Calcium Oxalate Crystals in Plants Author(s)

Calcium Oxalate Crystals in Plants Author(s): Vincent R. Franceschi, Harry T. Horner and Jr. Source: Botanical Review, Vol. 46, No. 4 (Oct. - Dec., 1980), pp. 361-427 Published by: Springer on behalf of New York Botanical Garden Press Stable URL: http://www.jstor.org/stable/4353973 Accessed: 19-03-2015 05:39 UTC

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BOTANICAL

THE VOL. 46

REVIEW

OCTOBER-DECEMBER, 1980

No. 4

CALCIUM OXALATE CRYSTALS IN PLANTS VINCENT R. FRANCESCHI1AND HARRY T. HORNER, JR. Department of Botany, Iowa State University Ames, Iowa 50011

Abstract-361 Introduction-364 Oxalic Acid in Plantsand Animals-365 Synthesis in Plants-365 Metabolismin Plants-370 Synthesis and Occurrencein Animals-371 Oxalic Acid as a Balanceof Calciumand OtherIons in Plants-374 NitrogenAssimilationand Oxalic Acid Synthesis in Plants-377 Calciumin Plants-379 Function---------------------------------------Absorptionand Transport-379 Shape and Distributionof CalciumOxalate Crystalsin Plants-381 Analyses for Oxalic Acid, Calciumand CalciumOxalate-396 Oxalic Acid-396 Calcium-403 CalciumOxalate-403 Developmentand Ultrastructureof Crystal Idioblastsin Plants-409 Function(s)of CalciumOxalateCrystal Idioblastsand Crystals-411 Discussion-414 AcknowledgmentsLiteratureCited-416

379

416

ABSTRACT

Calcium(Ca) oxalate crystals occur in many plant species and in most organs and tissues. They generally form within cells althoughextracellular crystals have been reported.The crystal cells or idioblasts display

1 Presentaddress:Departmentof

Botany, Universityof California,Davis, CA 95616.

The Botanical Review 46: 361-427, October-December,

(?) 1980 The New York Botanical Garden

1980.

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ultrastructuralmodificationswhich are related to crystal precipitation. Crystal formation is usually associated with membranes,chambers, or inclusions found within the cell vacuole(s). Tubules, modified plastids and enlargednuclei also have been reportedin crystal idioblasts.The Ca oxalate crystals consist of either the monohydratewhewellite form, or the dihydrateweddelliteform. A numberof techniquesexist for the identificationof calcium oxalate. X-ray diffraction,Ramanmicroprobeanalysis and infraredspectroscopyare the most accurate. Manyplantcrystals assumed to be Ca oxalate have never been positively identifiedas such. In some instances, crystals have been classified as whewellite or weddellite solely on the basis of their shape. Certainevidence indicates that crystal shape may be independentof hydrationform of Ca oxalate and that the vacuole crystal chamber membranesmay act to mold crystal shape; however, the actual mechanismcontrollingshape is unknown. Oxalic acid is formed via several majorpathways. In plants, glycolate can be converted to oxalic acid. The oxidation occurs in two steps with glyoxylic acid as an intermediateand glycolic acid oxidase as the enzyme. Glyoxylic acid may be derivedfrom enzymatic cleavage of isocitric acid. Oxaloacetate also can be split to form oxalate and acetate. Another significantprecursorof oxalate in plantsis L-ascorbicacid. The intermediate steps in the conversionof L-ascorbicacid to oxalate are not well defined. Oxalic acid formationin animalsoccurs by similarpathways and Ca oxalate crystals may be producedundercertain conditions. Various functions have been attributedto plant crystal idioblasts and crystals. There is evidence that oxalate synthesis is related to ionic balance. Plant crystals thus may be a manifestationof an effort to maintain an ionic equilibrium.In many plants oxalate is metabolizedvery slowly or not at all and is consideredto be an end productof metabolism.Plant crystal idioblasts may function as a means of removingthe oxalate which may otherwise accumulatein toxic quantities. Idioblast formationis dependent on the availabilityof both Ca and oxalate. Under Ca stress conditions, however, crystals may be reabsorbedindicatinga storage function for the idioblastsfor Ca. In addition, it has been suggested that the crystals serve purely as structuralsupports or as a protective device against foraging animals. The purpose of this review is to present an overview of plantcrystalidioblastsand Ca oxalate crystals and to include the most recent literature. ZUSAMMENFASSUNG

Calciumoxalat-Kristallekommen in vielen Pflanzenartenund in fast allen Teilen und Geweben vor. Im allgemeinenwerden sie innerhalbder


CALCIUMOXALATECRYSTALSIN PLANTS

363

Zellen gebildet, doch sind auch extrazellulareKristallebeschrieben. Die Kristallzellen oder Idioblasten zeigen besondere ultrastrukturelleSpezialitaiten,die mit der Kristallbildungzusammenhiingen.Diese ist meist mit Membranen,Raumenoder Einschliissenin der orderden Vakuole(n) verbunden. Tubuli, modifiziertePlastiden sowie vergroBerteZellkerne sind ebenfalls fur die Idioblasten erwahnt. Die Kristalle bestehen entweder aus der Monohydrat-(Whewellit)oder der Dihydrat-form(Weddellit). Mit verschiedenenMethodenkonnen die Calciumoxalat-Kristalle identifiziertwerden, die sichersten sind Rontgenstrahlen-Diffraction, Raman Mikroprobe-Analyseund die Infrarot-Spektroskopie.Viele pflanzliche Kristalle, die als Kalciumoxalat-Kristalleangesehen werden, sind nie eindeutigbestimmtworden. In einigen Fallen wurdensie lediglichauf Grundder Form als Whewellitoder Weddellitklassifiziert;es gibt aber Hinweise, dab die Kristallformunabhangigvom Hydratationsgradist und daBdie Membranender Kristallkammerin der Vakuole die Form beeinflussen. Der eigentliche Kontrollmechanismusist noch unbekannt. Oxalsaurewird auf verschiedenen Wegen in den Zellen synthetisiert, in Pflanzen kann Glycollat in Oxalsaureumgewandeltwerden. Die Oxidation erfolgt in zwei Schritten mit Glyoxylsaure als Zwischenprodukt und dem Enzym Glycolsaure-Oxidase.Die Glyoxylasaurekonnte durch enzymatische Spaltungder Isozitronensaureentstehen. Auch kann Oxalazetat in Oxalat und Azetat splitten. Eine weitere wichtige Ausgangssubstanz fur Oxalat in Pflanzenist die L-Ascorbin-saiure,die Zwischenproduktedieser Umbildungsind noch nicht eindeutigbekannt.OxalsaureBildungerfolgt bei Tieren uiberahnliche Stoffwechselwegeund Calciumoxalat-Kristallemogen in ahnlicherWeise entstehen. VerschiedeneFunktionenwerden den pflanzlichenKristall-Idioblasten und Kristallen zugeschrieben. Es besteht Gewif3heit,daB3die OxalatSynthese mit der lonen-Balancekorreliertist; so konnen die pflanzlichen Kristalle die Anstrengungenbelegen, ein Ionen-Gleichgewichtzu erhalten. In vielen Pflanzenwird das Oxalat nur in geringem MaBeoder gar nicht weiter metabolisiert,es wird als Endproduktdes Stoffwechsels angesehen. Die Kristall-Idioblastenfunktionierenwohl zur Entfernungdes Oxalats, das sonst toxische Konzentrationerreichenwurde. Die Bildung hangt von der Verfiigbarkeitsowohl des Calciums wie des Oxalats ab. Unter Calcium-Mangelkannes zur Reabsorptionvon Kristallenkommen, ein Hinweis auf eine gewisse Speicherfunktionder Idioblastenfur Ca. Zusatzlichwird vermutet,dal3die KristallemechanischeFunktionhaben und ein Schutz gegen fressende Tiere darstellen. Die Absicht dieses Review ist es, eine aktuelle Ubersicht uber die Kristall-Idioblastenund CaOxalat-Kristallezu geben, die neueste Literaturberucksichtigt.


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THE BOTANICALREVIEW INTRODUCTION

Several reviews withinthe past ten years have dealt with calcium (Ca) oxalate and oxalic acid in plants and animals(Arnott and Pautard,1970; Arnott, 1973, 1976; Hodgkinson, 1977). There have been a number of more recent studies in these areas which suggest continued interest in the general field. The purpose of this present review is to complement the earlier reviews and to use the more recent studies to increase the understandingof the presence of plant Ca oxalate crystals. We also have included a brief section and assorted references on animalCa oxalate to emphasize some of the similaritiesand differences associated with plant Ca oxalate. Many plants and animalsproduce oxalic acid, and it is of interest that they share some commonpathwaysof oxalic acid synthesis. Oxalatemay be present as the soluble sodium or potassium salts or as insolublecrystalline Ca oxalate. Calciumoxalate crystalformationin animalsis generallyconsideredto be pathological and extracellular. A good example of this situation is representedby urinarycalculi (stones) which are often partly or entirely composed of Ca oxalate. In contrast, Ca oxalate recently has been shown to be a structuralcomponent of the gizzard plates of certain gastropods (Lowenstam, 1972)and thereby serves a useful function. Calciumoxalate in plants is not considered to be pathological,and its formationis typically intracellular,with the crystals formingin specialized cells called crystal idioblasts. The shape, size, and numberof crystals within a crystal idioblastvary considerably.The numberand location of crystal idioblasts within the plant body also vary among taxa. Little is known about the function(s) of the crystals and the crystal idioblasts. Two possible explanafionsfor their presence are that the crystals serve as a protectivedevice againstforaginganimals,or, alternatively, as a means of removing excess oxalic acid from the plant system. Oxalateis generallyconsideredto be an endproductof metabolism(Vickery and Abrahams,1950;Crombie, 1954;Schneider, 1961)and not usable to the plant. Although there have been numerousstudies on oxalate metabolismin plants and animals, very little work has been done on Ca oxalate crystal formation.This form of biologicalcalcificationis quite commonin higher plants and may parallelsimilarprocesses in animals. It is not understood why plant Ca oxalate crystals are formed. It is known that this calcificationprocess involves changes in cell development and formationof specialized structures, but the factors which induce these changes are poorly understood.Manyfactors may be involved in the induction of Ca oxalate crystal idioblasts and, concomitantly, in



365

the crystallization process. A number of the recent papers on crystal formation deal with idioblast ultrastructure and the developmental changes which occur. Crystal idioblast formationhardly has been looked at with respect to the metabolism or translocationof the constituents which make up the crystals. A review of the literaturein such areas as oxalate synthesis and metabolismand their relationshipto Ca uptake and mobility, along with the structuraland developmentalstudies, is necessary in order to obtain an overall perspectiveof the crystallizationprocess. Factorswhich contol oxalate synthesis and Ca uptake and mobility may be expected to affect crystal idioblast inductionand formation. Before elaboratingon the specific aspects of Ca oxalate, it should be mentionedthat Ca combines with a numberof anions besides oxalate to form a variety of Ca salts. Calciumcarbonatedeposits are characteristic of certain angiospermfamilies, occurringeither in a diffuse state within cells or as large stalkedbodies termedcystoliths within specialized cells, the lithocysts (Pireyre, 1961; Arnott and Pautard, 1970; Arnott, 1973). Other Ca salts that have been identified in plants are those of sulfate, phosphate, silicate, citrate, tartrate and malate (Arnott and Pautard, 1970). OXALIC ACID IN PLANTS AND ANIMALS

Synthesis in Plants

Organicacid synthesis and metabolismin plants has long been studied and much informationis available. Oxalic acid, often considered an end product of metabolism,has been investigatedin some detail. A number of investigators have elucidated the pathways of oxalate synthesis, the majorones of which are given in Figure 1 (see also Hodgkinson, 1977). Early studies on plant organic acids, includingoxalic acid, produced evidence that the productionof these acids was relatedto photosynthesis and carbohydratemetabolism. Steinmann(1917) found continuous formation of oxalic acid in rhubarbleaves in the light. Later, Myers (1947) noted that oxalate concentrationin rhubarbleaves increasedthroughout the growing season, which he correlatedwith the season of most active photosynthesis. Because of the magnitudeof the increase, he concluded that the oxalate arose in a synthetic process involving photosynthesis. Pucher et al. (1939)reportedthat in buckwheatoxalic acid concentration increased duringilluminationof the plant. Also, the oxalate concentrationcontinuedto increasein the dark,thoughat a lower rate, indicating a transformationof some substance, probably photosynthetically-derived, into oxalic acid.


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THE BOTANICALREVIEW

CARBOHYDRATES GLYOXYSOM

CARBOHYDRATES PHOSPHOPYRUVATE-*

PYRUVATE

__GLYOXYSOME__

OXALIC ACID

CHLOROPLAST

I

OXALOACETATEPHOTOSYNTHESIS

FA

MALATE

ACETATErXALOACETATE A

ACETYL COA L-ASCORBIC ACID /_|_|DEHYDRO

____/__

GLYCOLIC ACID GLYCOLIC --ACID.,

L-ASCORBIC ACID.

