T H E F U N C T I O N OF CALCIUM I N PLANTS 1

R. G. W Y N JONES ANB O. R. LUNT

D e p a r t m e n t of B i o c h e m i s t r y & Soil Science, University College of N o r t h W a l e s , Bangor, W a l e s , and D e p a r t m e n t of Botanical Sciences University of California L o s A n g e l e s , California, 9 0 0 2 4

Introduction ................................................................................................................................................................................................ 407 The calcium requirement of plants ................................................................................................................................ 408 Anatomical and cytological effects of calcium deficiency ........................................................................410 Gross defects ................................................................................................................................................................................... 410 Cellular and subeellular effects ...................................................................................................................................... 411 Physiological and biochemical evidence for the role of calcium ................................................. 412 Cell wall ................................................................................................................................................................................................. 412 Ion absorption and membranes ...................................................................................................................................... 414 Ion absorption by whole cells ...................................................................................................................................... 414 Mitoehondria ............................................................................................................................................................................... 416 Membranes and model systems ................................................................................................................................417 Nucleic acids and chromosomes ......................................................................................................................................418 Enzyme activity .............................................................................................................................................................................. 418 Concluding discussion .................................................................................................................................................................... 420 Literature cited ....................................................................................................................................................................................... 421 INTRODUCTION The problem of the precise biochemical function of some of the essential nutrient elements in plant metabolism, including that of calcium, has proved extremely intractable. Calcium was recognized as an essential element in higher plants during the middle of the last century (see True 1922) although the use of limestone and marl in agriculture dates back to pre-Christian times (Tisdale & Nelson 1966). However, the role of calcium in agriculture and soil management involves many factors, a number of which are only indirectly related to the nutritive value of the element (Chapman 1966). The difficulties experienced in investigating the role of calcium may be illustrated by quoting from Ludwig Jost (see True 1922), who wrote in 1907 that " . . . we are bound to admit that its function (i.e. calcium) has not yet been discovered," and from a recent review by Nason and McElroy (1963), who concluded that " . . . the role of calcium is not entirely clear." However, considerable evidence, some of recent origin, has accumulated about the possible 1. The work reported in this paper and the preparation of the manuscript were supported by National Science Foundation Grant B5-1226 to Dr. O. R. Lunt. 407

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functions of calcium and it appeared worthwhile to attempt to integrate the cytological, physiological, and biochemical data. During this process we have been drawn to three main areas of plant metabolism, namely the cell wall, membranes, and chromosomes. In addition, there is evidence of calcium involvement in the maintenance of the configuration of specific enzymes. T H E CALCIUM R E Q U I R E M E N T OF PLANTS The analyses of a great variety of crop plants under many environmental conditions indicate that calcium levels of several per cent on a dry weight basis are commonly found in the foliage of legumes, tomato, tobacco, and other dicotyledons (Beeson 1941) (Table I ) . Grasses and cereals generally have somewhat lower concentrations of the element; 0.2-0.5 per cent dry weight (approximately 10 m moles/kg, wet weight) is usually reported. In both monoand dicotyledons the roots ordinarily contain significantly less calcium than the tops. In keeping with the high values of the ion reported, the solutions used for hydroponic culture (Hoagland & Arnon 1950) and for callus or root-tip culture (White 1943) contain 200-300 p.p.m, calcium salts or about 2-3 mM Ca + + ions. In view of these observations it is natural that calcium should be regarded as a macronutrient element. However, several indications have appeared recently TABLE I
CALCIUM LEVELS IN PLANTS UNDER VARIOUS GROWTH CONDITIONS PLANT ORGAN GROWTH CONDITION CA CONTENT % DRY WT. REF.

Corn

Wheat Oats Alfalfa Potato Tobacco

mature leaves grain leaves roots straw grain above ground portion above ground portion tuber leaves leaves remainder of shoot roots leaves

field field solution, 2 p.p.m. Ca solution, 40 p.p.b. Ca field field field field field field solution, 200 p.p.m. Ca and 50 p.p.m. Mg solution, 200 p.p.m. Ca and 50 p.p.m. Mg solution, 200 p.p.m. Ca and 50 p.p.m. Mg solution, 2 p.p.m. Ca

0.34 0.015 0.01 0.005 0.29 0.05 0.2 2-3 0.043-4 0.41.1 0.2 0.08

1 1 2 3 1 1 1 1 1 1

2 2

References: 1) Beeson (1941), 2) Wallace, Frolich, & Lunt (1966), 3) Jones & Lunt (1968).

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which suggest that the calcium requirement of many higher plants has been overestimated. Pharis, Barnes, and Naylor (1964) reported a very low requirement in Pinus taeda L. whereas Wallace, Frolich, and Lunt (1966) were able to grow both tobacco (Nicotiana tabacum LOaM corn (Zea mays L.) in a solution containing only a few parts per million of Ca, provided other potentially toxic, although essential, ions such as Mg, Cu, Fe, Mn were kept at a low balanced level. Under these conditions the leaf tissues of corn contained 0.01-2 per cent dry weight of calcium, or 0.1 per cent in tobacco. Tall wheat grass (Agropyron elongatum (Host) Beauv.) grew normally in low calcium media and the shoots contained only 200 p.p.m. (0.02 per cent dry weight) (Lunt & Jones 1968). The normal development of corn roots required only 3-10 [zM Ca ions in the external solutions in the absence of other ions (Jones & Lunt 1968), and healthy roots contained only 0.007 per cent Ca dry weight. However, the presence of toxic ions, e.g., 0.1 [zM cupric ions, increased the calcium required in the external solution to 100-200 lzM. The ability of Ca ++ to counteract the adverse effects of increasing H + concentration on root elongation (Biirstrom 1952, 1954) and ion absorption (Jacobson, Moore, & Hannapel 1960, Rains, Schmid, & Epstein 1964) is well documented. The ability of calcium to protect against heavy-metal toxicity was observed in the early studies by True (1914), and more recently with Chlorella (McBrien & Hassall 1965), yeast (Chester 1965), and a variety of bacteria (Nicholas 1963). The observations clearly imply that the high levels of calcium utilized in plant growth are required primarily to ameliorate toxicities and the role of calcium under ideal conditions may approach that of a micronutrient. This situation more nearly approaches that found in lower plants where numerous examples exist of a very low calcium requirement (Nicholas 1963, Bollard & Butler 1966). A number of reports have appeared suggesting that calcium is not required for the growth of several species of yeasts and fungi. The literature in this field is well documented in the excellent review by Bollard and Butler (1966). In fungi it appears that there is a higher calcium requirement for sporulation than for vegetative growth. A close relation between calcium and other divalent cations and the structure and viability of spores has also been reported in several bacilli (Slepecky & Foster 1959). In general, algae require calcium, although in Chlorella this requirement is tess than a part per million in the medium. As in higher plants, it may be assumed that the observed response to calcium is greatly influenced by the other ions in the external medium. O'Kelley and Herndon (1961), in discussing the calcium requirement of algae, suggested that it may be related to the quantity of pectic material secreted by the cells. Although the requirement for calcium has been widely studied in plants, little is known about the distribution of the ion within the cell. Many technical difficulties are involved in the use of soluble isotopes for radioautography. Gielink, Sauer, and Ringoet (1966) have recently reported that freeze-substitution using acetone yields satisfactory autographs in which the calcium is localized mainly in the cell wall. Freeze-dried paraffin-embedded material has been

