The Vacuole: a Cost-Benefit Analysis

The Vacuole: a Cost-Benefit Analysis

J . A . RAVEN

Depurtment of Biological Sciences, University of Dundee, Dundee DDl 4HN, UK

I.

Introduction

........................................................................

50

11. Demonstrated and Hypothesized Functions of Vacuoles. and Alternative Means of Performing these Functions .........................................

62

Demonstrated and Hypothesized Costs o f Producing and Maintaining Vacuoles, and of Costs of Alternative Means of Performing Vacuolar Functions ..............................................................................

62

A Case History: ”Vacuolate” (Eu)bacteria

62

111.

IV. V. VI.

.................................

Another Case History: Vacuoles and Buoyancy

...........................

Costs and Benefits of Vacuolation: Simple Analyses and the Allocation of Costs Among Various Benefits ..............................................

75 78

VII.

........................................

81

VIII.

Conclusions . .............................................................. Acknowledgements ......................... ..... References .............................................................................

82 82 81

1.

INTRODUCTION

A largc number of functions have been demonstrated, or suggested, for the aqueous vacuoles of plants (including algae) and fungi (including the “pseudofungal” oomycetes), as well as vacuole-like structures (not “gas

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J. A. RAVEN

vesicles”) in prokaryotes (e.g. Raven, 1987; Fassing el al., 1995). If we are to understand the distribution of vacuoles, then these functions of the vacuole which yield quantifiable benefits must be considered in the context of the costs of synthesizing and maintaining the vacuoles, including any consequential requirements of vacuolation such as increased resource allocation to cell walls. Such cost-benefit analyses are predicated on maximizing the inclusive fitness of the organisms by optimal acquisition and allocation of resources (Osmond et al., 1980). The costs and benefits must be expressed in the same resource units: the appropriate units in a given environment will be determined by the resource which is most limiting for growth (Liebig, 1840) as determined by sensitivity analysis. When light is limiting the growth rate then energy (or carbon) is the appropriate resource to concentrate on in cost-benefit analyses (Raven, 1984a,b, 1985). If water is limiting for a land plant then reduced carbon, whose production from atmospheric C 0 2 involves the expenditure of water in transpiration, may be an appropriate unit to use (Raven, 1985, 1987), remembering, of course, the water retained during growth in the vacuoles or their alternatives. Similar arguments apply to limitation by phosphorus or nitrogen (Raven, 1987). These considerations related to the role(s) of vacuolation in the acquisition of limiting resource, i.e. alleviating stress sensu Grime (1979). We must also consider biomass retention in the sense of the costs and benefits of vacuoles in reducing biophagy, including parasitism, i.e. overcoming biotic disturbance sensu Grime (1979). Considerations so far all relate to biomass production or retention. Inclusive fitness involves successful reproduction, so we must also consider biotic resources needed for pollination (more generally, fertilization) or for dispersal of propagules. Finally, the multiple proposed benefits, and known costs, of vacuolation mean that multiobjective optimization methods must eventually be used (Farnsworth and Niklas, 1995). Having considered what is meant by cost-benefit analyses, the end-product of such analyses over a wide phylogenetic range of organisms, metabolic types and of life-forms (sensu Raunkaier, 1934; Luther, 1949) should be some rationale for the extent of vacuolation (vacuolar volume per unit cytoplasmic volume) in different organisms. The range of vacuo1e:cytoplasm among phototrophs is from 0.05 (many microalgae; host invertebrates in various symbioses with microscopic photolithotrophs (or chemolithotrophs)) to 250 (giant-celled macroalgae; photosynthetic cells of CAM (crassulacean acid metabolism) plants) (Raven, 1987, 1993a, 1995a; Winter, Robinson and Heldt, 1993, 1994). Such a rationale requires consideration of alternatives to vacuolation in providing non-cytoplasmic fluid phases (coelom, haemocoel, coelenteron in various metazoa; apoplasmic water in plants) (Raven, 1987).

THE VACI!OLE: A COST-BENEFIT ANALYSIS

61

Space does not permit a detailed quantitative cost-benefit analysis of all vacuolar functions. Such detail is given for many functions by Raven (1OX7), and the conclusions are briefly summarized here as an introduction to the tabulated material (Tables I-IV) and to the two case histories (text, and Fig. 1) presented in this article. An inescapable effect of vacuolation of a cell of a given shape is to increase the surface area per unit volume of cytoplasm. This increases the rate of resource (gaseous or dissolved chemicals: photons) acquisition from resourcelimited environments on a unit cytoplasm basis. Although extra material and energy costs are incurred in building and maintaining a vacuolate cell as compared to a non-vacuolate cell with the same volume of cytoplasm, the benefits generally exceed the costs under resource-limited conditions. However, under resource-saturated conditions, vacuolation may mean a lower maximum specific growth rate due to resource diversion from catalytic apparatus to the additional wall as well as vacuolar material in the vacuolate cell. Alternatives to vacuolation in achieving additional resource acquisition rates per unit cytoplasm under resource-limited conditions include changes in cell shape (less closely approximating to a sphere) or the occurrence of apoplasmic volume in multicellular organisms. These alternatives do not exclude vacuolation, and themselves have material and energy costs similar to those of vacuolation which partly offset the benefits which accrue at low resource availability. Vacuolation also has other costs and other benefits. These further costs only apply in particular habits, e.g. slow turgorholume adjustment of large vacuolate cells in relation to the rate o f change of external osmolarity in estuaries, and a possible decrease in desiccation tolerance as a result o f vacuolation which mainly affects "lower" plants in desiccation-prone environments. Benefits in addition to these geometric effects related to resource acquisition in resource-deficient environments include the storage, manipulation and protection of already acquired resources, the accumulation o f end-products of (for example) acid-base regulation, and contributions t o outbreeding and dispersal, as well as further contributions to resource acquisition in CAM and in rhizospherc acidification attendant on accumulation of endogenously produced organic acids R S their salts. While some o f these benefits may be attained in other ways (e.g. storage of hcxose as polysaccharide in plastids or cytosol rather than mono- or disaccharidcs in thc vacuole) others cannot (e.g. die1 changes in low-M, organic acids in CAM). Overall these additional benefits outweigh the additional costs of vacuolation and may account for vacuolation in those plants (if such there be) which invariably grow naturally in resource-rich habitats and in which vacuolation might be expected to diminish fitness by decreasing the achieved specific growth rate.