SERINE TARTAI OXALOACETATE ACONITATE MITOCHONDRION \ GLUTAMTACID t

MALATE

/

G GLYCINE E 2, 3 DIKETO L-GULONIC ACID

CITRATE

ISOCITRATE

FUMARATE o' ___________________________________

>

CO2

j

.EROXISOME LYAS/ ISOCITRATE

{ACETYL/

l

-------

GL~~~~~.-XATHN

~OXIDASE

C

CID OXALIC AIC ACID E

VACUOLE C?2 INSOLUBLE CALCIUM SOLUBLE OXALATE

Y

+ FORMATE

OXALATES

Fig. 1. Summaryof metabolicpathwaysof oxalic acid productionin plants.

Later experimentson rhubarband other plants (Stutz and Burris, 1951) supportedthe idea that oxalate synthesis was related to photosynthetically-derived carbon compounds. Through labeling studies, Stutz and Burris(1951) were able to show that CO2was quickly incorporatedinto malic acid during short exposure to light. When the plants were placed in the dark, the label began to appear in oxalic acid, which gradually increased in concentration.These studies indicatedthat oxalate was not a direct product of photosynthesisbut was formed from precursorssynthesized in the light. Tavant(1967)obtainedsimilarresults with Begonia. A very close relationshipbetween oxalate formationand photosynthesis was also found in Oxalis corniculataby Seal and Sen (1970). Oxalate concentrationwas found to increase duringthe hours of peak photosynthesis and decrease duringthe evening and night. A direct dependence on photosynthetically-derivedsubstanceswas implied.Recently, ZindlerFrank (1976b) tried to find a correlationbetween the type of photosynthetic pathway and oxalate formation. She could find no evidence for a difference in the ability of C3 and C4 plants to synthesize oxalic acid. In 1947, Nord and Vittucci reportedthe conversion of glycolic acid to oxalic acid by a fungus. They postulated that glyoxylic acid was an intermediate. Glycolic acid is one of the first formed products of photosynthesis in higher plants (Norris et al., 1955;Tolbert, 1973)and, thus,



367

confirmedthe earlierevidence that oxalate synthesis could be related to photosynthesis. Much work was done on the glycolate system to determine the enzyme involved in the conversion process. Zbinovsky and Burris(1952)found that tobacco and buckwheatleaves infiltratedwith labeled glycolic acid converted it to oxalate. At about the same time, Kenten and Mann (1952)isolated a plant a-hydroxy-acidoxidase which in the presence of catalase could oxidize glycolate to glyoxylate and glyoxylate to oxalate. The catalase was necessary to remove hydrogenperoxideproducedfrom the oxidationof glycolate, which could nonenzymaticallyoxidize glyoxylate to formic acid and CO2. A highly purifiedglycolic acid oxidase was preparedby Zelitch and Ochoa in 1953 from spinach leaves. The enzyme was a flavoproteinand could catalyze the conversion of glycolate to glyoxylate. The enzyme did not catalyze the conversion of glyoxylate to oxalate, possibly due to the conditions under which the experiments were run. A glycolic acid reductase was later isolated which convertedglyoxylate to glycolate (Zelitch, 1953).An oxalic acid cycle based on these enzymes was proposed by Carles and Assailly (1954).

Seal and Sen (1970) outlined the probable relationshipbetween photosynthesis and oxalate formation.They proposed that photosynthetically-derived glycolate is oxidized by glycolate oxidase to glyoxylate. The glyoxylate is furtheroxidized to oxalic acid via the same enzyme. Oxygen liberated by photosynthesis is used as an electron acceptor during the oxidation. This scheme would help explain the reports of light stimulated oxalate synthesis. Noll and Burris (1954) reported the presence of glycolic acid oxidase in 16 common plant families. They also found the enzyme in etiolated seedlings and noted that exposure to light resulted in its increase. It is now generally accepted that at least in some plants glycolate and glyoxylate are the immediateprecursors of oxalic acid and that the enzyme glycolic acid oxidase catalyzes both oxidations. A number of labeling experiments have provided further proof for this scheme (Bornkamm, 1965;Changand Beevers, 1968;Kpodaret al., 1978;Millerdet al., 1963a, b; Richardsonand Tolbert, 1961;Vickery and Palmer, 1956). The physical and catalytic properties of glycolic acid oxidase appear to vary somewhatamongdifferentplants. Richardsonand Tolbert(1961) found that the flavin-linkedglycolic acid oxidase isolated from spinach was inhibited by oxalic acid. This was especially true for the oxidation of glyoxylate to oxalate, and the inhibitionwas shown to be competitive. They suggested that this providedfor the regulationof oxalate synthesis. The enzyme also had a greateraffinityfor glycolate than for glyoxylate. Millerdet al. (1963d)found the flavin-linkedglycolic acid oxidase of Oxalis pes caprae to have some differentproperties.Oxidationof glyoxylate This content downloaded from 14.139.172.163 on Thu, 19 Mar 2015 05:39:28 UTC All use subject to JSTOR Terms and Conditions

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THE BOTANICALREVIEW

was not affected by the presence of oxalate and the affinityof the enzyme for glycolate and glyoxylate was the same. Bornkamm(1969), upon examinationof six plant species, found two forms of glycolate oxidase. One form was activatedby FMN (flavinmononucleotide), while the other was substrate-activated.Another form of glycolate oxidase was found in etiolated wheat seedlings (Bakerand Tolbert, 1967).It was shown not to be a flavinenzyme and was not activated by FMN but was stimulatedby ferredoxin.The enzyme was most prevalent in young tissue and it was suggestedthat it may later be converted to the type found in older tissues (FMN-activated).Evidence for this was found by treatingthe young form of the enzyme with (NH4)2SO4which converts it to a form requiringFMN. Carbon compounds not directly related to photosynthesis have been shown to produce oxalate in some plants. Millerd et al. (1963a) found that sucrose was a precursorof oxalate in Oxalis. Oxalate in spinach seedlings was reportedto come from two sources: degradationof stored foods in the endosperm or perisperm of the seed or from products of photosynthesis (Kitchen et al., 1964).Tavant (1967) showed that U'4-Cglucose can give rise to oxalate in Begonia. Nonphotosyntheticetiolated seedlings can also produce oxalate (Baker and Tolbert, 1967;Kitchen et al., 1964). Oxalate synthesis involving compounds not directly produced from photosynthetic carbon fixation does not necessarily exclude glyoxylate as the immediateprecursorof oxalic acid. Carbonfrom sugarsand certain amino acids can be channelled into the Krebs cycle and at least one Krebs cycle intermediateis known to give rise to glyoxylate undercertain conditions. Kornbergand Krebs (1957) demonstratedthat isocitric acid can be enzymaticallycleaved by isocitrate lyase to give glyoxylate and succinic acid. The glyoxylate thus producedcould conceivably be used for oxalate synthesis. Isocitrate lyase is an importantenzyme of the glyoxylate cycle which is involved in lipid breakdownin lipid-containingseeds (Stumpf, 1965). Carpenterand Beevers (1959) studied the distributionand propertiesof isocitrate lyase in plants. They concluded that it was limited to certain species and confinedto those tissues in which active breakdownof lipids is occurring. However, since Carpenterand Beever's report, a number of studies have demonstratedan association between isocitratelyase activity and oxalic acid synthesis in leaves and shoots. Millerdet al. (1963b, c, d) and Mortonand Wells (1964)demonstrated the formationof glyoxylate by the action of isocitratelyase in Oxalispes caprae. Carbonfrom labeled isocitrate was rapidlyand efficientlyincorporated into oxalic acid. The presence of glyoxylate and glycolic acid oxidase was also shown, and they furtherfound that glycolate could be



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converted to oxalic acid. For short incubation periods (2-6 hr), incorporationof label from isocitrate and glycolate was equal while for longer periods the contributionfrom glycolate was greater. This may reflect the relative need of the two compoundsfor other synthetic reactions. Osmond and Avadhani(1968) found that in Atriplex short-termincorporation of 14CO2in the dark led to labeled Krebs cycle intermediates. Oxalatewas slowly labeledand this labelingwas inhibitedby malonate(an inhibitorof the Krebs cycle), suggestingthat oxalate was producedfrom some Krebs cycle intermediate.Glyoxylate was also readilyconverted to oxalate in the dark. They later identifiedisocitratelyase and glycolic acid oxidase in the leaves indicatingthat oxalate synthesis occurred via isocitrate duringacid metabolismin the dark. Isocitrate lyase also has been found in the oxalate-producingfungus Sclerotium rolfsii (Maxwell and Bateman, 1968). Presumably,all of the oxalate is derived from isocitrate since the enzyme which oxidizes the glyoxylate will not oxidize glycolate. Oxalic acid is present as the free acid in some plants (Begonia, Oxalis) or as a salt and accompanied by other acids (Ranson, 1965). Ranson suggests that the differences may be related to whether oxalate is produced via isocitrate or from photosynthetically-derivedglycolate. Crombie (1960) proposed that the role of oxalic acid in metabolismmay vary among the two types of plants. Glyoxylic acid is not the only substance which has been found to be an oxalate precursorin plants. It appearsthat in many plants the metabolism of L-ascorbic acid, which can be synthesized from D-glucose, is involved in oxalate formation(Wagnerand Loewus, 1973).Loewus et al. (1975) showed that in Pelargoniumcrispum the number 1 and 2 carbons of L-ascorbic acid are converted into oxalic acid while the rest of the molecule gives rise to (+) tartaricacid. However, in Vitis labrusca and Parthenocissus inserta, ascorbic acid gives rise to (+) tartaricacid and hexoses but not oxalic acid (Wagnerand Loewus, 1974).Yang and Loewus (1975) reportedthat oxalate accumulatorssuch as spinach, begonia, and Oxalis, developed substantialamounts of labeled oxalate when fed ascorbic acid labeled at the number1 or 2 carbon. Non-accumulatorsdid not convert ascorbic acid to oxalate. More recently, Nuss and Loewus (1978)examined a numberof plant species for their abilityto accumulate oxalate via ascorbic acid metabolism. They also found that oxalate-accumulatingplantscould readilyconvert partof the ascorbicacid molecule (carbons 1 and 2) to oxalate. Although glyoxylate and L-ascorbic acid appear to be the majorprecursors of oxalic acid in plants, some other possible pathwayshave been reported. Gentile (1954) found evidence that oxalic acid in the fungus Botrytis cinerea was produced by direct oxidation of malic acid. It was This content downloaded from 14.139.172.163 on Thu, 19 Mar 2015 05:39:28 UTC All use subject to JSTOR Terms and Conditions

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proposed that oxalate formationserved as a mechanismfor the removal of excess malatefrom the system. This would permit the Krebs cycle to operate at a very rapid rate. In support of this hypothesis, Gentile reported that a decline in 02 uptake and glucose utilization, which he felt was indicativeof a slowingdown of the Krebs cycle, was coincidentwith the cessation of oxalic acid formation. Oxalic acid also may be producedby the enzymatic cleavage of oxaloacetate, a Krebs cycle intermediate.Hayaishi et al. (1956) found the enzyme oxaloacetic hydrolase in Aspergillus niger which catalyzed the cleavage of oxaloacetateto acetic acid and oxalic acid. The enzyme also may be functioningin higher plants. Chang and Beevers (1968) showed that Krebs cycle inhibitorsinhibitedoxalate synthesis in red beet discs and spinach leaf. They further showed that oxaloacetate and succinate, malate, and aspartate,all of which are readilyconverted to oxaloacetate, are good oxalate precursors.It was felt that oxaloacetate was giving rise to oxalate via enzymaticcleavage. The authorssuggestedthat enzymatic cleavage of oxaloacetatewould be a valuablesystem for oxalate synthesis in non-greentissues which lack glycolate oxidase. Metabolism in Plants

Oxalic acid is most often considered to be an end productof metabolism. However, some plants are apparentlyable to metabolize oxalate under certain conditions as indicatedby fluctuationsor decreases in oxalate concentration.Bakerand Eden (1954)foundthat the oxalate content of sugarbeetleaves decreased as the season advanced while diurnalfluctuations of oxalate content were observed by Seal and Sen (1970) in Oxalis corniculata.