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used by L. Jacobson (personal communication, 1965) who found the isotope mainly in the cell wall and the nucleus. However, both these techniques may be criticized and a much more elaborate and detailed study is required to elucidate the problem. The actual cytoplasmic and vacuolar concentration of calcium has been reported in Nitella by Spanswick and Williams (1965). They found 8 mM calcium in the cytoplasm and 12 mM in the vacuole and suggested that there is no necessity for a calcium pump at the plasmalemma because the low cytoplasmic calcium content could be due to low permeability. Millikan and Hanger (1965) have reported extensively on the intercellular movement of Ca 45 in subterranean clover as influenced by ion levels and chelating agents and have shown the differential distribution of calcium during deficiency conditions. ANATOMICAL AND CYTOLOGICAL EFFECTS OF CALCIUM DEFICIENCY GRoss DEFECTS The visual effects of calcium and other deficiencies in many crop plants may be found described by Wallace (1961) and in "Diagnostic Criteria for Plants and Soils" (Chapman 1966). A great deal of valuable information may also be found in Hewitt's excellent review (1963). Three principal symptoms commonly result from calcium deficiency, although other defects have also been reported. Low calcium status may result in a blackening and curling of the margins of the apical leaves, ultimately leading to acute necrosis and the cessation of growth. The physiological foundation for this deformation may be complex, however, as it is closely related to the magnesium status and occurs more readily at high magnesium levels (Hewitt 1963; E. FroIich, unpublished observations 1966). It may also be noted that in many plants the symptoms of calcium deficiency closely resemble those of aluminum toxicity (Bollard & Butler 1966). Since the typical ecological conditions in which these abnormalities would occur are so similar, it may be difficult to distinguish between them. Poor root development is also commonly associated with calcium deficiency. However, there are various indications that two phenomena, which may or may not be related, are involved. There is evidence of an intracellular physiologicaI requirement for calcium, as we shall discuss. In addition, there is evidence of an external requirement for divalent cations, usually calcium, in the medium surrounding the roots. In early studies True (1914) found that distilled water was toxic to plant roots and suggested that calcium was required in the external medium to maintain the selective permeability of the cell membrane and prevent leakage. Haynes and Robbins (1948) similarly concluded that calcium and boron must be present in the external medium for root development. Our own studies (Jones & Lunt 1968), although not in fundamental disagreement with these observations, suggest that further investigation is required. Corn roots exposed to distilled-deionized water (max. 15 p.p.b. Ca) cease elongating within hours but show only little and very localized damage. There is no evidence of a major breakdown of selective permeability after 7-10 days, because little potassium is released and the rate of rubidium uptake is only

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about half that found in normal roots. However, there is almost total breakdown of the cytoplasm of the cells in the root apex. Roots placed in ca. 1.0 jam alkali earth ions elongate rapidly. The reason for the dramatic influence of calcium and related ions is not clear but it may be a calcium requirement of the membrane structure which may be more acute in dividing cells. As calcium is poorly translocated (Biddulph, Cory, & Biddulph 1959), it would also be expected that the apex would be more severely affected. The results of work on the influence of calcium on root development are also hampered by the close relationship between calcium status and the toxicity of other ions. As pointed out by Edwards (1936), the omission of calcium from an otherwise complete nutrient solution exposes the roots to a highly toxic environment of potassium, magnesium, and other micronutrient ions. A confirmation of this view is the recent demonstration (Wallace, Frolich, & Lunt 1966) of the normal growth of tobacco and corn with one-fiftieth the level of calcium in Hoagland's solution provided other ions are in balance. Thus it is probable that many early reports on the toxicity of distilled water reflect the extreme toxicity of many ions, e.g., 0.1 tzM Cu ++, in the absence of calcium. The third defect associated with calcium deficiency is a decrease in fruit and seed formation. Hewitt (1963) describes the collapse of the ovule, with irregular necrosis of the embryonic cotyledons and shrinkage within the integuments, as occurring in broad-bean, pea, and French bean as a result of calcium deficiency. Likewise, calcium was required for the differentiation of sporangia and gemmae in an Achlya species (Griffin 1966). An important role for calcium in the Jn v#ro germination of pollen and tube growth is indicated by the work of Kwak (1965) and De Bruyn (1966). In view of this response to calcium and tropic growth of pollen tubes of Antirrhdnum majus L. towards a calcium source (Mascarenhas & Machlis 1964), Mascarenhas (1966) measured the distribution of calcium in the gynoecium but the Jn vivo results did not support the idea of a calcium-directed growth of the pollen tube. Calcium deficiency readily induces "blossom-end rot" in tomato and may be associated with "black-spot" in apples. The detailed anatomical changes in the former are discussed by Spurt (1959). The growth of the peanut fruit (Arachis hy[;ogaea L.) also exhibits a marked calcium requirement, whereas Hewitt (1963) reports that grain formation is often suppressed in cereals during deficiency. CELLULARAND SUBCELLULAREFF;.CTS In the years before the Second World War the cytological effects of calcium deficiency received much attention but conflicting results were obtained by the various groups. With the advent of the electron microscope interest in this problem has renewed. In 1929 Sorokin and Sommer reported that the omission of calcium from a complete nutrient medium caused the rapid degeneration of the apical cells of Pisum sativ.m L. They found a decrease in cytoplasm and increased vacuoIation of the meristematic cells. Abnormal mitotic figures and