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11. DEMONSTRATED AND HYPOTHESIZED FUNCTIONS OF VACUOLES, AND ALTERNATIVE MEANS OF PERFORMING THESE FUNCTIONS Table I lists a number of vacuolar functions (benefits), and of alternative ways of performing these functions. From this mainly qualitative menu, two examples not considered by Raven (1987) are subsequently taken for analysis in the text.

111. DEMONSTRATED AND HYPOTHESIZED COSTS OF PRODUCING AND MAINTAINING VACUOLES, AND OF COSTS OF ALTERNATIVE MEANS OF PERFORMING VACUOLAR FUNCTIONS Table I1 lists a number of costs of producing and maintaining vacuoles. and of costs of alternative means of performing vacuolar functions.

IV. A CASE HISTORY: “VACUOLATE” (EU)BACTERIA Prokaryotes d o not have an endomembrane system, and thus cannot have a vacuole in the sense that eukaryotic cells do, i.e. a hypertropied lysosome, since a lysosome is a differentiated part of the endomembrane system. However, certain eubacteria do have aqueous P phases (sensu Mitchell, 1979) bounded by protein-lipid bilayer membranes which occupy a considerable fraction of the cell volume (Fassing et al., 1995; Jamasch, 1995). These prokaryotes are marine S*--oxidizing eubacteria of the genera Beggiatoa and Thioplaca and have filaments of vacuolate cells up to 100 pin or more in diameter. They obtain the energy needed for (chemolithotrophic) growth and maintenance by oxidizing S2- or So using 0 2 or N 0 3 - as an electron acceptor. Their habitat is ocean floor sediments in which the rain of reduced organic carbon from surface photolithotrophy exceeds the rate at which O2 can diffuse into the sediments. Microbial chemo-organotrophy oxidizes more moles of organic carbon than moles of 0 2 are available, so S042- is used as terminal electron acceptor instead of 0 2 . Sulfate is abundant in seawater: 25 rnol mP3 S042- (8 mol of electrons consumed upon complete reduction of 1 mol) relative to -0.25 mol m-3 O2 (4 rnol of electrons consumed upon complete reduction of 1 rnol), i.e. 200 times more electron acceptors per unit volume of seawater as S042- than as 02.Of course, S042- is an energetically much inferior electron acceptor than is 0 2 in terms of joules available per electron transferred from organic carbon to the acceptor. Beggiatoa and Thioplaca live near the chemocline separating the seawater containing O2 and NO3- from the anoxic sediment containing H2S.

Raven (1987)

Reed ct a / . (1980) Young et (11. (1987)

N o viable alternative which does not cause change5 in protein. metabolites or effector concentrations in the cytoplasm which would disturb metabolism

However, estuarine algae often have cell walls with a low bulk elastic modulus which means that changes in external osmolarity as a function of time in the tidal cycle are reflected initially (and. to a large extent. over the time between high and low tide) in protoplast wdunie changes. and hence changes in protein, mctabolite or effector concentrations

(13) \':icuoles x e iinportant in cells which undergo I-apid (seconds-minutes) changes in volume. e.g stomatal guard cells: pulvinar motor cells). Delegation of most of the volume change to the vacuole meam that concentrations of proteins. metaholitea or effectors in the cytoplasm are little altered.

~

References

Raven (1981. 1Y8la.b, 19x7. I9Y3a. lYY5c. 1996)

~

Alteration of cell shape from near-spherical to less spherical (can be combined with vacuolation), yielding evaginations (root, shoot. thallus hairs) or invaginatiom (transfer cells). Altered morphology at the supracellular level. yielding intercellular gas spaces in plants. gills/trachea/lungs/boo~ lungs in metazoa (can be combined with vacuolation in plants). Presence o f internal apoplasmic aqueous spaces (xylem. leptome in embryophytes; intercellular spaces in algae: blood. haemocoel. coelom. coelenteron in metazoa). Animal spaccs can, like vacuoles, transmit hydrostatic pressure and be part o f a hydrostatic skeleton: plant apopl;isrnic spaces generally cannot. Photosynthetic (symbiotic) animals generally have an exoskeleton (corals. for arne ni fe ra . hiva lves : n o t certa I n h ydroids . or Cori I d i m )

~~

( a ) Increases the surface area of plasmalemma exposed to the cnvironnient per unit volume o f cytoplasm. Increases the potential for light absorption per unit cytoplasmic volume from a given radiation field. Increases the potential for solute and water influx across the plasmalemma by lipid solution passive flux with a given concentration difference, or mediated passive o r active transport at thc plasmalemma. Increases the volume o f substrate exploited (important if thc solute has a lo\\ diffusion cocfficicnt in the suhstratc. e.g. phosphate in oxidized soil)

( I ) Geometric

~~

TABLE I Vacitolar ficnctions and alferiiativc wrzy of performing these fiinction.r Function pel-foi-med by vacuole

Increased absorption of photosynthetically active radiation via smaller package effect in photosynthetic cells (see (1))

(a) Radiation absorptiotilscattering

(2) Physical

Changed cell shape (see (1))

Raven (1987)

Fahn (1974) Putz et a / . (1995)

Some contractile roots lack this collapse and death of cell

Contractile roots operate (in part) by collapse and death of highly vacuole cell layers between the cell layers

layers; all contractile roots depend on cortical cell growth, which increases the radial extent of the cells while decreasing their axial extent

Fahn (1974) Niklas (1992) Walker er al. (1995)

An internal aqueous apoplasmic phase containing the seeds which is pressurized by solute accumulation and contained by the pericarp could cause explosive seed dispersal; no examples known. Proposal is analogous to '.jet propulsion" of cephalopods. opposite of firing of the Utriciclaria trap