Franke et al. (1943) reported that a number of different angiosperms were able to oxidize oxalate to formate and CO2.An enzyme catalyzing the same reaction in a moss was isolated and found to be a flavoprotein (Datta and Meeuse, 1955).The enzyme which catalyzes the oxidation of oxalic acid is called oxalic acid oxidase and its activity has since been demonstratedin a number of different plants (Bornkamm, 1969; Nuss and Loewus, 1978;Sasaki, 1963;Srivastavaand Krishman1962).It has been implicated also in the rapid decarboxylationof oxalic acid during the climacteric in bananafruit (Shimokawaet al., 1972). There is evidence that the oxalic acid oxidase of plants is associated with an organelle.Arnonand Whatley(1954)and Finkle and Arnon(1954) found that the oxalic acid oxidase of sugarbeet leaves was associated with particles from the chloroplastfraction of their isolation procedure. Sasaki (1963) reportedthat the enzyme was associated with the chloroplast in Begonia and Nagahisa and Hattori (1964) found the same rela-



371

tionship in Chenopodium. Meeuse and Campbell (1959) reported that oxalic acid oxidase in beet root is associated with mitochondrial-type particles. Giovanelli and Tobin (1964) have studied an enzyme in pea seeds and cotyledons which catalyzes the oxidation of oxalic acid in a somewhat differentmannerthanoxalic acid oxidase. The decarboxylationof oxalate by this enzyme is ATP- and coenzyme A-dependent. It was proposed that oxalate is first activated via a thiokinaseto give oxalyl CoA. Decarboxylation of this product yields CO2 and formyl CoA. It was further suggested that the formyl CoA may act as a source of 1-carbonunits in the plant. However, no evidence of such utilizationcould be shown. Enzymatic decarboxylationof oxalic acid has been found to occur in the wood-destroyingfungus Collybiavelutipes (Shimazonoand Hayaishi, 1957). Various bacteria have also been shown to decarboxylateor otherwise metabolizeoxalate (Hodgkinson,1977;Jakobyet al., 1956;Janota, 1950;Yara and Usami, 1968). There is very little in the literatureabout reutilizationof oxalate as a carbon source. Zbinovsky and Burris (1952) found a slow incorporation of carbon from labeled oxalate into glycolate, succinate, and malate in buckwheat leaves but not in tobacco. Joy (1964) reported that oxalate could be used for amino acid and sugar formation in sugarbeet discs. DeKock et al. (1973) suggested that oxalate may be reduced and condensed with acetyl CoA to form malic acid. In Virginiacreeper, labeled oxalate suppliedin situ can be recoveredas intermediatesof the glycolate pathway such as glycolate, glycine, serine and glycerate (Calmes and Piquemal, 1977). Generally, where reported, the process of conversion of oxalate to other carbon compounds is sluggish and is probablynot a significantsynthetic pathway. Synthesis and Occurrence in Animals

Oxalic acid synthesis in man involves some of the same intermediates as in plants (Baker et al., 1966;Hagler and Herman, 1973;Hodgkinson, 1977;Hodgkinsonand Zarembski,1968;see Fig. 2). It has been estimated that the normalrangeof synthesis of oxalate in mammalsis 26-46 mg per day (Haglerand Herman,1973).Glyoxylateis one of the majorprecursors of oxalate in man and may be derived from various amino acids or from glycolate. L-ascorbic acid also has been shown to be a majorprecursor of oxalate. There is evidence that high ascorbic acid intake increases urinaryoxalate levels andcould lead to the formationof Ca oxalate stones in the kidneys and other regions of the urinarysystem (Roth and Breitenfield, 1977). Ascorbic acid and glyoxylate each have been found to account for slightly less than half of the endogenous oxalate.


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ETHYLENE GLYCOL NE HYDROXYPROLI

ETHANOLAMINE

GLYCOALDEHYDE.* HYDROXYGLUTAMATE OAKETO-

GLYCOLIC oxACID OXIDASE

|(KEIO-HYDROXYGLUTAMATE

HYDROXYPYRUVATE

GLYCOLIC ACID XANTHINE OXDXIDASE ,-- TRANSAMINASES

FORMIC ACID+ CO2

ACID_ GLYOXYLIC .

