In the classical formulation of Munch (1930), plasmodesmata are considered to form simple cytoplasmic bridges between neighbouring plant cells to create the symplasm. This concept has dominated, if not monopolized, the thinking of plant biologists and in particular plant physiologists over the last few decades. Recent advances in ultrastructural, physiological and molecular studies on plasmodesmata indicate that this simple view is in need of revision. Structurally, the higher plant plasmodesma has been revealed to be a supramolecular complex consisting of membranes and proteins. Functionally, evidence is at hand that this complex structure appears to have evolved not only to control the size exclusion limit for intercellular diffusion of metabolites and small molecules, but also to potentiate and regulate intercellular trafficking of macromolecules, including proteins and nucleic acids. In this regard, plasmodesmal transport may share parallel regulatory mechanisms with nucleocytoplasmic transport. Based on these findings, we advance the hypothesis that plants function as supracellular, rather than multicellular, organisms. As such, the dynamics of the plant body, including cell differentiation, tissue formation, organogenesis and specialized physiological function(s), is subject to plasmodesmal regulation. Plasmodesmata presumably accomplish such regulatory roles by trafficking informational molecules which orchestrate both metabolic activity and gene expression. Current and future studies on the evolutionary origin(s) of plasmodesmata are likely to provide valuable information in terms of the genetic and molecular basis for the supracellular nature of plants. Contents Summary 435 I. Introduction 436 II. Plasmodesmal formation, structure and biochemistry 436 III. Evolution of plasmodesmata 445 IV. Symplasmic dynamics 452 V. Plasniodesmal trafficking of macromolecules: parallels with nucleocytoplasmic transport 457 VI. Role of plasmodesmata in plant development 464 VII. Concluding remarks 469 Acknowledgements 470 References 470.

Plasmodesmata and the supracellular nature of plants

Plastids are an important group of plant cellular organelles and comprise one of the primary features that distinguish plant cells from those of other eukaryotes. Plastids are thought to have arisen as a result of an endosymbiotic event in which an early photosynthetic prokaryote invaded a primitive

Plastid division and development

Three D-cyclin genes are expressed in the apical meristems of snapdragon (Antirrhinum majus). The cyclin D1 and D3b genes are expressed throughout meristems, whereas cyclin D3a is restricted to the peripheral region of the meristem, especially the organ primordia. During floral development, cyclin D3b expression is: (a) locally modulated in the cells immediately surrounding the base of organ primordia, defining a zone between lateral organs that may act as a developmental boundary; (b) locally modulated in the ventral petals during petal folding; and (c) is specifically repressed in the dorsal stamen by the cycloidea gene. Expression of both cyclin D3 genes is reduced prior to the cessation of cell cycle activity, as judged by histone H4 expression. Expression of all three D-cyclin genes is modulated by factors that regulate plant growth, particularly sucrose and cytokinin. These observations may provide a molecular basis for understanding the local regulation of cell proliferation during plant growth and development.

The expression of D-cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene.

SUMMARY
Many plant cells are characterized by the presence of a vacuole (hypertrophied lysosome) occupying a large fraction of the protoplast. The possible benefits of the presence of the vacuole can be divided into those which are related merely to the increased volume of the protoplast, independent of the nature of the vacuolar contents, and those which depend on the nature of the solutes in the vacuole.

Increasing the volume of the protoplast per unit volume of cytoplasm increases the total surface area of the cell per unit of cytoplasmic volume. This area amplification has potential benefits in terms of resource acquisition under resource-limiting conditions. At a given chromophore concentration in the cytoplasm, dispersal of a given volume of cytoplasm at the periphery of a vacuole increases the efficiency of photon absorption of an ‘average’ chromophore molecule relative to a comparable non-vacuolate cell, unless the chromophore concentration is low arid/or the cell is very small (no package effect) or the total chromophore per unit projected area is such that almost all light is absorbed, regardless of vacuolation. The area amplification also increases the plasmalemma area per unit cytoplasmic volume through which‘lipid solution’, or mediated transport of chemical resources can occur on a cytoplasmic volume basis and decreases the impediment to resource transfer from a bulk aqueous phase to the cell surface, again on a cytoplasmic volume basis. The increased capacity for uptake of chemical resources from low external concentrations due to vacuolation can increase overall solute acquisition, except in cells of picoplankton size, although other remedies for restricted capacity for chemical nutrient uptake per unit cytoplasmic volume are possible (e.g. evaginations of the cell surface).