Pressurization o f internal tissues leading to explosive ("squirting") seed dispersal ( Echalliiim, Echiriocystis. Itnyatiens. Oxalis), a result of vacuolar accumulation of solutes (sugars, glycosides) in highly vacuolate tissue (cytoplasmic pressure = vacuolar pressure)

References Sydenham and Findlay (1975)

Alternative means of performing the function Rapid increase in volume of traps of Utricularia caused by pumping ions (hence water) out of the apoplasmic lumen of a trap. followed by "firing", yielding an inrush of water (plus. with luck. prey)

Function performed by vacuole

TABLE I (continued) Vacuolar functions and alternative ways of performing these functions

Lee (1991)

Lee et al. (1979. 1990) Lee ( 1986)

Colour production by lipophilic pigments in plastids (does not produce identical spectral range to the vacuolar pigments). Colour production by thin-film interference in cell walls (does not reduce photosynthesis underlying fruit tissue as much as does pigment) Back-scattering via thin-film interference in cell walls of ahaxial epidermal cells

Focusing in non-vacuolate cells of given cytoplasmic volume involves different cytoplasmic volume/focal length relation. Solid lens in some single-celled eukaryotes (Dinophyceae). and metazoa focuses light on photoreceptor. e.g. retina in some metazoa

Production o f colour attractive to pollinatorsipropagule disperser5 caused hy the presence of pigments in the vacuole (anthocyanins. betalains)

Increased absorption of adaxially incident photosynthetically active radiation by back-scattering from anthocyanin-containing vacuoles o f abaxial epidermal cells of leaves

Focusing o f radiation related to phototaxidphotosynthesis by the difference between the refractive index of wallicytoplasmivacuole and medium

Vogelman (1993)

Raven ( 1987)

Ramus (1978) Raven (1987. 1996) Vogelman (1993)

Increased pathlength for photons in photosynthetic organ due to the presence of an intercellular pas space with a much lower refractive index than cell wall/cytoplasm/vacuole. or intercellular CaCOi

Increased photon absorption via an increased pathlength of photons in photosynthetic celliorgan if vacuoles have a large difference in refractive index relative to cell wallskytoplasm (not proven)

Raven (1987. 1991. 1995~) Raven and Johnston (1992) Marchant et al. (1991) Garcia-Pichel and Castenholz (1991) Edwards et al. (1996)

Presence of UV-B-absorbing material in extracellular matrix (cyanobacteria, Phaeocystis). Presence of UV-B-reflecting cuticle. (External UV-B barriers can protect all cell components)

Increased absorption of UV-B radiation caused by the presence of UV-B-absorbing solutes in vacuoles (cannot protect DNA unless it is suspended in vacuole on cytoplasmic strands. as i n Mougeotia or is always on shaded side in a vector radiation field. as in leaves with UV-B absorbers in the epidermal vacuoles)

Decrease in density to value lower than that of fluid medium, hence positive buoyancy. via exclusion of Ca’+, Mg’+, ( K + ) . S042- from, emphasis on NH4+, Ht in, vacuoles when cells are isomolar with/hyperosmolar to medium (only works in high-osmolarity environments such as seawater). Occurs in both planophytic (sensu Luther, 1949) organisms, permitting upward movement in water column and halophytic (sensu Luther, 1949). related to posture

(b) Change in overall celllorganisrn density

Function performed by vacuole

Walsby (1994) Dromgoole (1982, 1990) Walsby (1972) Raven (1996)

Positive buoyancy in high- or low-osmolanty environments achieved by extracellular gas spaces in multicellular aquatic planohapto- or rhizophytic plants (Enterornorpha, Codiurn, Dumontia. many brown algae in Fucales, Laminariales, Durvillaeales, Scytosiphonales; aquatic vascular plants) and in some metazoa (teleost swim bladder)

Denton (1974) Dromgoole (1982) Lambert and Lambert (1978) Luther (1949) Villareal (1992) Villareal and Carpenter ( 1994) Villareal et al. (1993)

References

Positive buoyancy in high- or low-osmolarity environments achieved by prokaryote gas vesicles

Positive buoyancy in high-osmolarity environments achieved by H+/NH4+, C1- in apoplasmic phases of some metazoa. Positive buoyancy (in high-osmolarity environments) by the use of lipid rather than polysaccharide as the energy store

Alternative means of performing the function

TABLE I (continued) Vacuolar functions and alternative ways of performing these functions

Raven and Richardson (1984) Raven (unpublished science fiction) Gibor (unpublished science fiction) Walsby (1972) Bauman et al. (1978)

Raven (1995~) Clifford et al. (1989) Kiss (1994) Severs et al. (1989) Raven (unpublished science fiction)

Flagellar (dyneidtubulin) or muscular (myosinlactin) motility permits organisms which are denser than the aqueous medium upwards in media of high or low osmolarity

Hypothetical positive buoyancy in air of hydrogenic or methanogenic organisms

Dense components of cytoplasm (stored polysaccharide, protein, polyphosphate) or cell wall (Si02, CaC03).

Effective premortem retranslocation of N , P to living part of the plant, i.e. dead “wings” etc. could have originated from non-vacuolate (low C:N, C:P) cells

Graviperception by amyloplasts in vascular plants, CaS04 (etc.) crystals in animal cytosol

Hypothetical graviperception by movement of gas vesicles of prokaryotes (away from gravitational atractor) within cells

Increase in density relative to a highor low-osmolarity medium due to high-density solution andor solid components of vacuole

Large surface area per unit volume of wind-dispersed seeddfruits; gas-filled dead cells derived from vacuolate cells with high C:N, C:P ratios

(c) Graviperception by high-density vacuolar components moving (towards gravitational atractor) within cell (BaS04 crystals in characean rhizoids)

