116 F. G. E. Pautard 



survey of other forms of calcification shows at once that in many cases the cell is 

 entirely responsible for salt formation and leads us to alternative suggestions as to 

 how mineralization might take place. 



Before reviewing some recent experiments on bone, however, the results of some 

 of our present studies on the formation of calcium oxalate and calcium carbonate are 

 of interest. 



Calcium oxalate 



A common form of calcium oxalate in plants — the monohydrate — is found 

 within specialized cells in bundles (often as many as 2500) of lath- or needle-shaped 

 crystals up to 200 microns long and about 1 micron square in section. The bundles of 

 raphides are remarkably regular and each crystal is roughly parallel to its neighbour. 

 It is usually supposed that these deposits are "waste products" and it is believed 

 generally that the crystal arrangements arise within vacuoles, each crystal being 

 enclosed within some sort of sheath (see Scott, 1941). Since the crystal cells develop 

 rapidly, often in the root tip soon after it emerges from the seed, development of the 

 mineral can be traced. The present ultrastructural and biophysical studies of raphides 

 in plants (Arnott and Pautard, in press; Arnott, in press) suggest that the crystals 

 do not arise in a haphazard manner but are laid down by a complex cell organization 

 which involves the formation of chambers in which the inorganic material develops. 



In Yucca, for example, these chambers are irregular when they are first formed 

 and they soon become filled with electron-dense material which does not give an 

 electron diffraction pattern. Later, as the chambers assume the characteristic tapering 

 profile of the oxalate crystals, the contents become crystalline and there is evidence 

 that the vacuole in which the chambers develop contains a detailed fine structure of 

 tubes and membranes. In Lemna, the chambers occur in sequence at regular intervals 

 between two membranes; here again the system becomes loaded with an electron- 

 dense, non-crystalline substance during the earlier phases of growth. 



The large intracellular crystals of calcium oxalate, then, might not arise by 

 continuous growth from a single site nucleated by some fibrous orienting surface, but 

 by the rearrangement of a mobile inorganic mixture within compartments which 

 have been prefabricated in the vacuolar substance. 



Calcium carbonate 



Most of our information about the molecular basis of calcium carbonate for- 

 mation comes from the studies of shell development in molluscs, crustaceans, and 

 echinoderms. The evidence (for example, in the sand dollar Echinarachnius [Beve- 

 LANDER and Nakahara, 1960), in Crassostraea (Jodrey, 1953) and in the crustacean 

 exoskeleton (Travis, 1963) suggests that the epidermal cells play a major role in 

 laying down the organic phase. In Mytilis, the whole of the mantle calcium has been 

 reported (Rao and Goldberg, 1954) to turn over in 24 hours. The method by which 

 the mineral is transported to the developing shell is, however, a matter of dispute. 

 Some authors (for instance Wilbur, 1964) assume that the ions are extruded by the 

 cell into the extrapallial fluid; in some cases, the presence of crystals within the 

 mantle cells has been reported (Bevelander, 1953). 



Our present studies of the ultrastructure of the cells in apposition to the de- 

 veloping shell in Pinna and Pedalium (Arnott et al., in press) show that the mantle 

 edge extends during the active phase into long filaments which have a detailed fine 