LYCINE

'SERINE

- 'GLYC INE OXIDASE

ACID L-ASCORBIC OXALI C ACID PUR INES 1, L-DEHYDRO ASCORBIC ACID

2,3-DIKETO GULONIC ACID

~~~~~~~~~~~TRYPTOPH THREONIC

C

Fig. 2. Summaryof metabolicpathwaysof oxalic acid productionin man and animals.

Oxalic acid can be found in many parts of the animalbody such as the blood, cerebrospinalfluid, soft tissues, and bones (Hodgkinson, 1977). Oxalic acid is also found in the feces, to some extent, and to a greater degree in the urine. The excretion of oxalic acid in the urine and the potentialfor it combiningwith Ca ions to form kidney and bladderstones (Figs. 3-5) representa majorarea of importance. Normal oxalic acid urinaryexcretion usually does not exceed 40 mg per day in adults (Dempsey et al., 1960) and is lower for children. Increased oxalic acid excretion in man has been related to substantialuptake of Ca from various sources such as milk, vitaminD which regulates the phosphorus-Cametabolism,vitamin C (ascorbic acid), ethylene glycol, and oxalates from various plant sources (Oke, 1969). In both man and animals,concentrationsof mineralmatter(calculi)are common in the urinarysystem (Gibson, 1974).Animalsforagingon plants which are high in oxalates, such as Halogeton (Kingsbury, 1964),Amaranthus, and Chenopodium(Buck et al., 1966; Marshallet al., 1967), quite often develop perirenaledema, with Ca oxalate crystals formingin the tubules of the kidneys (Figs. 6, 7). The toxicology of oxalic acid in humans has been reviewed (Jeghers and Murphy, 1945; Polson and Tattersall, 1959; Hodgkinson, 1977). This content downloaded from 14.139.172.163 on Thu, 19 Mar 2015 05:39:28 UTC All use subject to JSTOR Terms and Conditions

CRYSTALS IN PLANTS OXALATE CALCIUM

373

?~~~ 70~~~~ 6.i55

;

4i

i,~~~~~~~~~~I

Figs. 3-7. Aggregationsof calcium oxalate crystals associated with man and cat: Figs. 3-5, scanningelectron micrographs;Figs. 6, 7, light micrographs.Fig. 3. Fractureface of kidneystone taken from a 25 year-old,white female. Fig. 4. Upper surfaceview of Fig. 38 showing tetrahedralcrystals. Fig. 5. Central view of fracture face from Fig. 3 showing sphericalcrystals. Fig. 6. Brightfield image of cat kidney showing tubularmatrix.Fig. 7. Polarizedlight view of Fig. 6 demonstratingpresence of tubularcalcium oxalate crystals producedby feeding cat ethylene glycol, a precursorof oxalic acid. Line scales on Figs, 3, 6, 7 equal 100,m; Figs. 4, 5 equal 10,m.


374

THE BOTANICALREVIEW

Symptomsof the poisoningare expressed by either local corrosive action or from absorptionand excretion of soluble oxalates. The occurrence of tetany is attributedto the combiningof ionic Ca (probablyfrom blood) with oxalic acid (about 600 mg of oxalic acid is equal to total blood Ca). A marked fall in blood/plasma Ca is noted and appears to be due to deposition of crystalline Ca oxalate in soft tissues such as the kidneys and myocardium(Bennettand Rosenblum,1961)and non-crystallineform of Ca oxalate and lipid in liver and other tissues (Zarembskiand Hodgkinson, 1967). One additionalrelationshipthat recently has been studied is the associationbetween uric acid metabolismand Ca oxalate stones (Hodgkinson, 1976, 1977). It appearsthat most uric acid abnormalitiesin patients with Ca stones are relatedto consumptionof purine-richfoods. It is interesting to note, however, that xanthineoxidase, an enzyme mediatingconversion of xanthineto uric acid, may also aid in the conversion of glyoxylic acid to oxalic acid. Calcium oxalate has also been reportedin some marineinvertebrates as microarchitecturalcomponents (Lowenstam, 1968, 1972)and as nonpathologicaldeposits (Saffo and Lowenstam, 1978). OXALIC ACID AS A BALANCE OF CALCIUM AND OTHER IONS IN PLANTS

Organicacids have been found to influenceion uptake and balance in certain plants (Dijkshoorn,1973;Jacobson and Ordin, 1954).Oxalic acid is a majoracid in many plants and as such may play a special role in ion balance. There are a numberof early studies which deal with the relationship of oxalate and Ca content of plants. Dunne in 1932 noted that high Ca concentrationslead to an increase in oxalate in buckwheat. At high potassium levels, less oxalate was formed than at low potassium levels. Dunne concluded that potassium was interferingwith Ca absorption and thus lowering oxalate formation.Chandler(1937) examined the leaves of various forest trees and found that in all the species examined total oxalate never exceeded total Ca. He also noted that most, but not all, of the oxalate was combinedwith Ca as the insoluble Ca salt. Pierce and Appleman (1943) reported that plants with low amounts of oxalate had large amounts of free Ca in the sap. Plants high in oxalate had only trace amounts of free Ca in the sap. These and other early studies established the fact that oxalate could influenceion balance and that inorganic ions such as Ca and potassiumcould influenceoxalate formation. The relationshipbetween oxalate and ion uptake or availabilityhas been implicatedas a factor in certain plant disorders. McGeorge(1949) showed that lime-inducedchlorosis in citrus trees caused a drop in both oxalate and active ion concentrationswhile citric acid concentrationin-



375

creased. Blossom end-rot in tomatoes has been found to be at least partiallydue to an insufficientsupplyof Ca requiredfor cell wall synthesis and maintenance(Evans and Troxler, 1953).It is suggested that the high concentrationsof oxalate found in the stems were causing precipitation of Ca before it could reachthe actively growingleaves, shoots, and fruits. Similarly,Brumagenand Hiatt (1966)found that oxalic acid was probably responsible for Ca deficiency in certain varieties of Nicotiana tabacum. The oxalate content of Ca deficiency susceptible varieties was generally greaterthan that of unsusceptiblevarieties. Also Ca accumulatedby the plant top was not related to susceptibility since both susceptible and nonsusceptiblevarieties accumulatedapproximatelythe same amountof Ca. It was furtherdemonstratedthat the young leaves and upper stalks of the susceptible varieties had high levels of oxalate. The high oxalate levels were thought to be responsible for Ca deficiency by interfering with the translocationand utilization of absorbed Ca. In a later report, increasingtemperaturealso was found to induce Ca deficiency (Changet al., 1968)though the mechanismof this effect was not determined,other than showing that there was an immobilizationof Ca as previously reported. Lettuce tip burnwas found to be relatedto the relative amountsof Ca and oxalate in the leaves (Thibodeauand Minotti, 1969).Oxalateand Ca, when sprayedon lettuce plants, were foundto be picked up by the leaves. Oxalate sprayinginducedtip burn while Ca inhibitedtip burn. If oxalate was sprayed on the plant in the evening and followed by a Ca spray the next morning,tip burn was reduced or absent. It was concluded that tip burnwas not due to an overall Ca deficiencybut to a temporarylocalized shortageof soluble Ca duringa periodof need. It was proposedthat rapid growthand ensuinghighrespiratoryrates caused tip burnby immobilizing soluble Ca as salts of organic acids, particularlyoxalate. Lotsch and Kinzel (1971) showed that, in Ca-precipitatingplants, the presence of oxalate may cause an especially urgent need for Ca. When Tradescantiaviridis, an oxalate-accumulatingplant, was grown on low Ca substratean increasein soluble oxalates occurred. Calciumdeficiency necrosis became apparentafter four to five weeks and was relatedto the inhibitionof Ca supply to the young parts of the plant, supposedly because of precipitationby oxalate. Plants which do not have high levels of soluble oxalates, such as Zebrina pendula, did not develop Ca deficiency symptoms when grown on low Ca substrate. It was concluded that high oxalate levels can lead to precipitationof needed Ca while in low oxalate plants Ca was kept in a soluble and physiologically active state, even at low levels. Other studies provide further evidence that a relationshipexists between oxalate and Ca and other ions. Scharrerand Jung(1954)found that


376

THE BOTANICALREVIEW

increasing the Ca concentrationof the substrate caused an increase in oxalate in the leaves of sugarbeet and Swiss chard. Rasmussenand Smith (1961) showed that incubationof Valencia orange leaves on high Ca solutions caused an increasein Ca content of the leaves and that the oxalate concentrationincreased proportionately.An increase in potassium also resulted in an increase in oxalate but not Ca, and the oxalate was found as the insoluble Ca salt. At low potassiumlevels, 15%of the leaf Ca was found as the Ca salt of oxalate. At high potassiumlevels, 30%of the Ca was in oxalate. It was proposed that potassium caused a release of Ca from some of its functions so that it could be used for neutralizationof the increased oxalic acid. In Lemna, an induced Ca deficiency caused a decrease in insoluble oxalate while soluble oxalate increased and total oxalate only slowly declined over time (Bornkamm, 1965). Bornkamm felt that this suggestedthat ionic Ca was in equilibriumwith less soluble forms of Ca, such as Ca oxalate. Austenfeld and Leder (1978) reportedthat in Salicornia europaea oxalate is importantin the maintenanceof ionic equilibrium.They demonstrated that with increasing Ca supply water-insolubleCa compounds were predominantand oxalate was presentprimarilyin acid-solubleform, probably as Ca oxalate. At low Ca supply oxalate was mostly in the water-soluble form. The total amount of oxalate present did not show any dependence upon concentration of CaCl2in the nutrient medium. Chlorideion (Cl-) absorptiondid show a relationshipto the CaCl2supply. They concluded that at moderate Cl- accumulationsexcess cations are offset by oxalate but at high CaCl2supply and subsequent high Cl- absorptionoxalate is precipitatedby excess Ca ions and substitutedfor by Cl- ions. They also suggested that oxalate may act as a decontaminating agent for excess Ca (by precipitationas Ca oxalate). DeKock et al. (1973)found that when oxalate was suppliedto Lemna in nutrientsolution without Ca, the growth rate was reducedabout 50o. When oxalate was added in the presence of Ca, the growth rate was greater than the controls. Uptake of both oxalate and Ca was found to increase in each other's presence. EDTA caused reductionof absorption of both Ca and oxalate, probably by binding Ca. They proposed that oxalate acted as a carrierion for Ca and that it was later reduced and condensed with acetyl CoA to form malic acid. In a study of regulationof oxalate synthesis in Atriplex,Osmond(1967) found that Ca absorption and oxalate synthesis varied independently. Enough oxalate was producedto balance 75%of the excess cations and he suggested that oxalate and excess cations accumulatedin the vacuole. Osmondfelt that oxalate productionwas gearedto the total excess cation content of leaf tissue and not just Ca content. However, Zindler-Frank (1975),in a study on Ca-oxalatecrystalformationin Canavalia, suggested



377

that Ca was responsiblefor the inductionof oxalate biosynthesis (Frank, 1969b). Various studies on the effect of substrate pH on organic acid accumulationlend furthersupport to the idea that oxalate helps to maintain ionic balance in plants. Wadleighand Shive (1939)found that the organic acid content of corn plants increased as substrate pH was increased. Brown and Wadleigh(1955) showed large accumulationsof Na and oxalate in beet leaves culturedon bicarbonatesolutions, while Ca concentrationwas not affected. Tobacco leaves culturedin solutionsof glycolate at pH 3 to pH 6 were found to take up and metabolize glycolate more rapidlyat the higher pH, with subsequent increase in oxalate. It is apparentthat pH can affect ion balance and oxalate synthesis in plants. Bangerth(1976)reportedthat inducedCa increases caused an increase in ascorbic acid content of apple, pear, and tomato fruits. Ascorbic acid could conceivablyinfluenceion balancesimilarlyto thatshownfor oxalate, or possibly be convertedto oxalic acid. Apple fruitsare known to contain oxalate (Liegel, 1970; Wieneke and Fiihr, 1973) and Liegel (1970) has demonstratedthat oxalate synthesis even occurs in stored apples. However, the informationon changes in ascorbic acid duringfruit ripeningis limited and often conflicting(Spencer, 1965). NITROGEN ASSIMILATION AND OXALIC ACID SYNTHESIS IN PLANTS

Ruhlandand Wolf (1934), after investigatingthe organic acids of rhubarband Begonia, proposeda correlationbetween organicacid formation and nitrogenmetabolism.They felt that organicacids helpedto neutralize the toxic action of ammoniawhich was liberated duringmetabolismof nitrogenouscompounds. However, Clark(1936) found that the total organic acids, includingoxalate, in tomato increasedwhen nitratewas used as a nitrogen source as compared to ammonia. He suggested that the absorptionand assimilationof nitrogenwas related to organic acid synthesis. Similarresults have been obtained with many other plants (Carafigal et al., 1954;Crombie, 1954;Kpodaret al., 1978;Pepkowitz et al., 1944;Scharrerand Jung, 1954;Wadleighand Shive, 1939). Olsen (1939) also found an increase of oxalate in plants cultured on nitratemediumversus ammoniummedium. In a numberof plants which he tested, the amountof Ca absorbedalso was found to influenceoxalate formation. Olsen felt that in the plants cultured on ammoniummedium the ammoniumions were interferingwith Ca absorptionand thus oxalate formation. The nitrogen source has been found to influence uptake of other ions besides Ca. Cotton leaves cultured on nitrate accumulated greater amountsof Ca, Mg, K, and Na than those on ammoniumcultures (Ergle


378

THE BOTANICALREVIEW