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polyploidy were observed. However, the authors specifically denied any adverse effects on the cell wall. They felt that their results did not substantiate the concept of calcium pectate as a cell-wall binding agent. These results were severely criticized by Edwards (1936) both because of histological artifacts and because of the toxic effects of the other ions in the medium, as previously mentioned. Bamford (1931) regarded the chromosomal defects as a terminal effect of calcium deficiency and ionic imbalance. However, since this time various reports have appeared indicating a close association between calcium deficiency and chromosomal abnormality (Steffensen 1958, Hyde & Paliwal 1958; see Hewitt 1963). In contrast to Sorokin and Sommer (1929), Read (1907) had found irregularities in the cell walls of calcium-deficient cells and noted an inability to form a new cell plate during cell division, which he attributed to the absence of calcium. The recent electron microscopic studies by Marinos (1962) on barley stem apex revealed extensive membrane and mitochondrial disintegration in deficient cells. Florell (1956) previously reported fewer mitochondria in wheat roots low in calcium whereas comparable membrane and cytoplasmic breakdown has also been observed in barley roots (Marschner & Gunther 1964, Marschner, Handley, & Overstreet 1966). The latter work also illustrated cytologically the ability of Ca ++ to protect against H + toxicity which had previously been observed from leakage and growth measurements. In our work (Galey, Jones, & Lunt 1968) we have also observed the substantial break-up of the endoplasmic reticulum and other cytoplasmic membranes in the cells of corn root apices exposed to a divalent-ion-free environment. It would appear therefore that cytoplasmic structure is adversely affected by calcium deficiency although this process is greatly worsened by further ionic imbalance (Sorokin & Sommer 1929, Marschner, Handley, & Overstreet 1966). THE PHYSIOLOGICAL AND BIOCHEMICAL EVIDENCE FOR THE ROLE OF CALCIUM The evidence regarding the physiological function of calcium falls largely into four categories: the cell wall, membranes, chromosomes, and enzyme activation. As in any compartmentalization of this type, it is a simplification which tends to blur certain relationships and is at best rather arbitrary. However, we hope that in adopting this layout the field will be enlightened rather than obscured. THE CELL WALL The hypothesis of calcium pectate acting as a cementing agent in the cell wall of plant cells was originated during the last century (see True 1922) and has found general acceptance in plant physiology texts. However, the attempt by Bennet-Clarke (1956) to relate the proposed role of calcium as an ionic bridge to auxin action has been subjected to considerable criticism. It was

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proposed that IAA" stimulated cell elongation by complexing calcium in a manner comparable to EDTA and thus increasing the plasticity of the cell wall. However, neither Cleland (1960) nor Burling and Jackson (1965) detected any release or redistribution of Ca ++ during IAA-stimulated growth of coleoptiles, as would be expected from the calcium-bridge hypothesis. Considerable evidence has accumulated that the formation of calcium pectate increased the rigidity of the cell wall (Tagawa & Bonner 1957, Rasmussen 1966, Cormack 1965). The process of calcification increases the resistance of tissue to infection probably by increasing the resistance of the walls to polygalacturonidase (Bateman & Lumsden 1965). Further evidence for a role of calcium in the cell wall may be deduced from the use of EDTA and EDTA with protein or lipid-dissolving agents to increase tissue maceration and to produce isolated cells (Ginzberg 1958). EDTA treatment also causes the removal of Ca ++ and pectin and increases cell plasticity (Taylor & Wain 1966). A more complex relationship between cell rigidity, elongation, and calcium is indicated by the detailed and extensive studies of Burstr6m (1952, 1954, 1957). He concluded that root cell growth occurs in two stages: a) an increase in plasticity and elasticity of the cell wall, and b) the biosynthesis and laying down of new cell-wall material. The first stage is enhanced by auxin but antagonized by calcium, whereas the relationship is reversed in the second stage. Burstr6m evolved this postulate to explain the differences between his observation of Ca+ +-induced elongation of root cells and the effect of Ca ++, noted by Cormack (1949), of hardening and producing shorter cells. Burstr6m's proposal for the role of IAA in root tissue differs somewhat from the recent work of Morr6 and Bonnet (1965), who find that IAA inhibited root growth by shortening the period of cell elongation. Little change in pectin and hemicellulose contents of Ca-deficient roots was found (Burstr6m 1958), although a 20 per cent decrease in cellulose content occurred. In comparable studies on coleoptile cell-wall constituents, Ray and Baker (1965) found that the synthesis of matrix polysaccharides except ~-cellulose was induced in coleoptile segments by IAA when elongation is inhibited by Ca + +. In the absence of Ca + + an increased synthesis of ~ -cellulose was also observed. The comparison of these results is complicated by opposite effects of ions on root and coleoptile elongation. The former is induced by Ca ++ whereas the latter is stimulated by monovalent cations but not by divalent ions (Purves 1966). It is difficult to determine whether these interactions are related to a direct influence on the cell wall or to a more subtle alteration in metabolism. IAA at low concentration has been shown by Sarkissian and McDaniel (1966) to increase the respiration and the P:O ratio of maize-scutellum mitochondria 2. The following abbreviations are used: EDTA, ethylene diaminetetraacetic acid; IAA, indoleacetic acid; RNA, ribonucleic acid; sRNA, soluble RNA; RNAase, ribonuclease; DNA, deoxyribonucleic acid; NADPH, reduced nicotinamide adeninedinucleotide phosphate; ATP, adenosine triphosphate; ATPase, adenosine triphosphatase; K,, Michaelis constant.