(a) Storage of water, and watersoluble compounds (storage = deposition with stochastic or deterministic likelihood of withdrawal and further use). Strict limits on the extent to which cytoplasm can be diluted (water storage) or to which the concentration ( 5 a few moIm-3) of inorganic phosphate, total Ca’+, NH4+ or NO3- can vary in cytoplasm; any additional storage of these resources (and of K + , Mg2+, etc.) can occur in the vacuole. Low energy costs of storage of NO3- in vacuoles relative to storage in a reduced form. Vacuolar storage of “CO2” for -12 h as organic acid in the vacuole is the only method of “COf” storage used in CAM plants. Vacuolar storage of free Ca2+ at a higher electrochemical potential than that of free Ca2+ in the cytosol, thus permitting (downhill) efflux across the tonoplast to act as a means of signalling, may not be consistent with accumulation of large amounts of oxalate (see (b) below)

(3)Chemical

Function performed by vacuole Apoplasmic water is stored in some saccate intertidal algae during emersion, and in the wood of trees. Nutrient solutes are apparently less frequently stored apoplasmically. Nutrient storage in desiccation-tolerant seeds, vegetative organs/organisms as polymers (C, energy as polysaccharides, lipids; N , C, energy as protein; P as polyphosphate, phytic acid). Storage of fuel for thermogenesis in aroid spadices as starch (and lipid) rather than soluble sugars: relatively non-vacuolate nature of spadix cells related to need for large heat production (a function of cytoplasmic (mitochondrial) volume) per unit volume if temperature is to be significantly above ambient. Extracellular mucilage of the colonial marine alga Phaeocysfis serves inter alia as a store of organic C and energy. Metazoan nutrient storage generally involves insoluble material. No known method of storing “CO,” for -12 h except as organic acids stored in the vacuole

Alternative means of performing the function

TABLE I (continued) Vacuolar functions and alternative ways of performing these functions

Raven (1982, 1984a, 1987) Zhen et al. (1991, 1992) Raven and Spicer (1996) Welbaum et al. (1993) Canny and Huang (1993) Trebacz et al. (1995) Lee and Ratcliffe (1983) Mimura (1995) Mimura et al. (1990) Rebeille et al. (1983) BermadingerStabentheiner and Stabentheiner (1995) Lancelot and Rousseau (1994) Sanders et al. (1992) Kinzel (1989) Leigh and Wyn Jones (1984)

References

(b) Accumulation of defence materials (nuclear, chemical, biological) and optical attractants, UV-B absorbers, photosynthetically active radiation in back-scatterers (accumulation = synthesis and sequestration without (generally) remetabolism). Defence compounds includes free radical scavengers (active against ionizing and UV-B radiation, chemicals) and anti-biophage agents. Also include solutes which offset the effects of (stable) material necessarily produced in metabolism, e.g. OH- from NO3assimilation neutralized by organic acid production with a salt of the organic anion accumulated in the vacuole (generally soluble, sometimes precipitated, e .g. Ca( COO)2). Vacuoles with total oxalate in excess of total Ca2+ may have free Ca2+ concentrations too low to allow downhill Ca2+ efflux to the cytosol to participate in signalling. Also in this category are salts moved to the shoot in emergent halophytes; one fate for these is vacuolar accumulation Alternatives to vacuolar storage of solutes interacting with (non-ionizing) radiation were considered in 2(a) above. Free radical scavengers occur mainly in cytoplasm where free radicals are (metabolically) produced and there is a higher concentration of damageable components. OHneutralization can include apoplasmic C a C 0 3 precipitation (could not occur in vacuoles of “normal” pH) and (like H + disposal) direct or indirect efflux to the environment. Emergent halophytes can exclude un-needed salts from their roots, or excrete them via salt glands or (?) in abscised leaves

Raven (1987, 1995c) Osmond ei al. (1980) Kinzel (1989)

Echeverria (1990)

Raven (1997)

Extracellular H+-catalysed conversion of HC03- to C 0 2 in acid zones on the surface of certain freshwater macrophytes, with C 0 2 entry and fixation. Extracellular catalysis of HC03- to C 0 2 conversion by carbonic anhydrase with COz entry and fixation. HC03- transport into cell with intracellular conversion to C 0 2 and then fixation. All of these mechanisms ‘‘leak’’ C 0 2

Possible occurrence of H+-catalysed, H+-consuming conversion of HC03to C 0 2 in vacuoles of photosynthetic cells provided vacuole is large enough or has a low enough pH relative to rate of C 0 2 consumption in photosynthesis. Involves transport of exogenous HC03- across plasmalemma and tonoplast, with C 0 2 transport from vacuole to plastids (and some leakage to medium!)

References

Acid invertase in vacuoles with a higher pH than that of Citrus juice vesicles, or enzymes in other cell compartments (including acid invertase in low-pH cell walls)

Alternative means of performing the function

(c) Non-enzymic catalysis by H + of reactions involving inorganic and organic compounds. Hydrolysis of sucrose in Citrus juice vesicles is initially enzymic, later (as vacuolar pH decreases to pH 2.5) the enzyme activities disappear and all hydrolysis can be accounted for by H + catalysis

Function performed by vacuole

TABLE I (continued) Vacuolar functions and alternative ways of pegorming these functions

The diversion of resources to synthesis of vacuoles and the attendant additional plasmalemma and cell wall may reduce the maximum specific growth rate (p.,,,) relative to that of a non-vacuolate cell with the same cytoplasmic volume per cell; however, this has not thus far been demonstrated

(1) Synthesis of vacuoles and of concomitant additional quantities of other cell components. Costs of making the additional tonoplast and plasmalemma membrane, and of filling the vacuole with inorganic salts, is only -1% of the cost of synthesizing the rest of the cell for a vacuo1e:cytoplasm volume ratio of 10. Laplace’s principle requires that the turgor-resisting wall has not only a greater area, but also a greater thickness. in the vacuolate cell with the same volume of cytoplasm as a non-vacuolate cell. The additional wall cost may double the overall cost of cell synthesis per unit cytoplasm

Costs of vacuolation Change of cell shape as an alternative (and addition) to vacuolation as a means of increasing surface area per unit cytoplasmic volume involves (as does vacuolation per se) a need for more wall synthesis. Polymer synthesis (for storage in cytoplasm) may cost more (per unit energy, N or P stored) than transport into the vacuole of the monomers of this polymer synthesis. However, the cytoplasmic storage structures (if any) may have lower synthetic costs (energy, C, N, P) than vacuoles and (by not increasing cell volume as much as vacuoles storing the same quantity of material) lower resource costs of synthesis of walls (where present)