and Eaton, 1949).The increased cation concentrationwas compensated for by an increase in organicacids. In tomato, as the ratio of nitrate:ammoniumnitrogen in the medium was increased from 0-80%, K content increased greatly while Ca, Mg, Na, citrate, malateand oxalate content increasedto a lesser extent (Merkel, 1973). From 80-109o0 nitrate, K content remainedalmost constant while Ca, Mg, citrate, malateand oxalate content increasedconsiderably. Merkelfurtherdeterminedthat the value of the cation content minus the inorganicanion content correlatedwell with the total amountof organic anionspresent, whichconsisted of aboutequalamountsof oxalate, citrate and malate. Osmond (1967) found that cation excess in Atriplex arose principally from the metabolic incorporationof nitrate and felt that the increased oxalate content which occurredunder such conditions was geared to the excess cation content. Joy (1964) obtained similarresults with sugarbeet leaves and also felt that accumulationof oxalate was related to the assimilationof nitrateand preservationof cation-anionbalance. Recently, however, Roughanet al. (1976) examined the effects of nitrogen source on oxalate accumulationin Setaria and suggested that anions other than nitratewere responsiblefor the inductionof cation excess which prompts oxalate synthesis. Gilbertet al. (1951) showed that oxalate formationin tung leaves was directly related to nitrateconcentrationand uptake as well as Ca supply and uptake. Potassiumand Na were found to decrease oxalate formation by a repressingeffect on Ca uptake. They proposed that oxalate formation in tung plant was a byproduct of the process of carbohydrateoxidation which furnishesthe energy for the reduction of nitrateto protein nitrogen. The influence of nitrogen metabolismon oxalate formationmay be a pH effect. Nitrate assimilationleads to the formation of hydroxyl ions (OH-; Dijkshoorn, 1962;Raven and Smith, 1974)which, if not removed from the cytoplasm, would cause large pH changes. Duringnitrateassimilation in shoots some of the OH- generated can be neutralizedby formation of strong organic acids such as oxalic acid (Raven and Smith, 1976).This would account for the observed increase in organicacid concentrationduringgrowthon nitrate substrates. In supportof this, BaillyFenech et al. (1979)found that oxalate levels in Fagopyrumesculentum M., grown on nitrate substrate, were highest in areas where nitrate reductase activity was greatest. They concluded that oxalic acid plays a majorrole in cellularion balance. Withammoniumas the nitrogensource, hydrogen ion (H+) is produced duringnitrogen assimilation(Raven and Smith, 1974), and thus organic acid synthesis would not be helpful for maintenanceof pH. Much of the ammoniumcan be assimilated in the



379

roots and the H+ generated may be lost to the soil solution (Raven and Smith, 1976). CALCIUM IN PLANTS

Function

Calcium is an essential element in plants and a majorcomponent of plant crystals (see Jones and Lunt, 1967; Burstr6m, 1968; Arnott and Pautard, 1970, for comprehensive reviews). Only a brief summary of calcium function, translocation,and metabolismis presented here. Calciumions are known to affect variouspropertiesof biologicalmembranes (Manery, 1966)and may be associated with proteinsor be present as Ca phosphatidesin plant membranes(Sutcliffe, 1962).Calciumis also associated with the activation and/or stabilization of certain enzymes (Mandelsand Reese, 1957;Dodds and Ellis, 1966;Chrispeelsand Varner, 1967;Hewitt and Smith, 1975). The element is also an importanthigher plant cell wall component and is essential for formation of the middle lamella (Ca pectate). Serious abnormalitiesresult from Ca deficiency (see Wallace, 1961; Chapman, 1966; Hewitt and Smith, 1975); some may arise from membrane and organelle disruption caused by a lack of Ca necessary for stabilization(Marinos, 1962;Marschnerand Gunther, 1964),others from cell wall changes (see Hewitt and Smith, 1975)and mitotic abnormalities (Sorokin and Sommer, 1940). Other reports indicate an association between chromosomalabnormalityand Ca deficiency (Hyde and Paliwal, 1958;Steffensen, 1958). Absorption and Transport

Absorption of Ca and other ions generally occurs at the root. The mechanism of Ca absorption and translocationis not certain (see Sutcliffe, 1962 and references therein), although some evidence indicates that absorption is dependent on metabolism (Lowenhaupt, 1956; Sutcliffe, 1962). However, Ca absorptionis also influencedby transpiration rate (Wright, 1939;Hymlo, 1953)and would not rely only on active transport but could be driven by mass flow of solution from roots to aerial organs. The relative importanceof active and passive absorptionis most likely dependent upon the environmentalparameters under which the plant exists (Sutcliffe, 1962). Calciumprobablymoves throughthe xylem sap. However, it has been reportedthat 45Ca2+ movement throughbean stems includes a reversible exchange phase and an irreversible adsorption phase (Biddulphet al., 1961; Bell and Biddulph, 1963). These data and those of Jacoby (1967)


380

THE BOTANICAL REVIEW

Figs. 8-14. Scanning electrom micrographsof fresh, isolated calcium oxalate single crystalsand crystalaggregationsfromplants. Fig. 8. Singleraphidecrystalfromleaf raphide bundle of Psychotria punctata. Fig. 9. Leaf raphid crystal bundle of Psychotria punctata.



381

suggest that Ca movement through stems may be assisted by negative exchange sites in the xylem. Although there is evidence that Ca can be transportedthrough the phloem (Guardiolaand Sutcliffe, 1972), the generally accepted view is that Ca cannot enter sieve tubes (Zeigler, 1975).Epstein (1973)suggested that the exclusion of Ca from sieve tubes may be the reason for the special cytological differentiationthat occurs in maturingsieve cells. Raven (1977) examinedthe general restrictionson the low concentrationof free Ca in the phloemcytoplasm, which results in the relativeimmobility or low Ca transportcapacity observed. Calcium moves easily in xylem because of its largervolume of water and fewer restrictionsgoverningCa concentrationin extracellularfluids (Raven, 1977). Calciumaccumulatesin variousregionsof the plantas metabolic,structural(wall), or crystallinedeposits (Arnottand Pautard,1970).The degree to which the Ca in these deposits can be remobilizedand utilizedin other parts of the plant is not clear. It appears that in annualplants remobilization of Ca does not occur; however, in perennialsat the beginningof each season, Ca is obtainedfrom undesignatedregions for development of new leaves (Arnott and Pautard, 1970).The possible reabsorptionof Ca oxalate crystals duringperiods of calcium stress (deficiency) will be discussed in a later section. The absorption and utilization of Ca is an importantprocess which affects various aspects of a plant's physiology. The formationof Ca salts is a part of this process and should be looked at with respect to its relationshipto the other facets of Ca absorptionand function. SHAPE AND DISTRIBUTION OF CALCIUM OXALATE CRYSTALS IN PLANTS

The most commonlyencounteredforms of calcium oxalate crystals are (Haberlandt,1914):(1) the raphide, a needle-shapedcrystal occurringin bundles of many crystals per cell; (2) the styloid, an elongated crystal with pointed or ridgedends; (3) variouslyshapedprisms;(4) crystal sand, a mass of many tiny, individualcrystals in a single cell; and (5) the druse, a spherical aggregate of individual crystals (Figs. 8-13). Other shapes appearto be variationsof these forms (Fig. 14). Calciumoxalate crystalsare widely distributedin plants. McNair(1932) listed 215 plantfamiliesin which at least some species containCa oxalate

Fig. 10. Styloid crystal from leaf spongy parenchymaof Peperomia sp. Fig. 11. Druse crystalfromleaf of Opuntiasp. Fig. 12. Crystalsandfrompetiole of Nicotianaglauca. Fig. 13. Prismaticcrystalfromleaf of Begonia sp. Fig. 14. Aggregatecrystalcomplexfromleaf spongy parenchymaof Peperomiaastrid. All line scales equal 5 ,um.


382

THE BOTANICALREVIEW

*i7

Figs. 15-20. Scanningelectron micrographsof plant calcium oxalate crystals. Fig. 15. Seed of Oxalis stricta showing seed coat morphology.Dots between ridges are prismatic crystals. Fig. 16. Enlargedview of Fig. 15 in region of crystals. There is one crystal per



383

crystals. The shape and location of these crystals within a given taxon is often very specific and some investigatorshave used them in classification (Chartschenko, 1932, Gulliver, 1864; Heintzelman and Howard, 1948;Jacardand Frey, 1928). Calciumoxalate crystals occur in a large numberof different tissues (Table I). Chattaway(1953, 1955, 1956)described crystals in the heartwood and other woody tissues of tropical woods and other timbers, including species distributedover more than 1000genera and 160families. Wattendorffand Schmid(1973)found crystals in the barkof Larix and Picea and in the bark and secondaryxylem of Acacia senegal Willd. (Wattendorff,1978). Crystals may also occur in floral organsincludinganthers(Buss and Lersten, 1972;Homer, 1977), ovaries (Dormer, 1961;Golazewska, 1934;Stebbins, 1940;Tilton, 1978), fruits and floral buds (Stebbins et al., 1972). They may occur in seed protein bodies (Buttrose and Lott, 1978;Guilliermond,1978)and in the seed coat (Biddle and Homer, unpublished;Figs. 15, 16). Raphide-sacs have been recently found in two-celled trichomes on the adaxial epidermis of the petals of Jubaeopsis caffra Becc. (Robertson, 1978). Dell and McComb(1977)found clusters of Ca oxalate crystals present in each cell of a maturingglandulartrichomehead in Eremophilafraseri F. Muell. Crystals are also present in roots (Arnott, 1966, 1976;Horner and Franceschi, 1978; Mollenhauerand Larson, 1966; Stebbins et al., 1972) and are quite common in leaf tissues (Arnott, 1973; Horner and Whitmoyer,1972;and many others). Crystalsare often observed in many different tissues of a single plant. In apple trees, crystals were found in the bark, petioles, callus tissue of wounds, old roots, fruits, pedicels, and elongation region below the apical meristem of shoots (Stebbins et al., 1972). Biddle and Homer (unpublished)have found that prismatic and some druse crystals occur throughoutthe plant parts of certain Oxalis species and are conspicuous on the outer surface of seeds such as in 0. stricta (Figs. 15, 16). Molisch (1918) has observed crystals in grasses. Wardet al., (1979)have shown that crystals of Ca oxalate, potassium oxalate, or a combinationof the two are present in alfalfa. Crystals are especially abundant in the one-cell thick parenchymatoussheath surroundingvascularbundles in the leaves. Crystals are less frequentin the stems. Some plants may accumulateenormous quantitiesof Ca oxalate. This

surfacecell. Cell wall surroundseach crystal. Fig. 17.Eichhorniapetiole transsectionwith styloididioblastsandtheircrystalsextendingintoairspaces.Fig. 18.TwoEichhorniastyloid crystals. Fig. 19. Pistia root transsectionwith raphideidioblasts extendingthroughsepta into airspaces. Fig. 20. Pistia raphideidioblastwith wrinkledouter wall. Line scales on Figs. 15, 17, 19 equal 100, m; Figs. 16, 18, 20 equal 10,um.


Table I Selected

anatomical

Region of plant body REPRODUCTIVE Anther

studies depicting

Taxon

Compositae Helianthus annuus L.

Petal

Specific location

Crystal type'

Prismatic

Home

Tapetal cells, connective tissues

Varies

Buss a

Compositae 7 spp.

Ovary wall

Varies

Dorme

Liliaceae Ornithogalum caudatum Ait.

Ovary wall

Raphide

Tilton

Embryo sac

Raphide

Golaze

Vascular tissue near pedicle

Druse

Stebbi

Trichomes on abaxial epidermis

Raphide

Rober

Druse

Stebbi

Aspidistra elatior Blume Fruit

and shape of crystals wit

Tapetal cells

Leguminosae 167 spp.

Ovary

location

Rosaceae Pyrus malus L.

Palmae Jubaeopsis caffra Becc.

Dormant Flower Rosaceae Buds Pyrus malus L.


Table I Continued. Region of plant body Seed

Taxon Betulaceae Corylus avellana L.

Specific location

Crystal typel

In protein bodies of cotyledon

Druse

Vaugh Lott a

Buxaceae Simmondsia chinensis Link (Schneid)


Druse

Buttro

Leguminosae Glycine max (L.) Merr. Phaseolus vulgaris L.

Cotyledon Testa, second layer

Prismatic Prismatic

Piper a Haberl

Myrtaceae Eucalyptus erythrocorys F. Muell


Druse

Buttro

Euphorbiaceae Tragia ramosa Tragia saxicola

Surfaces Surfaces

Styloid Styloid

Thurst Thurst

Leguminosae Canavalia ensiformis DC. I.

Upper and lower

Prismatic

Frank

Rutaceae Citrus aurantium L.

Subepidermis

Prismatic

Arnott

LEAF Epidermis


Table I Continued. Region of plant body Parenchyma

Specific location

Taxon

Crystal type'

Agavaceae

Agave americana L.

Mesophyll

Styloid

Watte

Agave americana L.

Mesophyll

Raphide

Watten

Palisade

Druse

Price (

Spongy

Raphide

Ledbet

Leguminosae Cercidium floridum Benth. Lemnaceae Spirodella oligorrhiza (Kurtz) Hegelm.

(197

Piperaceae Palisade

Druse

Homer

Rubiaceae Psychotria punctata Vatke

Palisade and spongy

Raphide

Homer (197

Vitaceae Vitis vinifera 0. Ktze.

Spongy

Raphide

Arnott

-

Raphide

Sakai e

-

Prismatic

Arnott

Peperomia spp.

Air Canals

Araceae Xanthosoma sagittifolium (L.) Schott Nymphaeaceae Castalia Salisb. sp.


Table I Continued. Region of plant body

Vascular

Crystal type1

(1970 Lott (1 Amott (1970

Nuphar variegatum Engelm. Nymphaea L. sp.

Astrosclerids Astrosclerids

Prismatic Prismatic

Nymphaea tuberosa Paine

Astrosclerids

Prismatic

Homer (unp

Parenchyma around vein

Druse

Amott

Adjacent to secondary phloem fibers

Solitary

Scott (

Raphide Raphide

Sakai e Sakai e

Raphide

Sakai a

Druse

Scott (

Ginkgoaceae Ginkgo biloba L.

Euphorbiaceae Ricinus communis L.

Unspecified

Specific location

Taxon

Araceae Alocasia cucullata Schott Alocasia macrorrhiza (L.) Schott Colocasia esculenta (L.) Schott Euphorbiaceae Ricinus communis L.

-



Taxon

Specific location

Onagraceae Oenothera hookeri Torr. and Gray PETIOLE

Araceae Alocasia cucullata Schott Colocasia esculenta (L.) Schott Xanthosoma sagittifolium (L.) Schott Rosacae Pyrus malus L.

Scitamineae Musa nana Lour. STEM

Boraginaceae Cordia subcordata Lam.

Crystal type'

Raphide

Schot

Raphide

Sakai

Parenchyma

Raphide

Sakai

Trichome

Raphide

Sakai

Phloem, parenchyma between sieve tubes and epidermis

Druse

Stebb

Aerenchyma, surface

Raphide

Lott

Wood, vertical parenchyma

Crystal sand

Scurf

Vertical parenchyma

Crystal sand

Scurf

Burseraceae Santiria tomentosa Blume



Taxon

Crystal type'

Specific location

Combretaceae Anogeissus acuminata Wall.

Wood, ray parenchyma

Prismatic

Scurfie

Geraniaceae Pelargonium sp.

Cortical parenchyma

Druse

Arnott

Septa and septal surfaces

Druse

Homer (197


Prismatic

Scurfi


Prismatic

Scurfi

Bark, near bast fibers Bark and secondary xylem Xylem

Twin crystal Twin crystal

Param Sch Watte

Prismatic

Czanin

Wood, vertical

Prismatic

Scurfi

Haloragaceae Myriophyllum spicatum L. var. exalbescens (Fernald) Jepson Lauraceae Cryptocarya angulata C. T. White Dehaasia microcarpa Blume Leguminosae Acacia senegal Willd.

Robinia pseudo-acacia DC. Myrtaceae Psidium guajava L.

parenchyma