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whereas Hanson, Malhotra, and Stoner (1965) have demonstrated that calcium rechannels energy from ATP formation to ion accumulation. Thus both compounds may substantially alter the metabolic balance within the cell. It is interesting to note that Lamport (1965) has proposed a hypothesis of cell-wall elongation based on the concept of IAA increasing the NADPH level and therefore stimulating the reduction of sulfhydryl bonds of the cell-wall protein. He suggests that IAA causes an increase in ATP levels which in turn channel glucose-6-phosphate into the NADPH-generating pentose phosphate cycle. Thus these metabolic interactions must aIso be considered as well as cell-wail depositions when interpreting the varying effects of calcium on cell elongation. The results suggest that the deposition of calcium pectate in the cell wall increases the rigidity of the structure. However, it is not clear how vital this process is to the growth of the plant in the absence of stress, as such low levels of calcium have been found in seemingly healthy tissue, particularly roots. The complex interactions thereto described cannot be resolved into a unified hypothesis, although it is possible that they relate more to the interaction of calcium with membranes than with the cell wall. ION ABSORVTIONAND MEMBRANES

a) Ion absorption by whole cells

As previously discussed, there is evidence from both light- and electronmicroscopy that calcium deficiency causes a breakdown of membranous structures and the decreased synthesis of mitochondria. A further substantial body of evidence relating calcium to membrane integrity comes from work on ion absorption. Under certain conditions calcium enhances the uptake of potassium by excised roots and other tissue--the Viers effect (1944). In a comprehensive study Jacobson and his collaborators (1960, 1961a, b) showed that the influence of Ca ++ on the uptake of monovatent ions is dependent on the pH. K + absorption is rapidly inhibited by increasing H + and calcium is able to counteract this effect. The rapid loss of ions from excised roots at low pH is also minimized by Ca ++ ions. However, at mildly alkaline or neutral pH, calcium either has no effect on potassium uptake or is slightly inhibitory. At neutral pH calcium enables roots preloaded with K + to retain the ion which would otherwise be leached out (Marschner, Handley, & Overstreet 1966). Epstein (1961) concludes from experiments on the competition between Na and K that calcium is required at 1 mM for the maintenance of the selectivity of the absorption process. Later results (Rains, Schmid, & Epstein 1964) showed that influence of Ca on Rb uptake was highly dependent on pH. These results imply that calcium decreases the efflux of ions, particularly in a toxic ionic environment, and is essential for the integrity of the boundary membranes, possibly the plasmalemma. However, Waisel (1962) maintains that calcium increases the rate of passive diffusion of the potassium across the plasmalemma. Handley and his colleagues (1965), in an attempt to dissociate the cytoplasm and vacuole, studied the influence of Ca on K and Na uptake and release by the first unvacuolated (0-1.8 ram.) and second vacuolated (1.8-3.8 ram.) segments of corn root. They suggest that K absorption into the cytoplasm is passive and that calcium inhibits this

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diffusion by decreasing the pore size. The active uptake into the tonoplast, however, is stimulated by calcium. These interpretations differ fundamentally in the concept of the place and mechanism of ion absorption. Epstein, Rains, and Elzam (1963) demonstrated a biphasic absorption isotherm of potassium by barley roots. The two phases have been attributed by Torii and Laties (1966) to a low Ks system operating at the plasmalemma and a high K, system operating at the tonoplast. At the higher concentration the plasmalemma system is saturated and diffusion becomes increasingly important. A further modification of these ideas has appeared recently (Pitman & Saddler 1967). Evidence is produced to show that the low K, system at the plasmalemma is a sodium/potassium exchange pump. In several animal tissues, e.g., red blood cells, nerve fibers, the uptake of potassium is accompanied by a metabolically mediated movement of sodium across the membrane against the electrochemical gradient. The movement is mediated by a K + and Na +stimulated ATPase enzyme which is usually inhibited by Ca ++ (Skou 1965). This type of sodium exclusion pump has not previously been reported as operating in root ion accumulation (see Skou 1965). In addition, an ATPase activity has recently been reported in plant cell walls which is stimulated by K + and is dependent on Ca ++ or Mg ++ (Dodds & Ellis 1966). The observations correlate very well and yield the attractive hypothesis that the influence of calcium on potassium uptake is due to its role as an activator of ATPase. A possible explanation for the anomalous results obtained by Handley, Metwally, and Overstreet (1965) may lie in the relatively high concentration of potassium, 1 mM, they employed. The low K~ system of Epstein and Laties is saturated at one-fifth to one-tenth of this concentration and may not be observed under the conditions employed. An alternative approach to the role of calcium in membranes and ion absorption has been the use of EDTA to remove a considerable proportion of the tissue calcium. Hanson (1960) found that treatment of excised roots with EDTA, RNAase, or KCI caused decreased respiration and the loss of nucleotides to the solution. Calcium and magnesium protected the cells from the damage. In later work (Foote & Hanson 1964) these treatments were found to cause an initial increase in potassium uptake which was attributed to a rapid flux of potassium into the vacated exchange sites of the cytoplasm. The loss of calcium and degradation of RNA was thought to increase the membrane permeability. Tanada (1955, 1956) had previously shown the adverse effects of ultraviolet light irradiation and RNAase on absorption. On this basis it was suggested that a Ca-ribonucleoprotein complex is involved in potassium absorption. The removal of calcium by EDTA also prevented chloride and phosphate absorption. The impaired uptake of phosphate, nitrate, chloride, and bromide has previously been demonstrated in calcium-deficient tissue (Foote & Hanson 1964, Hooymans 1964, Pitman 1965, Hyde 1966, Legget, Galloway, & Gauch 1965). The increased leakiness of beet root disks exposed to EDTA was reported by Van Steveninck (1965). However, somewhat different results were obtained by Hirata and Mitsui (1965), who found that both EDTA and MgEDTA treatment induced the loss of calcium and caused decreased potassium

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absorption. Only EDTA and not Mg-EDTA caused nucleotide leakage; thus no direct relation between the influence of CA on K uptake and RNA metabolism was postulated. The difference between the results of the Japanese workers and those of Hanson and his group are probably due to the specific conditions employed, as Foote and Hanson noted that longer exposure to EDTA decreased potassium uptake. Possibly a tenuous balance exists between destruction of cytoplasm and increasing the number of exchange sites.

