with each chromosome as a single DNA molecule and ending with each as two identical DNA molecules — sister chromatids. In G2, the events that control entry of cells into mitosis occur. Duration of the G1, S and G2 phases varies enormously depending on the cell type, normally being measured by applying a pulse of [3H]thymidine, which is specifically incorporated into DNA, followed by autoradio-graphy. Root apical meristems are a conveniently accessible rapidly dividing tissue for such studies. In maize (Zea mays) root tips, Barlow (1973) found that G1 lasts 3 h, the S phase lasts 5 h and G2, 6 h. Mitosis itself lasts around one hour, in which time the cell goes through the familiar prophase, metaphase, anaphase and telophase (Figure 10.2). Of course, the cell also needs to double in size otherwise cell dimensions would diminish with each division cycle. Cells can temporarily suspend the cycle and enter a rest or quiescent phase, some-times referred to as G0. Permanent exit from the cell cycle and progress into a differentiation pathway is possible at G1 or G2.

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Figure 10.3 The cell cycle in plants comprises four phases: mitosis (M), Gap 1 (G1), DNA synthesis (S) and Gap 2 (G2). Key transitions in the cycle (G1 → S and G2 → M) are regulated by cyclin-dependant kinases (CDKs) which in turn are influenced by several factors including the plant hormones auxin and cytokinin.

Control of progression through the cell cycle

How are the complex sequences through the cell cycle regulated? In plants, we have less complete evidence than is available from simpler organisms such as yeasts or the more extensively studied mammalian cells, but the principles appear similar. Using specific inhibitors, we can demonstrate that protein synthesis is required for progress at several points throughout the cell cycle. The key proteins are collectively known as cyclins, whose abundances change in very precise ways as the cell cycle operates. Cyclins activate regulatory enzymes called cyclin-dependent kinases (CDKs), a group of enzymes that add phosphate groups to other proteins. Two key cyclin-controlled points in the cell cycle are the G1→S and G2→M transitions (Van’t Hof 1985). Entry into the S phase requires activation of the replication machinery onto a series of specific sites along the DNA called ‘origins of replication’. Importantly, DNA must replicate only once per cycle, and mitosis, of course, should not proceed until the S phase is complete. Control of the S and M phases enables the processes of each to be mutually exclusive, and to have an obligate alternating sequence.

The G1 control point is called ‘Start’. At this point, a cell becomes committed to proceeding through the cell cycle. Provided no essential cell cycle component is limiting, the complete cycle will continue from here (Chasan 1995). Induction of a specific cyclin may precipitate a cell’s capacity to divide, and also initiates the subsequent sequence of events regulated by its CDKs. In plant cells, as in other organisms, there are links between cyclins, CDKs and cell cycle control (Jacobs 1997). Here we focus on one CDK called p34cdc2, a key player in the plant cell cycle (Gorst et al. 1991). The cdc (cell division cycle) gene which encodes the p34cdc2 protein was originally identified in yeast, then a homologue was found in Arabidopsis, designated cdc2a. In this species, cdc2a expression is linked to competence for cell division (Hemerly et al. 1993). Differentiated leaf mesophyll cells, when induced to recommence division, show increased cdc2a expression. Very large terminally differentiated cells are commonly endo-polyploid, and this condition appears to relate to failure of cyclins to be activated, so leading to DNA replication without mitosis, and hence doubling of the chromosome complement.

Several plant cyclins and CDKs have now been identified, and many more are deduced to exist. They each relate to control through one of the four key phases of the cell cycle. D and E cyclins are associated with initiation and completion of DNA replication, while A and B cyclins function in mitosis. There are also questions of how biosynthetic control of cell growth and plastid and mitochondrial division cycles are all integrated into the cell cycle.

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Figure 10.4 Chloroplasts contain multiple copies of their circular chromosome (solid circles), associated with thylakoid membrane (straight line). In this experiment on cultured spinach leaf discs, DNA with a [3H]thymidine pulse, followed by incubation in unlabelled thymadine ('chase'). Data indicate that label (dashed circles) was present on every new DNA strand and that equal numbers were segregated to each daughter chloroplast during division. In the next round of synthesis, each DNA molecule was replicated again, this time with unlabelled thymidine present.