Taxon Pinaceae Abies magnifica A. Murray Abies nordmanniana Spach. Larix decidua Mill

Picea excelsa Link.

Polygonaceae Triplaris surinamensis Cham. Rosacae Pyrus malus L.

Specific location

Crystal type'

Wood ray cells Cork (bark) Cork (bark)

Prismatic

Cork (bark)

Solitary

Fibers in wood

Prismatic

Scurf

Bark, shoot apex, region of

Druse

Stebb

Styloids Varies

Scurf

Varies

Chatt

Solitary Solitary

Scurf Watte Watte Watte (19 Watte Watte (19

elongation Verbenaceae Gmelina arborea Roxb.


39+ spp. woody stems 100+ spp. woody stems

Rays and parenchyma


Scurf

Table I Continued. Regionof plant body ROOT

Agavaceae Yucca sp.

Crystal type'

Zone of active mitosis

Raphide

Arno

Tips

Raphide

Aerenchyma

Raphide

Molle (19 Hom (19

Adventious root apex

Raphide

Molle (19

Rosacae Pyrus malus L.

Druse

Stebb


Druse

Stebb

Araceae Monsteradeliciosa Liebm. Pistia stratiotes L. Orchidaceae Vanillaplanifolia Andr.

CALLUS

Specific location

Taxon

Rubiaceae Psychotria punctata Vatke

Raphide

Franc

Fran (19


Table I Continued. Region of plant body EXTERNAL

Nymphaeaceae Nymphaea tuberosa Paine Pinaceae Tsuga canadensis L. Carr.

UNSPECIFIED

Specific location

Taxon

Araceae Monstera deliciosa Liebm.

Crystal type'

Outer surface of leaf mesophyll

Prismatic

Horn (un

Outer surface of leaf mesophyll

Prismatic

Gamb (19

-

Raphide Al-Ra and conglomerate

Begoniaceae Begonia spp.

Chenopodiaceae Beta vulgaris L. Spinacea oleracea L.


Prismatic Al-Ra and conglomerate

Varies

Al-Ra

Solitary

Chan


Specific location

Taxon

Crystal type'

Dioscoreaceae Dioscorea alata L.

Raphide and barrel

Al-Ra

Labiatae Coleus spp. Phlomis viscosa Poir

Varies Elongated

Al-Ra Al-Ra

Lemnaceae Lemna minor L.

Raphide

Al-Ra Arnot


Druse

Stebb

Conglomerate

Al-Ra

Tiliaceae Sparmannia africana L.

1 2

-

Crystal Type designation may represent description presented in citation. LM = light microscope; TEM = transmission electron microscope; SEM

=


scanning electron m

394

THE BOTANICALREVIEW

mPK K-~~~~~~~~~~


CALCIUM OXALATE CRYSTALS IN PLANTS

395

is especially true of some of the cacti. Cactus senilis has been found to be 85%Ca oxalate by dry weight (Cheavin, 1938). The cells in a tissue which producecrystals and are specializedfor this task are referred to as crystal idioblasts (Foster, 1956). The idioblasts often become much largerthan other cells of the tissue. An unusualtype of crystal idioblast is sometimes found associated with aerenchyma,and may contain large single crystals (styloids; Eichhornia, Figs. 17, 18), bundles of raphids(Pistia, Figs. 19, 20) or druses (Myriophyllum,Figs. 21, 22). The crystals may protrudeinto the air spaces (Hornerand Franceschi, 1978;Rothert, 1900;Sakai and Hanson, 1974). In some plants only certain cells are able to develop into crystal idioblasts. The paired crystal idioblasts in Canavalia are both derived from a mother cell and occur only in the epidermis (Frank, 1969a;ZindlerFrank, 1976a).In Psychotria callus, any parenchymatouscell seems capable of developinginto a crystal idioblastprovidedit is given the proper inductive stimulus (Franceschiand Homer, 1979). Calcium oxalate crystals may be found also in other tissues such as collenchyma (Thoday and Evans, 1932) and between the primaryand secondary walls of astroscleridsin Nymphaea (Figs. 23-25; Arnott and Pautard, 1970; Franceschi and Homer, unpublished;Gaudet, 1960)and Nuphar (Lott, 1976). Calciumoxalate crystals are typically described as intracellularinclusions. However, there are reports of "extracellular"Ca oxalate crystals. Solereder (1908) described a number of plant species in which crystals occur in or on the wall of cells in various tissues. Chamberlain(1935) describedcrystal-encrustedsurfaces of the spiculatecells of Welwitschia bainesii. However, upon closer examinationusing SEM, Scurfieldet al. (1973)found that the crystals of Welwitschiasometimes are covered with a sheet-like layer. Gamblesand Dengler(1974)describedcrystals on the outside surfaces of the palisade parenchymain Tsuga leaves, but Homer and Franceschi (1978)noted that these crystals have a covering of fibrous material.Thus there is some doubtaboutthe natureof the "extracellular"crystalswhich have been reported. It is also possible that they are produced intracel-

Figs. 21-25. Scanningelectron micrographsof plant calcium oxalate crystals. Fig. 21. Myriophyllumstem transsectionwith surfacedruse crystals associatedwith septa and airspaces. Fig. 22. Surfaceview of Myriophyllummaturedruse crystals covered by cell walls alongside developing druse crystals. Fig. 23. Nymphaea petiole airspace showing septa astrosclereidarm with manyprismaticcrystals. Fig. 24. Portionof astrosclereidarm with many prismaticcrystals. Fig. 25. Fracturethrougharm of astrosclereidshowing crystal sandwichedbetween primaryand secondarywalls. Line scales on Figs. 21-23 equal 10gm; Figs. 24, 25 equal I l.tm.


THE BOTANICALREVIEW

396

lularly and during cell development are displaced to the outside of the cell protoplasmand into the surroundingcell wall. Calcium oxalate has been found in organisms other than the higher plants. The spines on the sporangiaof the fungi Mucor phimbesis and Cunninghamellaechinulata are composed of Ca oxalate (Jones et al., 1976). Urbanus et al. (1978) described tetragonalcrystals embedded in, and partly covered by, wall materialat the surface of zygophores and sporangiophoresof Mucormucedo Brefeld. Certainsoil fungi (Graustein et al., 1977)and wood rottingfungi(Scurfieldet al., 1973)have Ca oxalate crystals on the exterior of their hyphae. Some lichens have been found to accumulatelarge amountsof Ca oxalate (Erdmanet al., 1977).Calciumoxalate accounts for 66%of the dry weight of the lichen Lecanora (Crombie, 1960). Friedmannet al. (1972) reported that the green alga Penicillus and related genera had large amounts of Ca oxalate crystals. Observations of intracellularand extracellularcrystals are extensive. The many reports of endogenous oxalic acid in plants support the assumption that Ca oxalate crystals are common in the Plant Kingdom. Metcalfe and Chalk(1950)reportedthem from many families. However, these crystals are rarelyidentifiedas oxalate on the basis of precise methods (see Table II). This raises the possibility that crystals of other Ca salts or organic compoundsmay occur in additionto oxalate crystals. ANALYSES FOR OXALIC ACID, CALCIUM AND CALCIUM OXALATE

There are a wide variety of tests which directly or indirectlydetermine the presence and quantityof Ca, soluble oxalates, and Ca oxalate. The earliest procedureswere developed in the late 1700's. Hodgkinson(1977) extensively reviews most of the procedures used with both plant and animal materials. Oxalic Acid

Oxalic acid has been shown to be formed naturallyby several biochemical pathways in both plants and animals (see Figs. 1, 2). It is a dicarboxylicacid [(COOH)2]which has relatively strongacidic properties (pKa = 1.23;pK2 = 4.19). Oxalic acid is moderatelysoluble in water(8.7 g/100g of water at 20?C)and can be oxidized to carbondioxide and water by potassium permanganateand by ferric and ceric salts. When treated with zinc and hydrochloricacid, it is reduced to glyoxylic acid, then to glycolic acid. These various reactions can be used to test for oxalic acid (Hodgkinson, 1977). Oxalic acid can combinewith variouselements and compoundsto form


Table II Identification and analyses of calcium oxalate monohydrate and dihydra Crystal morphology and optical instrument used

Whewellite! Taxon

(mono)

Analysis method

(dihy)

Plan ce

Agavaceae Raphides (SEM)

Dracaena

Whe Whe

IR + x-ray diff. x-ray diff.

leaves

Whe

x-ray diff.

leaves

alata L.

Whe

x-ray diff.

leaves

Poir

Whe Whe

x-ray diff. x-ray diff.

leaves leaves

Whe

x-ray diff.

whole

Whe

?

stems

fragrans

Ker-Gawl. Raphides (?)

Yucca rupicola

Scheele

stems

Araceae Raphides (LM)

Monstera

deliciosa

Liebm.

Dioscoreaceae Raphides (LM)

Dioscoria

Labiatae Raphides (LM) Raphides (LM)

Coleus Phlomis

sp. viscosa

Lemnaceae Raphides (LM)

Lemna minor L.

Liliaceae Raphides (?)

Aloe sp.

Rubiaceae Raphides (SEM)

Hamelia

Jack

?

?

IR

wood, pare

Raphides (SEM)

Morinda citrifolia L.'

?

?

IR

Raphides (LM, SEM)

Psychotria punctata Vatke

wood, pare callus

patens

Whe

IR


Table II Continued. Crystal morphology and optical instrument used

Prismatic (SEM)

Prismatic (LM) Prismatic (LM) Prismatic (LM) Prismatic to cylindrical (LM) Prismatic (SEM) Prismatic (SEM) Prismatic (SEM)

Whewellite/ weddellite Taxon Annonaceae Goniothalamus sp.

(mono)

Whe

Begoniaceae Begonia maculata Raddi

Begonia manicata Cels Begonia metallica L. Sm. Chenopodiaceae Beta vulgaris L. cv. saccharifera Combretaceae Combretum lecananthum Engl. & Diels Combretum verticillatum Engl. & Diels Terminalia belerica Roxb.

?

Analysis method

(dihy)

IR

wood,

Wed Wed Wed

x-ray diff. x-ray diff. x-ray diff.

leaves

Wed

x-ray diff.

whole

-

wood,

IR + x-ray diff. IR + x-ray

wood, pare wood, pare

?

Whe Whe

diff. Prismatic (LM)

Prismatic (LM, TEM)

Labiatae Coleus sp. Leguminosae Canavalia ensiformis DC.

Wed

Whe

Pla ce

leaves leaves

x-ray diff.

leaves

x-ray diff.

leaves



Whewellite! Taxon

Analysis method

Plan ce

(mono)

(dihy)

?

?

IR

wood

?

?

IR

stock

Monimiaceae Prismatic (SEM)

Prismatic (SEM)

Tetrasynandra laxiflora Perkins Welwitschiaceae Welwitschia bainesii (Welw.) Carriere

Druse (LM)

Araceae Monstera deliciosa Liebm.

Wed

x-ray diff.

leaves

Druse (LM) Druse (LM) Druse (LM)

Begoniaceae Begonia maculata Raddi Begonia manicata Cels Begonia metallica L. Sm.

Wed Wed Wed

x-ray diff. x-ray diff. x-ray diff.

leaves leaves leaves

Wed

x-ray diff.

stems

Wed

x-ray diff.

stems

Wed

x-ray diff.

stems

x-ray diff.

stems

Druse (SEM) Druse (SEM) Druse (SEM) Druse (SEM)

Cactaceae Echinocactus horizonthalonius Lem. Echinomastus intertextus Engelm. Escobaria tuberculosa Britton & Rose. Opuntia imbricata Haw.

Whe



Taxon

Whewellite! weddellite (mono) (dihy)

Analysis method

Pla c

Haloragaceae Druse (LM, SEM)

Myriophyllum spicatum L. var. exalbescens (Fernald) Jepson

Druse (LM)

Labiatae Coleus sp.

Druse (LM)

Myrtaceae Tristania conferta R. Br.

IR

stem s

Wed

x-ray diff.

leaves

?

Pizzolato methods

lignotu

Wed

x-ray diff.

leaves

x-ray diff.

leaves

IR

wood,

Whe

?

Tiliaceae Druse (LM)

Styloids (?)

Sparmannia africana L. Agavaceae Yucca rupicola Scheele

Whe

Lauraceae Styloids (SEM)

Actinodaphne hookeri Meissner

?

?


pare


Crystal sand (?)

Whewellite/ weddellite Taxon

(mono)

Cornaceae Aucuba japonica Thunberg

Whe

Analysis method

(dihy)

Pla c

?

stem p

x-ray diff.

leaves

unashed, x-ray diff.

thallus

Chenopodiaceae Undetermined (SEM) Undetermined (-)

Spinacia oleracea L.

Lichen Parmelia chlorochroa Tuck.

Whe (2 forms) Whe



402

4'

'-

..~~~~~~~~~~~~~~~~~~~~~~~.~

by Scanningelectron micrographsof plant crystals within cells and exposed aIgs230. by a freeze fracturetechnique. Fig. 26. Psychotria leaf raphideidioblast. Fig. 27. Myrio-



403

acid and neutral salts, a monoamide(oxamic acid), and a diamide (oxamide). Alkali metal ions such as lithium (Li), sodium (Na), potassium (K), and iron (Fe+2) can combine with oxalic acid to form soluble salts. Both Na and K oxalates are prevalentin manyplants in varyingquantities and are potentiallytoxic to animals. Calcium

Determinationof Ca in biological materialat the light microscopelevel can be divided into two groupsof techniques.They are metal substitution techniques and dye-lake reactions (McGee-Russell, 1958). Von K6ssa's technique (von Kossa, 1901) is a classical method for Ca detection by formationof a silver precipitate.This has been used successfully on both animalsand plants (Bechtel and Homer, 1975). Other metal salts can be used also. In comparison, a variety of dye-like reactions result in the colorationof regionscontainingCa. X-ray microanalysisalso can be used to identify and locate Ca within the biologicalsystem in relativelyminute amounts. Non-microscopictests are treatmentof the materialwith ammonium hydroxide, ammoniumcarbonate, or dilute sulfuric acid; the flame coloration test also confirms the presence of Ca. Calcium Oxalate

Calcium oxalate can be rendered soluble by increasingthe hydrogen ion concentration,by addingurea, citrate, or magnesiumand to a lesser degree lactate, sulfate, sodium, and potassium and by adding ethylenediamine-tetraaceticacid (EDTA). In contrast, certain compounds will inhibit precipitationor crystallizationof Ca oxalate from aqueous solution. They are inorganic pyrophosphate, polyelectrolytes like heparin, and various naturally-occurringand synthetic dyestuffs. Determinationof Ca oxalate in biological material can be made by diverse procedures. Many investigatorshave used polarizingoptics and, more recently, scanningelectron microscopyto make a preliminaryidentification of Ca oxalate crystals. Among several histochemical identifications worth notingis an incinerationtechnique(JohnsonandPani, 1962; Wolmanand Goldring,1962)which converts Ca oxalate to Ca carbonate. Treatmentof sections with 5%acetic acid and 2%hydrochloricacid produces bubbles and a red color reaction with alizarin red S can be observed. Failure of a reaction of the incineratedCa oxalate with sodium

phyllum root druse idioblastin septum. Fig. 28. Capsicum(sweet pepper)anthercrystalsand idioblast.Fig. 29. Capsicum(sweet pepper)antherprismaticidioblasts.Fig. 30. Eichhornia petiole styloid idioblast in septum. All line scales equal 10 Am.


404

THE BOTANICALREVIEW

_

-~~~~~~~~~~~~~~~~4

_

2

S~~~~~~~~~

_a~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

-

.;At

tw.>

e

"10U

4

:

\

Figs. 31-36. Peperomia sps. leaf sections emphasizing photosynthetic palisade parenchyma containing druse crystals. Fig. 31. SEM freeze-fracture of leaf cross section. Upper large cells are multiple hypodermis; arrows depict palisade parenchyma; lower isodiametric cells are spongy parenchyma. Fig. 32. Palisade parenchyma with druse crystals. Fig. 33. LM of palisade parenchyma. Chloroplasts and crystals (arrows) are evident. Fig. 34. Po-



405