Costs of alternatives to vacuolation

TABLE I1 Vacuolar costs, and the costs of performing vacuolar functions by other meuns

Raven (1987) Bouma et al. (1994)

References

Raven (1987) Guggino and Gutknecht (1982)

No direct analogues in multicellular algae (or coenocytic algae with interwoven, small-diameter hyphae, e.g. Codium) with a hollow (saccate) thallus; essentially no turgor in the internal aqueous phase in these algae with similar fresh/dry weight ratio (including water in sac) to giant algal cells

( 3 ) Response to rapid changes in external osmolarity. Giant-celled algae with vacuo1e:cytoplasm volume ratio, of 20 or more require very large transtonoplast and trans-plasmalemma ion fluxes in these cells with low surface area per unit volume to adjust turgor after rapid changes in external osmolarity

Unknown costs of tolerance of bursting/resealing of certain giant-celled algae when exposed to rapid hyposmotic shocks

Raven (1987)

References

Maintenance of alternatives to vacuolation (e.g. pigmented chromoplasts; stored polymers; accumulated CaC03) individually involves less energy expenditure in solute homoiostasis than does a vacuole. This is probably also true of the alternatives to vacuolation in toto, unless there are extreme changes in shape in increasing surface area per unit cytoplasmic volume as an alternative to vacuolation of an isodiametric cell increasing costs of solute homoiostasis

Costs of alternatives to vacuolation

(2) Maintenance of vacuoles and concomitant additional quantities of cell components. The larger area of tonoplast (hypertrophied lysosomes) and plasmalemma membranes increases solute leakage intolfrom the cytosol; solute homoiostasis aspects of maintenance cost four times as much in a vacuolate cell (vacuo1e:cytoplasm ratio of 10) as a non-vacuolate cell with the same volume of cytoplasm and a similar shape

Costs of vacuolation

TABLE I1 (continued) Vacuolar costs, and the costs of performing vacuolar functions by other means

Raven and Richardson ( 1984)

Fahn (1974)

A similar volume of apoplasmic water per unit cytoplasm

Many alternatives to vacuolation relate to differentiated tissues which would normally have further cell division

( 5 ) Movement by dynein/tubulin (flagella, cilia), myosinkactin (muscles. amoeboid movement) or other mechanochemical transducers at a given velocity has a higher energy cost in a vacuolate than a non-vacuolate organism with the same kolume o f cytoplasm (more mass (corrected for density relative to medium) to move; greater surface area for frictional interaction with the medium)

(6) Alleged problems with cell division in highly vacuolate cells. Meristematic cells usually non-vacuolate; but many algal cells can divide despite being high vacuolate (e.g. Cludophoru)

(blood, haemocoel, coelom, coelenteron) would have a similar energetic cost for movement to that of vacuoles

Raven (1987) Kinchin (1994) Cavalier-Smith (1978)

There seems to be n o constraint on desiccation tolerance resulting from the various alternatives to vacuolation. However, there is a height limit of about 2 m (which may be exceeded in the Vreeziaceae) for desiccation-tolerant vascular plants, perhaps related to problems of refilling long-distance transport conduits, including the apoplasmic xylem. Absence of vacuolation may restrict length of conduit initials, thus imposing more resistance in long-distance transport pathways (more frequent cross-walls in phloem, xylem tracheids, or remains of cross-walls in xylem vessel elements)

(4) High vacuo1e:cytoplasm volume ratios seem to be incompatible with desiccation tolerance. Further work is needed here. Certainly a (relative) absence of vacuolation does not necessarily confer desiccation tolerance

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J. A . RAVEN

Jannasch (1995) suggested that Beggiatoa does not execute gliding movements between the more oxidized and more reduced ends of the chemocline, and that it relies on natural vertical movements of the chemocline to alternatively supply oxidant (02, N 0 3 - ) and reductant (S2-). Oxidant (NO3-) can be stored in the vacuole during an oxidizing episode, and (possibly) S2- and So can be stored in a reducing episode. This permits the cells to have a better supply of (internal) oxidant during reducing episodes and vice versa. Thioplaca exhibits gliding motility, and it has been suggested to migrate vertically across the chemocline (Fassing et al., 1995; Huetel et al., 1996; Schulz et al., 1996). At the oxidized surface of the sediment the NO3concentration is -25 mmol mP3. Thioplaca accumulates NO3- 20 000-fold to 500 mol mP3 in its “vacuole”, and then migrates downwards to the S2--rich reduced zone where S2- is oxidized, using NO3- as an electron acceptor, to So, then S042-, and N 2 0 and N2, respectively. Whether the cells migrate upwards with “vacuoles” full of So or S2- is not clear from Fassing et al. (1995); the retention of S2- is unlikely except at unrealistically high intracellular pH values in view of the probable high permeability of H2S. At all events, the downward transport of NO3- in motile filaments can move oxidant down to the S2- much faster than can diffusion. Nitrate is a less energetically desirable oxidant than is 02,but is easier to transport since it can be accumulated in vacuoles whence its leakage is slow because of its low permeability coefficient in the lipid parts of membranes (Raven, 1984a). Oxygen could be transported using a haemoglobin- or myoglobin-like carrier, but this would be a “leaky” transporter into anoxic regions as would the corresponding transport of S2-/HS-/H2S associated with haemoglobin, as in the pogonophoron Riftia (Childress et al., 1991). McHatton et al. (1996) showed similar NO3- accumulation in (motile) Beggiatoa, and suggested that it behaved as did Thioplaca (cf. Jannasch, 1995). At the moment the energetics of NO3- accumulation cannot be accurately estimated from a mechanistic point of view, although an estimate of the minimum thermodynarnic energy cost of NO3- accumulation is possible. This estimate is some 26 kJ (mol NO3-)-’. Minimum energetic costs of gliding mobility are increased by vacuolation due to the larger surface area per unit cytoplasm. Furthermore, the costs of vacuolation must be added to find the total costs of vacuolation including the related transport (of cells and of molecules) phenomena. The benefits of the transport can, of course, be expressed in the same units (energy conversion rate per unit of cytoplasm volume); in this case the computation involves the energy conversion rate based on diffusion of NO3- (and 0,) into the sediment and that based on the “active transport” of NO3- down from the sediment surface to the S2--rich zone. Alas, the information from which the latter “benefit” can be quantified is not readily come by.