(Based on Rose 1988)

(b) Plastid and mitochondrial division during the cell cycle

Plastid division cycle

All plastids (proplastids, chloroplasts, amyloplasts, chromoplasts, etioplasts, leucoplasts) develop from pre-existing plastids and are not produced de novo. Most commonly, differentiated plastids derive from proplastids. All plant cells contain plastids of some type and these are partitioned to daughter cells during cytokinesis. Because plastids carry their own genome and because of the need to maintain plastid number per cell, plastids conduct their own DNA replication and division cycle. Remarkably, this process seems to be tuned to the pace of the cell cycle. Chloroplasts are the most abundant and therefore the most important and best-studied plastid type. Replication of chloroplast DNA (cpDNA) and its partitioning at chloroplast division is monitored in a similar way to the nuclear genome, typically by application of a [3H]thymidine pulse. In spinach leaf discs, cpDNA was segregated in approximately equal amounts to daughter chloroplasts, consistent with semi-conservative DNA replication (Figure 10.4). Two points deserve mention: the 150 kilobase chloroplast chromosome is present in multiple copies per chloroplast, and each chloroplast has many nucleoids (Figure 10.5) containing a variable number of cpDNA copies. These characteristics mean that, unlike the cell cycle, the chloroplast division cycle does not have discrete G1, S and G2 phases. Instead, DNA synthesis occurs throughout the plastid division cycle. cpDNA segregation is possible because the nucleoids are attached to a membrane system which distributes DNA to daughter cells in a manner akin to mechanisms in prokaryotic cells.

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Figure 10.5 Flourescence micrograph of isolated tobacco mesophyll protoplast stained with DAPI which causes blue-white flourescence from DNA, visible both in the multiple nucleoids (small bright specks) present in each chloroplast and in the nucleus on the right. Background signal in chloroplasts is chlorophyll autoflourescence (x 1500).

(Based on Thomas and Rose 1983)

Chloroplasts divide by constricting into two (Figure 10.6), but how is this achieved? Under the transmission electron micrograph we can see a doublet ring associated with the constriction, consisting of fibrils on the cytoplasmic side of the outer chloroplast membrane and also on the stroma side of the inner membrane (Figure 10.6b). The fibrils are probably a cytoskeletal-like contractile protein that causes the constriction. Inhibitor and mutant experiments indicate that replication of cpDNA and chloroplast division are both under nuclear gene control and not regulated by cpDNA genes. Pyke et al. (1994) identified chloroplast division mutants with giant chloroplasts which probably have defects in these nuclear genes.

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Figure 10.6 Images of dividing plastids. (a) Transmission electron micrograph showing constriction prior to division (scale bar = 1 μm). (b) Detail and (ii) diagram of constricting doublet ring visible as densely stained areas inside and outside double plastid membrane, arrowed; oe, outer chloroplast membrane; ie, inner chloroplast membrane; c, cytoplasm; s, stroma (x 90 000; scale bar = 0.1 μm). (c), (d) Nomarski optic micrographs showing (c) accumulation of 'dumb-bell' shaped pre-division plastids caused by keeping spinach leaf discs under low light intensity for 5 d. (d) Normal division resumes when leaves are transferred to higher light intensity (x 1400).

(Based on Hashimoto 1986 and Possingham and Rose 1976)

Meristematic leaf cells contain approximately 10 chloro-plasts, which have developed from proplastids. In early leaf development, plastid number per cell and cpDNA content per plastid both remain constant. When cell division ceases, the cells grow rapidly giving room for dramatic increases in chloroplast numbers. In spinach, the final chloroplast number per cell can exceed 200. During the initial phase of cell enlargement, the rate of cpDNA replication exceeds the rate of chloroplast division, so the cpDNA copy number per chloroplast rises from about 50 to 200. Subsequently, cpDNA synthesis switches off but division continues, leading to a final copy number of about 30 (Figure 10.7). It is probably no coincidence that the maximum copy number coincides with the period of greatest demand for biosynthesis of chloroplast components. We also suspect that early completion of cpDNA production leaves the cell’s biosynthetic machinery free for later direction towards biogenesis of the photosynthetic membranes and proteins.

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Figure 10.7 Numbers of chloroplasts per cell () increase during leaf development but numbers of copies of cpDNA, expressed as plastomes per chloroplast (●) are maximal early in development, presumably coinciding with maximum demand for chloroplast gene expression.

(Based on Possingham and Lawrence 1983)

Chloroplast size and numbers per cell also depend on environmental factors such as light, temperature and mineral nutrients. Chloroplasts, unlike other plastids, require high light intensity in order to divide. Low light levels lead to large chloroplasts blocked at a partly constricted stage (Figure 10.6c), but transfer to high light leads to resumption of the normal division process (Figure 10.6d).

Mitochondrial division

We know much less about mitochondrial division, partly because these organelles are smaller than plastids, and therefore difficult to observe live under the light microscope. Nevertheless, studies on several species by Kuroiwa point strongly to a division cycle similar to the plastid process (Figure 10.8). The inner mitochondrial membrane has nucleoids associated with it, and division is again effected by constriction into two. Larger numbers of mitochondria are present in cells with high energy demand, such as phloem companion cells, anther tapetum and thermogenic spadix tissues of flowers in the Araceae.

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Figure 10.8 Division of mitochondria, from embryo of Pelargonium zonale, is similar in appearance to the process in plastids, with formation of a central constriction (x 29 000).

(Photograph courtesy H. Kuroiwa and T. Kuroiwa)

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