rhodizonate rules out the presence of barium or strontiumcarbonate. Wolmanand Goldring(1962) also showed a positive test for Ca oxalate with von Kossa's technique (1901) and the previous staining technique if incinerationof the biological sections occurs first. Pizzolato (1964)foundthat neutral30o hydrogenperoxideworkedwell in convertingoxalate to carbonate,to make it reactive to silver nitratein the presence of light to form black deposits in situ. Dell and McComb (1977) used this procedureto identifyCa oxalate crystals in head cells of resin secretingglandularhairsof Eremophilafraseri,and Silver and Price (1969) used it successfully with sectioned leaves of Cercidiumfloridum. Yasue (1969) described a histochemicalmethod for localizing calcium oxalate which he considered superiorto previously described methods. The basic procedure,first, involves removingCa salts of phosphateand carbonate with 5% acetic acid treatmentfor 30 minutes. The next steps are to treat materialwith 5% aqueous silver nitratefor 15 minutes, then with saturatedrubeanicacid (dithio-oxamide)in 70%oalcohol containing 2 drops of strongammoniumfor 1 minute.Calciumoxalate appearsblack to darkbrown. Yasue believed that this procedureis more consistent and is faster than the von Kossa technique. The method has been used to successfully localize Ca oxalate crystals in some plant trichomes(Thurston, 1976) and anthers of pepper (Horner and Wagner, 1980), and ovaries of Ornithogalum(Tilton and Homer, in press). Determinationof Ca oxalate by X-ray diffractionand infraredspectra is usually carriedout by isolating Ca oxalate crystals from the biological material. Both the monohydrateand dihyrate forms can be separated fromeach other on the basis of spacingand presence or absence of peaks. Arnott and Pautard(1970)presented evidence that, no matterwhat morphologicaltype of crystal they isolated from variousplant sources, it was always the monohydrateform. Al-Rais et al. (1971) showed that both the monohydrateand dihydrateforms were present in plant crystals with the former being incorporatedin raphides. X-ray analysis of animalcrystals indicates that both hydratedforms can be present (Bennett and Rosenblum, 1961; Elliot and Rabinowitz, 1976). Infraredspectra also provide anothermeans of identifyingCa oxalate (Scurfi'eldet al., 1973;Franceschi and Horner, 1979). Wardet al. (1979) recently have used Ramanmicroprobe analysis to identify Ca oxalate crystals from alfalfa fed to cows. This type of analysis allowedfor identificationof individualsmallcrystals

larized light optics. Crystals are anisotropicin palisade parenchyma.Fig. 35. TEM of a palisadeparenchymacell. Chloroplastssurrounddruse crystal in vacuole. Fig. 36. TEM of portion of palisade parenchymachloroplastsshowing large grana stacks. Line scales on Figs. 31, 32 equal 100t,m; Figs. 33, 34 equal lOLAm; Fig. 35, 36 equal 1 ,um.


406


Figs. 37-42. Scanningelectronmicrographsof fresh isolatedcalciumoxalatedrusecrystals from photosyntheticpalisade parenchymaof several different species of Peperomia. Note differencesin shape and angularityof individualcrystalfacets. Fig. 37. P. obtusifolia.



407

(6 x 6 gum).Their results showed that the composition of the crystals rangedfrom Ca oxalate to potassium oxalate to Ca potassiumoxalate. Isolation of Ca oxalate crystals from plants has been used to characterize the crystals by some of the previousmethods. Severalinvestigators have isolated the crystals from animal (Bennett and Rosenblum, 1961) and plant (Al-Rais et al., 1971)tissues. These methods involve homogenization of the tissue, filtration, and centrifugation(sometimes through cesium chloride or other suitable gradients). For SEM observations of isolated crystals, Franceschi(unpublished)developed a simple technique which merely involved cutting the plant tissue and dippingthe cut end into water on a stub. On drying,the crystals are usuallyfree from cellular debris, except for starch grains, which have similar densities. Crystals that are embedded in plant cell walls will requirecareful enzymatic and/ or chemical digestion for which no published technique is presently known to us. PlantCa oxalate crystals have been determinedby examiningthe chemical and optical propertiesof the crystals (Frey, 1925;Honegger, 1952; Pobeguin, 1943). More recently, X-ray diffraction(Arnott and Pautard, 1970; Arnott et al., 1965; Cocco and Sabelli, 1962; Philipsbom, 1952; Walter-Levyand Strauss, 1962) and infraredspectroscopy (Scurfieldet al., 1973)have been used for crystal structureanalysis. The results have been somewhatconflictingpartlydue to the difficultyof isolatingcrystals in pure form and partly because of the variabilityin the crystals themselves (Table II). Both crystallinetypes may occur in the same plant. Calciumoxalate in plants occurs in two principalforms, monohydrate (CaCQ04 H20) and dihydrate (CaC204 2H20). Each of these has a dif.

ferent crystal structurewith the monohydrate,known as whewellite, belonging to the monoclinic system of crystallization and the dihydrate, known as weddellite, belongingto the tetragonalsystem (McNair, 1932). The monoclinic system has three crystallographicaxes unequalin length with one intersection oblique and the other two intersections equal to 900. The tetragonalsystem has three axes at right angles to each other with the two lateral axes equal and the third, vertical axis longer or shorter. There have been conflicting reports on the hydrationcomposition of the tetragonalCa oxalate system (weddellite).The watercontent in moles per mole of Ca has been given as 3 (Frey-Wyssling, 1935; Pobeguin,

Fig. 38. P. caperata. Fig. 39. P. astrid. This species contains crystals with various shapes. Compare with Fig. 42. Fig. 40. P. argyreia. Fig. 41. P. marmorata. Fig. 42. P. astrid. All line scales equal I am.


408


1943), 2.5 (Honegger, 1952), 2.25 (Walter-Levyand Strauss, 1962), and 2 (Bannister and Hey, 1936). Based on an X-ray analysis of weddellite from a kidney calculus, Sterling (1964, 1965)proposed a structurewith a water content of 2 + x, the fraction portion (x) being zeolitic. This zeolitic bindingof water could account for the variationreported. The crystal shape may be related to the hydration form of the Ca oxalate (McNair, 1932).Raphides from various plants have been identified as the monohydrate,whewellite (Al-Rais et al., 1971;Arnott et al., 1965;Honegger, 1952;Scurfieldet al., 1973;Steinfinket al., 1965).Druses have been identifiedas the monohydrate,whewellite (Franceschiand Homer, 1979)or as the dihydrate,weddellite(Al-Raiset al., 1971;Rivera, 1973)as have been prismaticcrystals (Al-Rais et al., 1971;see Table II). The taxonomic value of crystal shape assumes that shape is under genetic control. However, it is interestingto note that, while some plants contain only one crystal type, others may have two or more different crystal types. Crystals of different morphologymay even occur in adjacent cells (Gibson, 1973;McNair, 1932;Scurfieldet al., 1973).This raises some question as to the natureof the control of crystal shape and composition by the plant. Various physical and chemical parameterssuch as temperature,pressure, and ion concentrationcan affect crystal growth, habit, and properties. Frey (1927) reportedthat Ca oxalate monohydrateforms at temperatures of 0 to 100?Cwhile the trihydrate(2 + x = dihydrate)forms at 0 to 30?C in vitro. In correlationwith these findings, McNair (1932) showed that the monohydrateCa oxalate was found mostly in tropical plants. Stahl (1920)found that increasingthe Ca level in Viscum resulted in the formation of single crystals, either in association with the druse crystals normallyformed by the plant, or separately. Pfeiffer (1925) has shown that in several plant species pH can influence crystal habit. A pH of 4.4 favored the monoclinic form of oxalate crystals while a pH of 7 favored the tetragonalform. Scurfield et al. (1973) suggested that impurities, which would most likely differ from cell to cell, may be a factor in the formationof different crystal types. This seems inconsistent with the fact that some plants contain only one type of crystal throughoutmany tissues and that the crystal-containingcells may be specifically located in a particulartissue. Some rather unusual treatments have been found also to affect Ca oxalate crystal formation. Nadson and Rochline-Gleichgerwicht(1928) reported that ultraviolet irradiationof mosses which did not normally produce crystals resulted in a rapid formationof crystals. After four to five days the crystals, which were determinedto be Ca oxalate, disappearedand at about the same time the cells began to die. Similarly,Biebl (1940)found that alpha rays induced formationof oxalate crystals in the



409

moss Hookeria lucens. Paupardin(1964) reporteddistortionsin Ca oxalate crystals of plant tissues culturedin the presence of 2,4-D, a synthetic plant auxin. Ultravioletlight and alpharadiationare also known to cause changes in DNA structure,and 2,4-D has been shown to have a possible regulatoryeffect on RNA synthesis, as have other auxins (Key, 1964; O'Brien et al., 1968). DEVELOPMENT AND ULTRASTRUCTURE OF CRYSTAL IDIOBLASTS IN PLANTS

Calciumoxalate crystal formationhas been found to be an intracellular process in those plants in which the developmentof the crystals has been studied (Figs. 26-30). Though some investigatorshave reported crystal formation to be associated with the cytoplasm (Rakov'anet al., 1973; Scott, 1941),more often crystals are found to form in the centralvacuole or vacuoles (Arnott, 1973; Arnott and Pautard, 1965, 1970; Frank and Jensen, 1970; Homer and Whitmoyer, 1972; Hurel-Py, 1942; Robyns, 1928).In some plants, there may be a mucilagewithinthe vacuole (Eilert, 1974; Robyns, 1928; Sakai and Hanson, 1974; Tilton, 1978;Tilton and Horner, in press; Wattendorff,1976b). An enlargementof the nucleus and nucleolus is one of the first changes observed in developingcrystal idioblasts (Hornerand Whitmoyer,1972; Hurel-Py, 1938;Rakovanet al., 1973).Schlichtinger(1956)observedploidy levels in raphidecells in Gibbaeumheathii rangingfrom diploidto 64ploid. The cytoplasm of a young idioblastis often very dense and rich in organelles and vesicles (Frank and Jensen, 1970; Mollenhauerand Larson, 1966;Rakov'anet al., 1973;Schotz et al., 1970). Plastids are often smallerthan those in surroundingcells and have few grana(Hornerand Whitmoyer, 1972;Price, 1970)or may be highly modified(Arnott, 1966; Mollenhauer and Larson, 1966). Invaginations of the plasmalemma, which produce structuresanalogousto the plasmalemmasomesdescribed by Marchantand Robards(1968), have also been described(Dengg, 1971; Hornerand Whitmoyer,1972).The overallappearanceof the young crystal idioblasts suggests a highly active cell. Crystal formationis associated with some type of membranesystem. In many raphidecrystal idioblasts studied, membranouscomplexes are formedin the centralvacuole which latergive rise to membranechambers in which the crystals develop (Arnottand Pautard, 1970;Chiu and Falk, 1975;Eilert, 1974;Homer and Whitmoyer,1972).However, Rakovanet al,. (1973) reported that in Monstera deliciosa the crystals may form in dictyosome-derivedvesicles or at the boundaryof largervesicles within the cytoplasm. Earlier,Mollenhauerand Larson(1966), workingwith the same species, reportedthat the crystals developed withina carbohydrate


410

THE BOTANICALREVIEW

matrixand were not membrane-bound.Wattendorff(1978) suggests that single crystals in the barkand secondaryxylem of Acacia senegal Willd. grow in extraplasmiccompartmentsassociated with an ER-like system. Membranesystems also have been found to be involved in the formation of other crystal types (Arnott, 1973;Arnottand Pautard,1970;Frankand Jensen, 1970). The crystal chamber is not always delimited by a single membrane. Chiu and Falk (1975) reported that the crystal element or chamber of Lemnaperpusilla was composed of a double membrane.Lamellatecrystal chambershave been reportedin Acacia (Parameswaranand Schultze, 1974), Agave leaves (Wattendorff, 1976b), Yucca (Eilert, 1974), Ornithogalum carpels (Tilton, 1978;Tilton and Horner, in press) and in young Typhaleaves (Albert Kausch, personalcommunication,Iowa State University). In some idioblasts, there is a "sheath" between the crystal and the wall of the crystalchamber(Parameswaranand Schultze, 1974;Price, 1970). The composition of the sheath has not been clearly determined, except that it stains for carbohydrates(Sakai and Hanson, 1974;Tilton and Horner, in press), proteins, and RNA (Tilton and Homer, in press). Various structures are sometimes found associated with the crystal chambersduringcrystal development. In Psychotria (Homer and Whitmoyer, 1972) numeroustubules 10-13 nm in diameter are found in the central vacuole, often oriented parallel to the developing crystal chambers. These same tubules are found in callus crystal cells of Psychotria (Franceschi and Homer, 1980). Frank and Jensen (1970) observed filaments associated with the developing prismatic crystals in Canavalia. Chiu and Falk (1975)reportedribosome-likeparticlesassociated with the crystal chambersand suggested that, if they were ribosomes, they might be involved in the productionof proteinsnecessary for crystal synthesis. They also found fibrils within the devloping crystal chambers. At maturity, most crystal idioblasts are probably still living cells. A thin layer of cytoplasm can usually still be seen aroundthe peripheryof the cell. An exception to this may be some bark and cork cells which form crystals, though they would not be considered to be crystal idioblasts since the primaryfunction of the cells is not crystal formation. Hornerand Franceschi(1978)have shown that the drusecrystalidioblasts associated with the air spaces of the stem of Myriophyllumare dead at maturity. In mature crystal idioblasts, the crystal also may become surrounded by wall-like material.Styloid crystals in Agave leaf (Wattendorff,1976a) are surroundedby a suberized sheath. Frankand Jensen (1970) reported that the crystals of Canavalia become enclosed in wall materialduring later stages of developmentof the idioblast.In Ricinus, cellulosic sheaths which are often partiallysuberizeddevelop aroundthe single druse crys-



411