b) Mitochondria
The major problem in the use of excised roots or storage tissue disks in studying ion absorption is the complexity of the cells and tissue employed. One method of simplifying the problem has been the use of isolated mitochondria. The work of Lehninger (1967) and others demonstrated that animal mitochondria can accumulate large quantities of Ca ++, Mg ++, and inorganic phosphate when an energy source is provided, and under certain conditions can also take up K + (Harris, Cockrell, & Pressman 1966). Hanson and his colleagues in a series of papers (Hodges & Hanson 1965, Truelove & Hanson 1966, Kenefick & Hanson 1966) have shown the mutually dependent absorption of Ca + +, Mg ++, and inorganic phosphate by mitochondria from corn seedlings. The process requires energy which may be furnished by a Krebs-cyde intermediate such as succinate, ATP, or the potential energy of the contracted mitochondrion. It has been suggested that a high energy phosphorylated intermediate is involved in the uptake of the divalent ion, although the idea of charge segregation, as advocated by Mitchell (1966), is not excluded. Mitochondria from red beet also accumulate Ca and inorganic phosphate and the inhibition of this uptake by monovalent ions was interpreted as evidence for the chemo-osmotic theory (Millard, Wiskich, & Robertson 1965). The main product of the accumulation is the precipitation of calcium phosphate within the mitochondrion. However, Grunwald (1966) has also found a less soluble Ca45-1abeled fraction associated with RNA which may be related to the carrier complex. In addition to mitochondria, the magnesium- and light-dependent uptake of calcium and phosphate has been demonstrated in isolated spinach chloroplasts when cyclic electron transport was coupled to phenazine methosulfate (Nobel & Packer 1964, 1965). The question arises whether the process of calcium and phosphate uptake by mitochondria and chloroplasts bears any relation to the mechanisms of assimilation of ions by intact cells. There are conflicting suggestions whether calcium uptake into roots is either active or passive. However, there is considerable evidence of calcium deficiency diminishing phosphate uptake. In view of the close relation between ion absorption and respiration, it appears desirable in many instances to study the situation in mitochondria, although the case for analogous mechanisms in both organelles and cells cannot be pressed at this time. As well as the use of mitochondria as a model system for examining ion absorption, there is evidence of calcium influencing respiration. The old observation of the Ca + + ion uncoupling oxidation phosphorylation may now be explained in terms of a redirection of energy from ATP formation to calcium

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phosphate accumulation. The stimulation of respiration by the low level of Ca ++ noted in animals has not been found in plant mitochondria. It appears likely the calcium is involved in the structural organization of mitochondria because of the cytological evidence previously discussed and a Russian report specifically describing the swelling of cristae in calcium-deficient plants (Bushueva & Semikhatova 1965).

c) Membranes and Model Systems In view of the evidence linking calcium with the stability of membranous structures, we shall consider briefly the current theories of membrane structure and possible roles that calcium may play. Until recently, thinking about membrane structure and function has been dominated by the bimolecular leaflet concept (Danielli & Davson 1935), which was developed into the unit membrane theory by Robertson (1959). This concept, which was applied to all membranes, proposed a bimolecular leaflet of phospholipid whose non-polar tails were directed inward perpendicular to the plane of the membrane. The polar phospholipid groups comprise the external surface which was covered by a layer of protein. This theory has recently been critically examined by Korn (1966), Kavanau (1966), and others who have found it inadequate in many respects. A more dynamic concept of membrane structure is now advanced and the idea of a universal unit membrane is specifically denied. In several of these suggestions, the membrane is composed of lipo-protein or protein globules embedded in a protein matrix or sandwiched between protein layers. These concepts are supported by the electron-microscopic evidence of Sj/Sstrand, Cedergren, and Karlsson (1964) and by freeze-etching replicas observed by Branton (1966). Kavanau in particular stresses the dynamic requirement of the various functions performed by membranes and proposes that the lipid "spherocylinder" of the leaflet is capable of alternating between a cylindrical and a disk form. A similar "corpuscular" theory of the structure of biological membranes has been proposed by Benson (1966). A precise incorporation of calcium into these structures is difficult at present, although there is considerable evidence of calcium interacting with phospholipids and neutral lipids in vitro. Shah and Schulman (1967) demonstrated that the binding of Ca ++ to monolayers of lecithin and sphingomyelin increases the surface potential. The binding is greater with dipalmitoyl lecithin than with sphingomyelin--this they attribute to steric interference from the 3-C hydroxyl of the latter. Calcium has been found to reduce the surface pressure of phospholipid monolayers as compared with K and Na. The presence of 10 mM K and Na does not affect this binding but the Ca is displaced by 100 mM of the monovalent ions (Rojas & Tobias 1965). The binding of Ca ++ with phosphatidyl serine has been demonstrated even in the presence of 145 mM Na or K. Bangham and Papahadjopoulos (1966) found an alteration in the binding of the Ca + + at 1 mM from a 1:1 ratio to a Ca + +n-I complex at higher concentration. In a study of the permeability of phosphatidyl serine liquid crystals to monovalent ions, calcium at above 1 mM was

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found to substantially increase the diffusion of the monovalent ions. Mg had a similar effect to Ca but at a higher concentration. Although these model systems do little to clarify the proposed role of calcium in the plasmalemma, they at least show the influence of calcium on the physical-chemical properties of the probable constituents of the membranes.
NUCLEIC ACIDS AND CHROMOSOMES

An important function of calcium in maintaining the structure of chromosomes may be implied from findings of Sorokin and Sommer (1929). The ability of DNA to form complexes with calcium may also account for the cytological observations of Steffensen (1958), Hyde and Palival (1958), and others (see Hewitt 1963), who found a close association between chromosomal aberration and calcium deficiency. Treatment of chromosomes with EDTA in a low ionic strength medium or citrate in water causes dispersion possibly due to the dissolution of ionic bridges. The formation of compounds between DNA and metal ions including calcium has been described by Kirby (1957) and more recently by Cheng (1965). There is also ample evidence of the ability of calcium to form a specific complex with ATP (Epstein & Whittam 1966). Allende et al. (1965) have recently proposed that calcium or another divalent cation is involved in the formation of threonyl-sRNA. The evidence does not tell us whether the ion confers a specific configuration on the sRNA or on the enzyme. As mentioned, the exposure of roots to EDTA causes the release of nucleotides. Furthermore, there are indications from the mitochondrial observation of Grunwald (1966) and from Foote and Hanson's (1964) work that calcium and nucleic acids are involved in membrane transport. It is interesting that the protoplasts prepared from Avena coleoptiles (Ruesink & Thimann 1965), although stable to various proteinases and lipases, are immediately lysed by RNAase. In view of these observations it is not inconceivable that the functions of calcium in nucleic acid metabolism and in membrane permeability are similar.
ENZYME ACTIVITY