In addition to these potential benefits of vacuolation which are essentially independent of the contents of the vacuole, and which relate almost entirely to the rate at which resources can be acquired, there are potentially beneficial effects of vacuolation (dependent on specific vacuolar contents) on the acquisition of resources, and on the storage, manipulation and protection of resources which have already been acquired. Effects of vacuolation which involve essentially irreversible deposition (within the life of the cell) of specific solutes in the vacuole embrace the acquisition, manipulation and protection of resources, but not, of course, true (‘reversible’) storage. Deposition-related increases in resource (at least phosphate and iron) acquisition capacity can, in attached rhizophytic macrophytes, result from rhizosphere acidification involving accumulation of cation salts of organic acid anions in the vacuole. Related to resource acquisition (CO2, H2O) in terrestrial halophytes is the disposal of ‘excess’ salt entering the roots by deposition in hypertrophied vacuoles. Resource manipulation involving deposition of salts of organic acid anions occurs as a means of OH− disposal in the reduction of NO3− in the shoot, a process which can increase the photon and water use efficiency of N assimilation. Resource protection in plants often involves the deposition of antibiophage solutes in the vacuoles. The possible osmotic problems engendered by cation organate deposition can be ameliorated by production of insoluble calcium oxalate.

Reversible accumulation of solutes in vacuoles is involved in CAM, which can increase the water (and nitrogen?) use efficiency of carbon assimilation, and may be involved in the storage of fermentation products during temporary anoxia. The storage of soluble compounds in vacuoles during CAM cannot, apparently, be replaced by that of insoluble materia; there is some scope for storage of insoluble waxes during fermentation. Storage of energy and carbon (as reduced carbon), phosphorus and nitrogen often involves soluble low-molecular weight materials in large vacuoles, but can also, in all three cases, involve polymers in smaller vacuoles or in other parts of the cytoplasm.

Other potentially beneficial effects of vacuolation include a (mechanistically unexplained) stimulation of the velocity of cytoplasmic streaming in large vacuolate cells and a role in buffering cytoplasmic volume from rapid, large changes in cells (e.g. stomata) undergoing large changes in protoplast volume.

Against these putative benefits must be set, in terms of natural selection, the costs of vacuolation. These costs include the costs (energy, solutes) of producing, and those (energy) of maintaining the vacuoles, and the costs (energy, carbon) of synthesizing the extra wall material. The additional costs (all on a unit cytoplasm basis) can be substantial relative to benefits in terms of additional energy and carbon acquisition resulting from vacuolation. Additional costs may be incurred in relation to the energetics of large cells maintaining a constant turgor pressure in environments (e.g. estuaries) with frequent and large changes in external osmolarity, and as colonization opportunities lost if vacuolation reduces desiccation tolerance.

The analysis conducted in the paper suggests that the specific growth rate of a cell of a given cytoplasmic volume under optimal growth conditions is decreased if it is vacuolated. Furthermore, the resource acquisition rate of the cell under conditions of low resource availability may be enhanced by vacuolation, even when the resource costs of vacuolation are taken into consideration, although this is not invariably so. Nevertheless, although the cost-benefit analysis of resource acquisition may not always favour vacuolation, the presence of vacuoles may still lead to an increase in inclusive fitness when benefits in relation to the storage, manipulation and protection of resources are also considered. At all events, most phototrophs are vacuolate, the major exception being many planktophytes (where the lack of vacuoles may be rationalized, at least for smaller cells) and essentially all microalga-invertebrate synthesis.