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V. ANOTHER CASE HISTORY: VACUOLES AND BUOYANCY Table I indicates that manipulation of the content of solutes in the “true” vacuole can regulate buoyancy. Positive buoyancy can only be achieved in this way in aqueous media of relatively high density and thus relatively high osmolarity. Positive or negative buoyancy can only be of use in controlling the position of planophytic (sensu Luther, 1949) aquatic organisms relative to the water surface if the vertical motion imparted by the buoyancy exceeds the vertical component of bulk water movement. Vacuole-related buoyancy can be achieved by manipulation of the ionic composition of the vacuole such that “heavy” ions (Ca2+, Mg2+, SO4*-, (K+)) are diminished while “light” ions (Na’, NH4+, H + ) are favoured. Granted the availability of the ions in the (seawater) medium, the production of a “buoyant” vacuolar sap need not cost any more than that of a “non-buoyant” sap. This is true at least in mechanistic terms with 1 mol of ATP (or its equivalent) used per mole of ions transported across a plasma membrane and 0.5 mol of ATP per mole of ions transported across the tonoplast: the highest ionic gradients required could be maintained by these ion-to-ATP stoichiometries (Raven, 1984a). However, although the manipulation of vacuolar ions can be considered “costless” granted the occurrence of the vacuole, the constraints on the ionic content of the vacuole related to buoyancy could restrict the use of the vacuole for storage of dense solutes (e.g. NO3- rather than NH4+). Furthermore, the vacuole-tocytoplasm ratio must be high for this mechanism to work, since the cytoplasm is denser than seawater; this argument applies a fortiori to the required ratio of vacuolar volume to that of dense, silicified walls of diatoms. The constraint on the nature of vacuolar storage materials is, to some extent, offset by the large volume of vacuole relative to cytoplasm needed to obtain buoyancy, although there are still constraints on the content of “dense” stored solutes. The high vacuole-to-cytoplasm ratio needed to obtain buoyancy would require a higher wall-to-cytoplasm ratio, which might offset in part the buoyancy effect. Data are available for the vacuolar fraction and wall fraction as a function of cell size in diatoms (Raven, 1987, 1995a). An alternative to favouring “light” ions in a “normal” vacuole in reducing density is the use of lipid (low density) rather than polysaccharide (high density) as the organic carbon and energy storage material. This option of lipid is, to varying extents, seen in diatoms in parallel with “light” ion accumulation in the vacuole. Another component which reduces overall cell density is the gas vesicle (Walsby, 1994). This mechanism is confined to certain prokaryotes, and has not been found so far in those with true (aqueous) “vacuoles” (Walsby, 1994; Fassing et al., 1995). The volume of gas vesicles needed to give a certain degree of buoyancy is, of course, much smaller than that of an aqueous vacuole since the gas has a density

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that of the average solid plus fluid cell contents, although the need to have pressure-resistant (proteinaceous) walls for gas vesicles involves a very substantial energy cost (Walsby, 1994). However, the small fraction of cell volume occupied by gas vesicles means that a given volume of cytoplasm has its surface area increased less when gas vesicles cause a given reduction in density than is the case for aqueous vacuoles with ‘‘light’’ ions. This in turn means a more rapid upward motion for the gas-vesicle-containing cells, as shown by the application of hydrodynamic principles, for a given difference in density between the cell and its medium. A rather different means of adjusting the vertical position of cells in a non-turbulently mixed water body is that of flagellar motility (Raven, 1982; Raven and Richardson, 1984). Walsby (1994) has shown that the energetic and nitrogen cost of the construction of gas vesicles (in cyanobacteria) greatly exceeds that of the flagellar apparatus (of eukaryotic algae) and that inclusion of running (energy) costs of operating the flagellar apparatus does not offset this difference in energetic constructional costs for any generation time attainable with balanced growth (20 times the minimum generation time) (Raven, 1986; Geider et al., 1985). Furthermore, the flagellar mechanism offers a more immediate (seconds) regulation of the direction of vertical movement. Adding ballast (carbohydrate as polysaccharide) to gas-vesiculate cyanobacteria to an extent which reverses the sign of the density difference between cells and medium takes tens of minutes or hours of net photosynthesis, as does accumulation of K+ salts (Walsby, 1994). It is likely that similar temporal considerations apply to alteration of buoyancy in algae with ‘‘light’’ions in their vacuole, granted the surface area per unit volume in these diatoms and the area-based net ion fluxes across the tonoplast and plasmalemma (Raven, 1984a, 1988). The more temporally flexible flagellar mechanism is not available to the cyanobacteria (with the exception of one marine strain, Synechococciis: Waterbury et al., 1985), with some means of swimming not associated with “normal” structures of bacterial flagella) o r to the walled (silicified) vegetative cells of diatoms (flagella only occur on the wall-less male gametes of centric diatoms). The discussion thus far of buoyancy regulation/flagellar motility has concentrated on the costs of the various “cell-positioning” mechanisms. What of the benefits? Raven and Richardson (1984) have performed a cost-benefit analysis of vertical diel migration by dinoflagellates growing in (normal) die1 light-dark cycles in stratified water bodies with a greater supply of nutrients (e.g. inorganic nitrogen and phosphorus) and depth from chemoorganotrophy than at the surface dominated by photolithotrophy. The strategy here is upward migration around dawn and downward migration around dusk, thus optimizing the acquisition of the “co-limiting” resources light (only available near the surface in the photophase) and the inorganic nutrients nitrogen, phosphorus, iron, etc. (available over the whole diel cycle but scarce near the surface).