The various reports of calcium involvement in enzyme activity are noted in Table II, although we cannot claim that this tabulation is exhaustive. Perhaps the most interesting enzyme systems in which calcium is well known to be involved are a-amylase and ATPase. However, in animals the interactions of the ion with trypsinogen (Smith 1951) and in prothrombin formation (Cole, Koppel, & Olwin 1964) are well documented. A variety of ATPases have been described from animal and plant sources which have very different responses to calcium. The classical mammalian enzyme is activated by Na plus K and inhibited by calcium (see Skou 1965), whereas the plant cell-wall enzyme requires Ca or Mg for activity (Dodds & Ellis 1966). Calcium- or magnesium-stimulated ATPases have also been described in synaptic vesicles of rat brain (Germain & Proulx 1965) and in myofibrils (Maryyama &

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TABLE II
SOME ENZYMATIC ACTIVITIES INFLUENCED BY CA + + .ENZYME SOURCE I N F L U E N C E OF CA ++ REF.

or-amylase

barley aleurones
Bacillus sublilis

q+ + + -"-b -+ q+ -qq-

Chrispeels& Varner 1967 Imanishi 1966 Takagi & Isemura 1965 Dodds & Ellis 1966 Brown et al. 1965 Germain & Proulx 1965 Skou 1965 Davidson & Long 1958 Einset & Clarke 1958 Keay & Crook 1965 Mildvan & Cohn 1965 Allende et al. 1965 Starr & Moran 1962

Cohn emend. Prazmowski

B. subtilis

ATPase

plant cell walls

drachis hypogaea

phospholipase esterase pyruvate k i n a s e threonyl-,RNA synthetase polygalacturonictranseleminase

rat brain many animal sources cabbage leaf carrot hog liver mammalian
Escherichia coil

(Migula) Castellani & Chalmers

Er~winia carotovora

(Jones) Holland.
B. polymyxa

(Prazmowski) Migula glucose-6-phosphate B. subtilis dehydrogenase arginine kinase } lecithinase pectin polygalacturonidase various sources adenyl kinase apyrase

q-

Marquet 1964

nt-

McElroy & Nason 1954

Ishikawa-Katsuki 1966). However, the enzyme from Arachis hypogaea is inhibited by Ca + + (Brown et al. 1965). Calcium is found as a component of a-amylase from a variety of animal and bacterial sources and is probably required for the stabilization of the enzymically active configuration (Imanishi 1966, Takagi & Isemura 1965). Recently Chrispeels and Varner (1967), in their work on gibberellic-acid-enhanced synthesis of ~-amylase in barley aleurones, have shown that 20 mM Ca ++ is required for the activity of the enzyme in higher plants. The suggested involvement of calcium in the maintenance of membrane function and cytoplasmic organization would of course profoundly influence a great variety of enzymic activities. It has also been suggested by Hewitt (1963) that an appropriate ratio of K to Ca is required for normal metabolism. A more elaborate hypothesis has been advanced by De Kock (1964) who impli-

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THE BOTANICAL REVIEW

cates the ratio of K/Ca and P in the control of the citric acid cycle and in determining the level of citrate, malate, and oxalate. CONCLUDING DISCUSSION The proposed role of calcium in protecting tissue, particularly roots, against toxic ionic environments raises some interesting questions. Even within commercially important plants and certainly within the higher plants, there exists a vast range of ionic habitats in which plant roots develop. A strain of Agrostis tenuis Sibth. was found well adapted to growth in a soil containing 1 per cent lead and 0.03 per cent zinc but low in calcium and phosphate. However, it did not grow well in a garden soil (Bradshaw 1952). Remarkable differences in resistance to copper, nickel, lead, and zinc have been detected in Agrostis species and these could be related to the original ecological habitat (Gregory & Bradshaw 1965). The results obtained by Wilkins (1960) on the lead tolerance of species of Festuca ovina L. imply a genetically determined "tolerance factor." A comparable problem is presented by the adaptation of certain plants to a high H + and A1+++ ion environment and others to calcifuge conditions. Clarkson (1965) studied the growth response and calcium uptake by five Agrostis species and found that the calciphobe species, A. setacea Curt., responded maximally to 0.125 mM Ca ++ whereas the calcicole species showed further response even over 1 mM. These results were taken to indicate that the calcium uptake mechanism of the calciphobe had a lower Ks. Since calcium is thought to play an important role in the stabilization of the plasmalemma under adverse conditions, this poses the question of the mechanism by which these plants adapt to toxic environments. Unfortunately, our knowledge of the composition of plant membranes is very sparse (Thompson 1965) and to date there has been no attempt to correlate ecological adaptation with membrane composition and structure. However, this would appear important for both scientific and agronomic reasons. In view of our profound ignorance of the chemical structure of the plasmalemma one can only speculate about the role of calcium in the structure. The role of calcium in cell membranes is most likely related to its binding to phospholipids, in view of the in vitro interactions, although there is little direct in vivo evidence. As it has been shown in a-amylase and lactic dehydrogenase that calcium can alter the allosteric structure of protein, this type of interaction cannot be discounted. An assessment of the role of the proposed Ca-ribonucleoprotein in ion uptake is difficult. The hypothesis is based on the similar effects of EDTA, RNAase, and ultraviolet light irradiation to which the sensitivity of protoplasts to RNAase, the association of Ca45 with mitochondria, and the chemical complexes of Ca with nucleic acid may be added as additional evidence. The use of EDTA causes such drastic effects that some doubt is cast on the validity of deductions made from the result. Furthermore, Foote and Hanson (1964), although explaining the rapid initial influx of K + as due to increased permeability and an excess of vacated cytoplasmic exchange sites, did not find a comparable influx of Ca ++ . However, one eagerly awaits chemical evidence for the presence or absence of RNA in membrane fractions.