The role of vacuoles

Accumulation patterns of mRNAs corresponding to histones H2A and H4, ribosomal protein genes rpL27 and rpL34, MAP kinase, cdc2 kinase and cyclin B were analyzed during growth-dormancy cycles in pea (Pisum sativum cv. Alaska) axillary buds. The level of each of these mRNAs was low in dormant buds on intact plants, increased when buds were stimulated to grow by decapitating the terminal bud, decreased when buds ceased growing and became dormant, and then increased when buds began to grow again. Flow cytometry was used to determine nuclear DNA content during these developmental transitions. Dormant buds contain G1 and G2 nuclei (about 3:1 ratio), but only low levels of S phase nuclei. It is hypothesized that cells in dormant buds are arrested at three points in the cell cycle, in mid-G1, at the G1/S boundary and near the S/G2 boundary. Based on the accumulation of histone H2A and H4 mRNAs, which are markers for S phase, cells arrested at the G1/S boundary enter S within one hour of decaptitation. The presence of a cell population arrested in mid-G1 is indicated by a second peak of histone mRNA accumulation 6 h after the first peak. Based on the accumulation of cyclin B mRNA, a marker for late G2 and mitosis, cells arrested at G1/S begin to divide between 12 and 18 h after decapitation. A small increase in the level of cyclin B mRNA at 6 h after decapitation may represent mitosis of the cells that had been arrested near the S/G2 boundary. Accumulation of MAP kinase, cdc2 kinase, rpL27 and rpL34 mRNAs are correlated with cell proliferation but not with a particular phase of the cell cycle.

Cell cycle regulation during growth-dormancy cycles in pea axillary buds.

The ultrastructure of the pea shoot apical meristem was examined quantitatively in longitudinal sections. Photographs were taken at eleven defined positions in the apex, at six developmental stages within a single plastochron. The only change in ultrastructure during the period of a single plastochron was the increase in the proportion of plastids with starch in the central regions of the apex and in the young leaf axils. This increase occurred midway in time between the emergence of successive leaves, at precisely the time that the orientation of growth changes in the region where a new leaf is to emerge. There were quantitative changes in ultrastructure associated with cell differentiation. In the sequence of cell development from the summit of the apex (central zone) to the incipient pith, cell enlargement was accompanied by an increase in the volume of endoplasmic reticulum, dictyosomes, microbodies and vacuoles per cell, an increase in the number of mitochondria, microbodies and vacuoles per cell, and an increase in the volume, but not the number, of plastids per cell. In the sequence of axillary development (before the axillary bud begins to grow) the number of mitochondria per cell decreased as cell volume decreased but the number of plastids per cell remained constant. The number of plastids per cell increased only in the developmental sequence leading to leaf development, in which the number of mitochondria and dictyosomes per cell also increased. There appeared to be no features of ultrastructure, qualitative or quantitative, which could be correlated with the different rates of cell division in different regions of the meristem. The differences in ultrastructure throughout the apex were mainly quantitative and seemed to be associated with cellular differentiation rather than with the plastochronic functioning of the apex during leaf initiation.

The quantitative ultrastructure of the pea shoot apex in relation to leaf initiation

Seven long days induce flowering inSilene coelirosa.
Francis andLyndon (1979) reported a strong synchronization of cell division in the apex on the eighth day. This could be due to a sharp fall in apical cell isolation, leading to syncytium formation or to increased cell isolation, leading to the derepression of cell division. To test the degree of cell isolation, a range of water soluble fluorescent probes was injected into either non-induced or induced apices just before, or at the time of the synchronous division. Probes of molecular size up to 665 daltons moved through vegetative apices. Those of size up to 536 daltons moved in the induced apices. Hence, as judged by this criterion the cells in the induced apices are more isolated than those in non-induced apices. Thus the induced synchronous division may be due to an increase in cell isolation.

Synchronisation of cell division during transition to flowering inSilene apices not due to increased symplast permeability

The length of the cell cycle was measured in the shoot apical meristem ofSilene coeli-rosa during the first day of an inductive photoperiod. The length of the cell cycle in the shoot apex of vegetative controls (those in short days) was about 18–20 hours. Exposure of plants to the long day resulted in an immediate shortening of the cell cycle to about 13 hours, roughly two thirds of that in short days. Measurements of the component phases of the cell cycle revealed that the shortened cycle in long days was the result of a decrease in the length of G 1 and perhaps S, whilst G 2 and M remained constant.

R. F. Lyndon

Papers

The quantitative ultrastructure of the pea shoot apex in relation to leaf initiation

Synchronisation of cell division during transition to flowering inSilene apices not due to increased symplast permeability

The cell cycle in the shoot apex ofSilene during the first day of floral induction