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The behaviour of certain of the planktonic diatoms which can regulate their density via control of vacuolar composition fits a similar paradigm (Villareal, 1992; Villareal et al., 1993; Villareal and Carpenter, 1994). However, the periodicity of the cyclic vertical migration is more than 24 h ; Villareal and Carpenter (1994) suggest 7-12 days for Erhmodiscus rex (cell volume m3). The “limiting” inorganic nutrient in the habitats investigated by Villareal and co-workers (central Pacific gyre) is nitrogen, as shown by the cellular C:N:P ratio (Villareal and Carpenter, 1994). The diatoms have more N03- in their cell (vacuolar) sap whcn ascending than when desccnding; for Rhizosolenia sp. (cell volume 10-“’m3) the concentrations are 9.7 k 2.9 mol m-3 when rising and 2.0 k 2.3 mol m - 3 when descending (Villareal et al., 1993). Natural-abundance “N/I4N studies show that Rhizosolenia obtains much of its nitrogen from subnutricline NO3- with a higher lsN/I4N than surface-water combined nitrogen (Villareal et al., 1993). The large size of the vacuole relative to the cytoplasm means that relatively modest NO3- concentrations (up to 27 mol m-3 in E. rex) can contribute up to 54% of the total (inorganic plus organic) cellular nitrogen quota (Villareal and Carpenter, 1994). The carriage of nitrogen from the nutricline to the surface waters as NO3 by ascending cells of these large diatoms illustrates the potential conflict between the storage function of the vacuole and its role in decreasing overall cell density. Nitrate is a “heavy” ion, and the buoyancy function would be better served by NO3- reduction t o yield the “light” ion NH4+ at the nutricline prior to or during ascent (Fig. 1 ) . However, energetic considerations may militate against this energetically costly reduction in a low-light environment. This appears to explain why the benthic marine brown macroalga Laminaria accumulates the NO3- available in winter (light energy available at the depth limit of its occurrence) as NO3- in its vacuoles, with reduction and assimilation later in the year (Raven, 1987). It may also explain why the freshwater red macroalga Lemanea, whose main growth occurs in winter with limited light availability, uses exogenous NH4+ as its nitrogen source and eschews the more abundant (at least in agriculturally influenced streams) NO3- (Raven, 1987; MacFarlane and Raven, 1990). At all events the absolute concentration of “heavy” NO3- is only a small fraction of the total ionic content of these large-celled diatoms, so that variations in the content of other ions can contribute to the conversion o f an NO3- -rich “floater” into an NO3-- poor “sinker”, possibly with contributions from the fraction of organic carbon and energy stored as polysaccharide rather than lipid. In addition to these and similar large diatoms, the “vertical migrations with NO3- transport” paradigm may well also apply to the large-celled, vacuolate phycoma stage of the green (prasinophyte) alga Halosphaera and to the large-celled, vacuolate but non-flagellate (in the vegetative state) dinoflagellates such as Pyrocystis (Villareal and Carpenter, 1994). Lest these migrations of large-celled vacuolate planktonic algae seem

78 1.050

-

1.045 -

1.040

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1.035 -

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E 0)

F 3

.-fn

$ n

-.-

J . A. RAVEN

NH,CI

-v-

NaCl KCI NaNO,

-4-

KNO,

-0-

-A-

1.030 1.025

-

1.0201.0151.010 1.005 7 0

100

200

300

400

500

600

700

Concentration/mol m-3 Fig. 1. Density of solutions of potential vacuolar solutes of large-celled diatoms as a function of concentration. (From tabulated data in Washburn (1928).)

remote from the preoccupations of most vacuoleers, a few comments on their global significance are in order. Planktonic algae in the ocean transform significantly more nitrogen each year than d o terrestrial (including cultivated) plants (Raven et al., 1993). Of this combined nitrogen taken up by marine planktonic algae, some 20% is as NO3- recycled from the ocean depths (Raven et al., 1993). While much of this moves upwards as macroscopic upwellings and microscopic eddy diffusion, there could be a very important role in parts of the open ocean for the N03--transporting vertical migrations based on regulation of vacuolar ionic composition; at least 1% of the oceanic nitrogen assimilation (i.e. more than 6Tmol N per year) could be moved upward by this mechanism.

VI. COSTS AND BENEFITS OF VACUOLATION: SIMPLE ANALYSES AND THE ALLOCATION OF COSTS AMONG VARIOUS BENEFITS The geometrically unavoidable consequence of vacuolation without a change in cell shape is an increase in the surface area per unit volume of cytoplasm (see Table I), which can increase the rate of resource acquisition, at least

800

THE VACUOLE: A COST-BENEFIT ANALYSIS

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TABLE 111 Costs and benefits of vacuolation for aquatic unicells growing under resource-saturating conditions, for a non-vacuolate cell 0.5 or 5 p m in radius and a vacuolate cell 1.11 or 11.1 p m in radius with a vacuo1e:cytoplasm volume ratio of 10 and the same cytoplasmic volume as for the 0.5 or 5 p m radius cell, respectively (from Tables 4-7 of Raven, 1987) Resource being acquired and used

Benefit (additional resource acquisition) of vacuolation

Cost (additional resource cost of cell synthesis) of vacuolation

Energy

Nil

U p to twice as much energy needed for cell synthesis per unit of cytoplasm

Carbon

Nil (except in the unlikely case of limitation of C acquisition by area of plasmalemma to house transporters, in which case the rate may be fivefold higher)

Up to twice as much energy needed for cell synthesis per unit of cytoplasm

Nitrogen

Nil (except in the unlikely case of limitation of N acquisition by area of plasmalemma to house transporters, in which case rate may be fivefold higher)

U p to 40% more N needed for cell synthesis per unit of cytoplasm

Phosphorus

Nil (except in the unlikely case of limitation of P acquisition by area of plasmalemma to house transporters, in which case rate may be fivefold higher)