FUNCTION OF CALCIUM IN PLANTS

421

The proposed function of calcium in membranes is not only related to the plasmalemma but may also account for the breakdown of mitochondria and endoplasmic reticulum and cytoplasmic disintegration observed in calciumdeficient tissue. The beneficial effect of calcium on cytoplasm was cited over 40 years ago by True (1914) and has since appeared in several guises. However, we are still some way from a biochemical or molecular understanding of this statement. It may be interesting to note a recent approach to this problem by Cerbon (1965). The 1.3 p.p.m, nuclear magnetic resonance signal relative to a tetramethyl-silane standard was found to be related in Nocardia asteroides (Eppinger) Blanchard to cellular lipils. Growing cells and those exposed to isotonic media gave no signal, but one was observed in cells exposed to hypoor hypertonic conditions. The presence of Ca or Mg induced the signal under isotonic conditions, indicating an alteration in the lipid phase. In addition to the influence of calcium on organization and possibly indistinguishable from it, the ion may be required specifically for the activation of certain enzymes. Possibly the most interesting of these is the ATPase found in cell-wall fractions. It is tempting to consider that the Viers effects may be explained by the activation of the ATPase by Ca or Mg. Despite the longevity of the calcium-pectate theory, certain difficulties remain. The major problem is the extremely low calcium content (Table I) now found in healthy roots. Preliminary studies have taken place on the distribution of calcium within cells and perhaps further investigation will provide more detailed information. Although there is evidence pointing to a calcium involvement in three areas of metabolism, no satisfying explanation can be advanced for the importance of Ca ++ and other divalent ions in root elongation. It has been the purpose of this article to bring together information relating to the function of calcium scattered throughout the biological and biochemical literature. In this study the evidence for the possible role of calcium in membranes, chromosomes, cell walls, and in the activation of certain enzymes has been most compelling and these topics would appear to be profitable lines for future study. LITERATURE CITED
ALLENDE, J. E., G. MORA, M. GATICA, & C. C. ALLENDE. 1965. The role of metal ions in the formation of threonyl-soluble RNA from threonyl adenylate-enzyme complex. Jour. Biol. Chem. 240: PC 3229-3232. BAMFORD~R. 1931. Changes in root tips of wheat and corn grown in nutrient solutions deficient in calcium. Bull. Torrey Bot. Club 58: 149-178. BANGHAM, A. D., & D. PAPAHADJOPOULO$.1966. Biophysical properties of phospholipids. I. Interaction of phosphatidyl serine monolayers with metal ions. Biochim. Biophys. Acta 126: 181-184. BATEMAN, D. F., & R. D. LUMSDEN. 1965. Relation of calcium content and nature of the pectic substances in bean hypocotyls of different ages to susceptibility to an isolate of Rhizoctonia solani. Phytopathology 55: 734-738. BEESON, K. C. 1941. Mineral composition of crops. U.S. Dep. Agr. Misc. Publ. 369, pp. 93-117. BENNET-CLARKE,J. A. 1956. Salt accumulation and the mode of action of auxin. A pre-

422

THE BOTANICAL REVIEW

liminary hypothesis. In: "The Chemistry and Mode of Action of Plant Growth Substances," ed9 by R. L. Wain & F. Wightman, Butter~orth Scientific Publ., London, pp. 284-291. BENSON, A. A. 1966. On the orientation of lipids in chloroplasts and cell membranes. Jour. Amer. Oil Chem. Soc. 43: 265-270. BXDDULPH, O., R. CORY, & S BIDbULPH. 1959. Translocation of calcium in the bean plant. Plant Physiol. 34: 512-519. BOLLARD, E. G., & G. W. BUTLER. 1966. Mineral nutrition of plants. Ann. Rev. Plant Physiol. 17 : 77-112. BRADSUAW,A. D. 1952. Populations of dgrostis tenuis resistant to lead and zinc poisoning. Nature 169: 1098. BRANTON, D. 1966. Fracture faces of frozen membranes. Proc. Natl. Acad. Sci., Washington, 5 5 : 1048-1056. BROWN, H. D., N. J. NF.UCE~e, A. M. ALTSCUUL,& W. J. EVANS. 1965. Activity patterns of purified ATPase from drachis hypogaea. Life Sci. 4: 143%1447. BURLING. E., & W. T. JACKSON. 1965. Changes in calcium levels in cell walls during elongation of oat coleoptile sections. Plant Physiol. 40: 138-141. Buasra6M, H. 1952. Studies on growth and metabolism of roots. VIII. Calcium as a growth factor. Physiol. Plantarum 5: 391-402. 9 1954. Studies on growth and metabolism of roots. X. Investigations of the calcium effect9 Physiol. Plantarum 7: 332-342. 9 1957. Auxin and the mechanism of root growth. In: "Biological Action of Growth Substances," Soc. Exp. Symp., Academic Press, New York, 11: 44-62. 9 1958. Influences of growth regulators on the composition of the cell wall. Kungl. Fysiograf. S~illskap. (Lund) Handl. 28: 53-64. (Chem. Abstr. 53: 20322e.) BUSHUF.VA,T. M., & O. A. SeMIKHATOVA.1965. Effect of calcium deficiency on the binchemical activity and structure of mitochondria from pea seedlings. Vestn. Leningr. Univ. 20 (Ser. Biol. No. 2.) : 106-112. (Chem. Abstr. 63: 15231e.) CV~RBON,J. 1965. Variations in the lipid phase of living organisms during the transport process. Bioehim. Biophys. Aeta 102: 449-458. CHAPMAN, H. D. 1966. Calcium. In: "Diagnostic Criteria for Plants and Soil," ed. by H. D. Chapman, University of California Press, Berkeley, pp. 65-93. CHENC, P-Y. 1965. Ultraviolet-rotatory dispersion as a probe for interaction between DNA and metal ions. Biochim. Biophys. Acta 102: 314-316. CHESTER, V. E. 1965. The role of calcium in the increase in flocculence of yeast growing in the presence of copper. Proc. Roy. Soc. (London) Ser. B 162(989): 555-566. CHRISPEELS, M. J., & J. E. VARNER. 1967. Gibberellic acid-enhanced synthesis and release of or-amylase and ribonuclease by isolated barley aleurone layers. Plant Physiol. 42: 398-406. CLARKSON, D. J. 1965. Calcium uptake by calcicole and calcifuge species of ,4grostis L. Jour. Ecol. 53 : 427-435. CLELANDjR. 1960. Effect of auxin upon loss of calcium from cell walls. Plant Physiol. 35: 581-584. COLE, E. R., J. L. KOPPEL, & J. H. OLWIN. 1964. Interaction of bovine autoprothrombin C with phospholipids and bivalent ions. Can: Jour. Biochem. 42: 1595-1603. CORMACK, R. G. H. 1949. The development of root hairs in Angiosperms. Bot. Rev. 15: 583-612. 9 1965. The effect of calcium ions and pH on the development of callus tissue on stem cuttings of balsam poplar (Populus balsami[era). Can. Jour. Bot. 43: 75-83. DANXELLI, J. F., & H. DAVsON. 1935. A contribution to the theory of permeability of thin films. Jour. Cell Comp. Physiol. 5: 495-508. DAWDSON, F. M., & C. LONG. 1958. The structure of naturally occurring phosphoglycerides 4. Action of cabbage-leaf phospholipase D on ovolecithin and related substances. Biochem. Jour. 101:31 P.

FUNCTION OF CALCIUM IN PLANTS

423

DE BRUYN, J. A. 1966. The in vitro germination of pollen of Setaria sphacelata. II. Relations between boron and certain cations. Physiol. Plantarum 19: 322-327. DE KOCK, P. C. 1964. The physiological significance of the potassium-calcium relation in plant growth. Outlook on Agriculture 4: 93-98. DODOS, J. A. A., & R. J. ELLIS. 1966. Cation-stimulated ATPase activity in plant cell walls. Biochem. Jour. 101:31 P. EDWARDS, J. K. 1936. Cytological studies of toxicity in meristem cells of roots of Zea mays. 1. Effect of the neutral salts. Amer. Jour. Bot. 23: 483-489. EINSET, E., & W. L. CLARKE. 1958. The enzymatically catalysed release of choline from lecithin. Jour. Biol. Chem. 231: 703-715. EPSTEIN, E. 1961. The essential role of calcium in selective cation transport by plant cells. Plant Physiol. 36: 437-444. , D. W. RAINS, & O. E. ELZAM. 1963. Resolution of the dual mechanisms of potassium absorption by barley roots. Proc. Natl. Acad. Sci., Washington, 49: 684-692. EVSTEXN, F. H., & R. Wrm"rAM. 1966. The mode of inhibition by calcium of cell-membrane ATPase activity. Biochem. Jour. 99: 232-238. FLORELL, C. 1956. The influence of calcium on root mitoehondria. Physiol. Plantarum 9: 236-242. FOOTE, B. D., & J. B. HANSON. 1964. Ion uptake by soyabean root tissue depleted of calcium by EDTA. Plant Physiol. 39: 450-460. GALEY, F., R. G. W. JONES, & O. R. LONT. 1968. Microscopic and histoehemical studies on calcium-deficient root apices of Zea mays. (In preparation.) GERMAIN, M., & P. PROULX. 1965. Adenosine triphosphatase activity in synaptic vesicles of rat brain. Biochem. Pharmacol. 14: 1815-1819. GIELXNK, A. J., G. SAUER, & A. RINCOET. 1966. Histoautoradiographic localisation of calcium in oat plant tissue. Stain Technol. 41: 281-286. GINSEERG, B. Z. 1958. A protein component of the middle lamella: a possible site for indole acetic acid action. Nature 181: 398-400. GRECORY, R. P. G., & A. D. BRAnSaAW. 1965. Heavy-metal tolerance in populations of Agrostis tenuis and other grasses. New Phytol. 64: 131-143. GRIFFIN, D. H. 1966. Effect of electrolytes on differentiation in Achlya species. Plant Physiol. 41: 1254-1256. GRUNWALn, C. 1966. Calcium uptake by potato tuber mitochondria. Isolation and identification of Ca 45 complexes. Physiol. Plantarum 19: 335-347. HANDLEY,R., A. METWALLY,& R. OVERSTREET.1965. Effect of calcium upon metabolic and non-metabolic uptake of Na and Rb by root segments of Zea mays. Plant Physiol. 40: 513-520. HANSON, J. B. 1960. Impairment of respiration, ion accumulation and ion retention in root tissue treated with ribonuclease and EDTA. Plant Physiol. 3,5: 372-379. , S. S. MALHOTRA, & C. D. STONER. 1965. Action of calcium on corn mitochondria. Plant Physiol. 40: 1033-1040. HARRIS, 2. J., R. COCKRELL,& B. C. PRESSMAN. 1966. Induced and spontaneous movement of potassium into mitochondria. Biochem. Jour. 99: 200-213. HAYNES, J. L., & W. R. ROBBINS. 1948. Calcium and boron as essential factors in the root environment. Jour. Amer. Soc. Agron. 40: 707-715. HEWlTT, 2. J. 1963. Essential nutrient elements for plants. In: "Plant Physiology," ed. by F. C. Steward, Academic Press, New York, 3: 155-172. HIRATA, H., & S. MITSUI. 1965. Role of calcium in potassium uptake by plant roots. Plant Cell Physiol. (Tokyo) 6: 699-709. HOAGLAND, D. R., & D. I. ARNON. 1950. The water-culture method of growing plants without soil. Univ. California, Berkeley, Coll. Agr. Circ. 347. HODGES, J. K., & J. B. HANSON. 1965. Calcium accumulation by maize mitochondria. Plant Physiol. 40: 101-109. HOOYMANS, J. J. M. 1964. The role of calcium in the absorption of anions and cations by excised barley roots. Aeta Bot. Neerl. 13: 507-540.

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