U p to 10% more P needed for cell synthesis per unit of cytoplasm

under resource-limiting conditions (Tables 111 and IV). The increase in the rate of resource acquisition is especially significant for photons, but can also apply to chemical resources (Table IV). These benefits are paralleled by the resource costs (see Table 11) of cell synthesis. Under resource-saturating conditions the resource costs of vacuolation outweigh the resource-acquisition benefits (see Table 111); the reverse can be the case under resourcelimiting conditions, most generally in the case of photon absorptiodenergy costs (see Table IV). This simple analysis deals in processes which all organisms carry out, i.e. resource acquisition and growth. However, Tables I and I1 show many more costs and benefits of vacuolation which generally (an exception is storage of resources) are not universal. Thus, the increased energy cost of motility of a given volume of cytoplasm at a given velocity as a result of vacuolation

TABLE IV

Up to 40% more N needed for cell synthesis per unit of cytoplasm Up to 10% more P needed for cell synthesis per unit of cytoplasm

Up to 40% more N needed for cell synthesis per unit of cytoplasm Up to 10% more P needed for cell synthesis per unit of cytoplasm

Up to five times as much C, N or P entry if influx is limited by plasmalemma area available for transporters

Up to five times as much C, N or P entry if influx is limited by plasmalemma area available for transporters

Nitrogen

Phosphorus

Up to twice as much C needed for cell synthesis per unit of cytoplasm

Up to twice as much C needed for cell synthesis per unit of cytop 1asm

Very small for extracellular diffusion of solutes bearing C, N or P

Zero for extracellular diffusion of solutes bearing C, N or P

Carbon

Up to twice as much energy needed for cell synthesis per unit cytoplasm

5 urn .~ (non-vacuolateY 11.1 p m (vacuolate)

Up to twice as much energy needed for cell synthesis per unit cytoplasm

0.5 um (non-vacuolateY . \ 1.11p m (vacuolate) ‘

Photon absorption 2.33 times that of non-vacuolate cell

5 urn .~ (non-vacuolateM 11.1 pm (vacuolate)

Photon absorption 1.15 times that of non-vacuolate cell

0.5 um (non-vacuolateY 1.i1pA (vacuo~ate)’

Cost of vacuolation for cell of radius

Energy (assuming 20mol of chromophore per 1 m3 of cytoplasmic volume)

Resource limiting growth

Benefit of vacuolation for cell of radius

Costs and benefits of vacuolation for aquatic unicells growing under resource-limiting conditions, for a non-vacuolate cell 0.5 or 5 p m in radius and a vacuolate cell 1.11 or 11.1 p m in radius with a vacuo1e:cytoplasm volume ratio of 10 and the same cytoplasmic volume as the 0.5 or 5 p m radius cell, respectively (from Fig. 2 and Table 2 of Raven, 1987)

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(see Table 2) is an exception among vacuole organisms, as is the benefit of increased visual attraction as a result of vacuolar pigments (see Table I). Quantitative analysis of the allocation of costs (see Table 11) among numerous benefits (see Table I), i.e. multiobjective optimization (Farnsworth and Niklas, 1995) is potentially possible, but the multiplicative effects of the increasing number of assumptions which must be made as more costs and benefits are included in the analysis limit the extent to which such analyses can be used for the multitude of costs and benefits in Tables I and 11. Eventually, with more quantitative knowledge, such multiobjective optimization analyses will become more realistic, and will contribute to the ultimate goal of relating vacuolation to include fitness (Osmond et af., 1990).

VII. EVOLUTIONARY ASPECTS In view of the lack of multiobjective optimization analyses noted above, it may be considered premature to consider evolutionary aspects of vacuolar costs and benefits. However, I wish to make two general points, one involving environmental variability over geological time, and the other to nonvacuolate, invertebrate-based phototrophic symbioses. The environmental variable considered is atmospheric C 0 2 . The first terrestrial embryophytes (bryophytes, tracheophytes) some 420 million years ago were confronted with C 0 2 partial pressures 10-20 times the present value of 36Pa (Raven, 1995b). Carbon dioxide partial pressures higher than the present value have predominated over the intervening 420 million years, with values probably as low as the extant C 0 2 level in the Upper Carboniferous, and even lower values in the interglacial and, especially, glacial episodes in the last million or so years (Raven, 1995b). Today there are advantages in vacuolation in homoiohydric plants (i.e. those which have cuticle, stomata and intercellular gas spaces, and thus can remain hydrated for some time in the absence of an adequate water supply to the roots and/or with a very large evaporative demand from the atmosphere) in terms of minimizing the resistance to C 0 2 diffusion in the aqueous phase (Raven, 1993b, and references therein). These advantages would have been greater at the last glacial maximum (18 000 years ago) with C 0 2 partial pressure at 18-20 Pa, but much less in the high-C02 environments of the Early and Mid Palaeozoic and the Mesozoic and most of the Tertiary. However, the light-absorption advantages of vacuolation would still have been present (see Table 1). At least as far as photosynthetic structures are concerned there might have been a smaller selection pressure for large vacuoles in the more distant past (except for the Upper Carboniferous) but larger selection pressure in the more recent past. Quantitative assessment of these suggestions is difficult except in terms of overall cell size: vacuoles do not fossilize well. The phototrophic symbioses between invertebrates and microalgae are

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non-vacuolate, yet they coexist with (vacuolate) macroalgae and higher plants (see Table I, and Raven, 1993a). Corals can achieve a large surface area per unit cytoplasmic volume by non-spherical shapes and the presence of a coelenteron; this is less readily achieved in the giant clams. These animalinvertebrate symbioses will form useful material for future evolutionary cost-benefit analyses of vacuolation.

VIII.

CONCLUSIONS

At the risk of being too Panglossian (Voltaire, 1759), it would be possible to conclude that the semiquantitative analyses currently possible show that the evolutionary costs of vacuoles are outweighed by their evolutionary benefits. However, much more quantitative analysis is needed to test the suggestion that we have currently identified all of the costs and benefits of vacuolation and their quantitative importance.

ACKNOWLEDGEMENTS Past and present colleagues have catalysed and refined my thoughts on the costs and benefits of vacuolation. Experimental work on the role of vacuoles in acid-base regulation of higher plants and of cell size in resource acquisition and storage by algae has been funded by the AFRC/BBSRC and NERC.

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