tals (Scott, 1941). The sheath is attached by stalks to the cell wall. A similarobservation was made by Price (1970) in druse crystal idioblasts of Cercidiumfloridum. The crystals were surroundedby a cellulose-like sheath which occasionally appearedto be connected to the cell wall by a stalk. Thurston(1976) found the prominent,grooved, single crystal at the tip of each Tragia trichome to be surroundedby a cellulosic wall. The current informationon crystal formationsuggests that it is a cellcontrolled process. The crystal idioblast undergoes many changes and develops a complex ultrastructure.Crystalchambersare producedwhich apparentlycontrol the shape and size of the crystals. The crystals themselves may develop structures such as barbs and grooves (Sakai and Hanson, 1974;Sakai et al., 1972;Thurston, 1976)which are inconsistent with a simple precipitationor crystallizationprocess. The metabolicmachinery of a cell destined to become a crystal idioblast must be altered to some extent to accommodatea process not normallyrequiredof parenchymatous tissue from which the idioblasts are usually derived. The evidence suggests that Ca oxalate crystal formationin plant cells is not a simple precipitationprocess but involves cellular specialization and direction. FUNCTION(S) OF CALCIUM OXALATE CRYSTAL IDIOBLASTS AND CRYSTALS

Little is known about the function of Ca oxalate crystals in plants thoughthere has been much speculation. Many believe that oxalate is an end-productof metabolismand in excess amounts may be toxic to the plant. As such, it is sometimes thoughtof as an excretory product. Thus it has been suggested that crystals and crystal-formingidioblasts are a means of isolating this product and Ca oxalate crystal idioblasts have been classified as "excretory idioblasts" (Foster, 1956). Similarly, the crystals may function in removingexcess Ca from the system. As previouslymentioned,a relationshipbetween oxalate formationand Ca uptake has been demonstratedin variousplants (DeKock et al., 1973; Dunne, 1932; Rasmussenand Smith, 1961; Scharrerand Jung, 1954). A relationshipalso has been demonstratedbetween the Ca and oxalate content of tissues and the numberof crystalsformed. Canavaliaplantsgrown on high Ca solutions have been found to form more crystals than control plants (Frank, 1972;Zindler-Frank,1975)while inhibitionof oxalate synthesis leads to a decrease in crystal formation(Zindler-Frank,1974). Franceschi and Homer (1979) found a direct relationshipbetween the numberof idioblasts formed in Psychotria callus and the amount of Ca available in the growth medium. It was also found that acHPMS(an inhibitorof glycolate oxidase; Inoue et al., 1978;Corbettand Wright,1971)


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and allopurinol(an inhibitorof xanthineoxidase; Kelley and Beardmore, 1970;an enzyme capable of oxidizing aldehydes; Feigelson et al., 1956), possible inhibitorsof oxalate synthesis, resultedin a decrease in the number of idioblasts formed. In an earlier study, Frank(1967)could not find a causal relationshipbetween the distributionof Ca and the localization of crystal idioblasts. Many plants containhigh concentrationsof soluble oxalates as well as the insoluble Ca salt and still others may contain the free acid (Crombie, 1960). This sheds some doubt on the toxic nature of oxalic acid to the plantand the need to remove it from the cell sap in the formof crystalline deposits. It is more likely that oxalate is not chemicallytoxic to the plant but representsa problemfor osmotic pressureregulation(and thus turgor and volume regulation)in the cells (Raven and Smith, 1976).This would be especially valid if oxalic acid synthesis occurs as a means of pH maintenance in response to OH- formationduringnitrateassimilation(as discussed earlier). Under such circumstancesthe precipitationof oxalate as the insoluble Ca salt would be a method of osmoregulation(Raven and Smith, 1976). There is some evidence that Ca oxalate crystals may function as a storage form for either Ca or oxalate. Alexandrow and Timofeev (1926) showed drawingsdepictingdruseandprismaticcrystaldegradationsimilar to starch grain degradation.In Ricinus, druse crystals in the stem, inflorescence, and fruitingaxis are degraded and replaced by starch grains (Scott, 1941). Assailly (1954) reportedthat the same process occurredin the stem of Euonymusduringbud growth. Calmes (1969)found that during leaf development in Virginiacreeper the crystals in the stem began to disappear.The Ca was recovered in the young stems while the fate of the oxalic acid was not determined.The plantcontainedboth raphideand druse crystals (Calmes et al., 1970) and, in a later paper (Calmes and Carles, 1970), it was reportedthat the raphides(weddellite)disappeared, possibly as a result of local Ca deficiency caused by rapidgrowthof buds (Calmes and Piquemal, 1977), and thus seem to be a storage substance while the druses (whewellite) are not degradedbut are partly eliminated by leaf fall. This indicates that the hydrationform of the crystal may affect the ability of the plant to breakdown the crystal and utilize the Ca and oxalate stored therein. However, complete or partialdegradationof both druse crystals (Rao and Mohana, 1972; Scott, 1941) and raphide crystals (Czapek, 1921;Franceschi and Horner, 1979;Paupardin,1964; Tilton, 1978)have been reported. Stevenson (1953) reportedan interestingrelationshipbetween Ca oxalate crystals and bacteriain certain Rubiaceousplants. Calciumoxalate crystals were often found beneath bacteria in young stipules, and the crystals largely disappearedas the bacteria aged and degenerated. She



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suggested that the oxalic acid was being utilizedby the bacteria,possibly as a startingpoint for amino acid synthesis, though it was not known if the bacteria were involved in nitrogen fixation. Stevenson furtherproposed that Ca oxalate crystals in plants may not be a waste productbut rather are connected with nitrogen metabolism. Van Hove and Craig (1973) reinvestigatedsome of the taxa studied by Stevenson and could not find either bacteriaor bacterialnodules. This latter study, therefore, casts doubt on Stevenson's work. However, Horner and Whitmoyer (1972) and Lersten and Horner (1976) noted that crystals are found in nodulatedand unnodulatedtaxa from Psychotria (raphides,Rubiaceae) and Ardisia (druses, Myrsinaceae)and found numerouscrystals present in matureleaves near nodules with degeneratebacterialpopulations. Another majorfunctionattributedto Ca oxalate crystals is that of protection against foraginganimals. The irritationand burningsensations of the mouth caused by eating crystal-containingplants is well known (Kingsbury, 1964) and may discourage foraging animals. Black (1918) found calcium oxalate crystals to be the sole cause of these symptoms through mechanicalpenetrationof the mucous membraneof the throat and mouth. Contactdermatitisfrom the juice of Ornithogalumcaudatum has been reportedto be due to penetrationof the skin by raphidecrystals (Pohl, 1965). Some plants have Ca oxalate crystals which are especially well adapted for a protectivefunction.The stingingcells of Tragiaramosa emergences have a large grooved crystal which is held in place by cell wall extensions around its base (Thurston, 1976). Upon contact with unprotectedflesh, the stingingcell wall is pushed back over the crystal, allowingsubsequent penetrationof the skin by the crystal tip. The groove apparentlyfunctions to channel a toxin from the rupturedcell lumen into the wound created by the crystal. Deep grooves also have been reported in raphides of Colocasia, Alocasia, and Xanthosoma (Sakai and Hanson, 1974; Sakai

et al., 1972).They suggestedthat the grooves allow materialto be carried into tissues upon penetration and may prevent tissues from healing around the crystals. These raphides also have surface barbs oriented away from the tips of the crystals which may help to anchorthem in the tissue. Most Ca oxalate crystals which have been examined do not have grooves or barbs as those in Tragia and the aroid plants mentioned. However, their oxalate content may itself be a protective substance. Oxalic acid is the only organic acid of plants which is toxic to livestock undernaturalconditions(Kingsbury,1964).Perirenaledema in swine has been linkedto ingestionof plants such as Amaranthusand Chenopodium, which are high in oxalates (Osweileret al., 1969).Ingestionof Halogeton, anotheroxalate-accumulator,by pigs results in extensive formationof Ca


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oxalate crystals in their kidneys (Buck et al., 1966). Ingested oxalates may interferewith Ca metabolism(Oke, 1969), and blood plasma oxalic acid and Ca levels have been found to change duringoxalate poisoning (Zarembskiand Hodgkinson, 1967). There have been reports of human deaths due to oxalate poisoning after ingestion of rhubarbleaves (Kallicate and Kauste, 1964; Tallqvist and Vaananen, 1960). Fasset (1973), however, in his investigationof the toxicity of oxalates, cocluded that there is no hard evidence that oxalates in foods are toxic to man. Althoughcrystal idioblasts are occasionally found on the exterior surfaces of plant organs as epidermalappendages (Robertson, 1978) or in trichomes(Rao and Sundararaj,1951;Thurston, 1976),they are generally scattered throughoutinternaltissues. Schneider(1901) surveyed the distributionof crystals in differenttissues and concludedthat, in the majority of instances, the presence of the Ca oxalate crystals pointed toward a function of mechanicalsupport. Still other functionsfor Ca oxalate crystals have been proposed. It has been suggested that some crystals may function as statoliths for gravity perception (Audus, 1962;Prankerd,1920). Schurhoff(1908), noting that crystals were associated with a thin layer of photosynthetic tissue in Peperomia leaf, felt that they might be involved in some way with the photosynthetic process (Horner, 1976; Hill and Homer, unpublished; Figs. 31-42). Other studies have implicatedthe unusual chloroplasts in these cells (Gausman, 1973) or the multilayeredhypodermis(Gausman et al., 1977)with the photosyntheticprocess. DISCUSSION

An understandingof the factors which induce Ca oxalate crystals to form in certain cells will be importantin determiningtheir function. It appears that idioblast formation is a complex process which involves various factors workingtogether or sequentially. The induction of crystal idioblast formation by Ca suggests that the crystals may serve as a storage form for Ca for future needs. Further supportfor this is the observationthat in some plants the crystals appear to be reabsorbedduringCa-deficientconditions(Scott, 1941;Calmes and Piquemal, 1977;Franceschiand Horner, 1979).However, it is not known how Ca brings about crystal idioblastformation.A relationshipbetween Ca absorptionand oxalic acid synthesis has been demonstratedin some plants and both processes appear to be important in the induction of crystal idioblasts. Which, if either, is the primaryinducer is uncertain. Perhaps Ca induces oxalate synthesis and, in those plants which are capable of producingcrystals, localized high concentrationsof oxalic acid may be the primaryinductive factor.



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The relationshipbetween Ca absorptionand oxalic acid synthesis may be due to an attemptby the plant to maintainionic balance (Rasmussen and Smith, 1961). Excess Ca could be removed by precipitationwith oxalic acid; crystalidioblastformationwould be the resultof maintenance of ionic balance, and a storage form for Ca could be a secondaryrole of the crystals. Although it has been suggested that calcium oxalate crystals are a means of detoxifyingexcess oxalic acid, the fact that manyplants contain high concentrationsof soluble and free oxalic acid indicates that oxalic acid is not especially toxic to plant tissues. However, accumulationof oxalic acid, which is not readily metabolized, may cause osmotic problems. Precipitationof the acid as deposits of the insoluble Ca salt would remedy this situation(Raven and Smith, 1976). If one takes into accountthe relationshipbetween oxalic acid synthesis, nitrogen metabolism, ionic balance and Ca absorption, a plausable hypothesis for the presence of Ca oxalate crystals in plants can be constructed. Plantsgrowingon substratecontainingnitratenitrogenwill produce net OH- duringnitrate assimilationwhich would tend to raise the pH of the cell sap. Strong organic acids such as oxalic acid could be produced to neutralizethe OH-, but would upset ion balance and also result in increasedosmotic pressure(endangeringcell turgorand volume regulation). Precipitationof oxalic acid as the insoluble Ca salt would restore ionic balance and osmotic pressure. Nitrate assimilationwould not be necessary for the crystallizationprocess to occur. An important factor is the formation of oxalic acid in response to unbalancedionic charge. This would explain formationof crystals duringgrowth on high Ca medium. Under such conditions large amounts of Ca would be absorbedresultingin ion imbalance.The imbalancecould be offset by oxalic acid synthesis and the resulting increase in osmotic pressure corrected by precipitationof calcium oxalate. However, the degree to which the crystallizationprocess would be importantto osmotic regulationmight be small since many plants handle pH, ionic or osmotic perturbations withoutthe formationof crystals. Also, it has been recently reportedthat in the calcicolous plant Dianthus luminitzeri,precipitationof Ca as Ca oxalate results in special problems in the osmoregulationof the plant (Albert et al., 1980). Removal of the dominant soil ion as potential osmoticumis compensatedby increaseduptakeof K+ and inorganicanions and accumulationof sugars. But perhapsthis strategyis aimedat keeping the concentrationof free Ca+2in the cell low (thus, ionic regulation)since high levels of free Ca+2can be deleterious to growth (see Bernstein and Hayward, 1958).It is worth noting that crystal idioblastformationoccurs in very young, growing tissues. At this period of growth regulationof ionic balance and cellularpH would be especially crucial for properde-


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velopment of the tissue or organ. Rapidly dividing meristematictissues would not be expected to be able to deal as effectively with largeinfluxes of ions as wouldmaturedifferentiatingtissues whichcoulddevote moreenof excess ions. Formationof cells (crystalidergy to compartmentalization ioblasts) specialized for this task would be advantageousto the plant. The shape of Ca oxalate crystals has been described as a propertyof the hydrationform of the Ca oxalate. Druses are typically described as being composed of weddelliteand raphidesof whewellite (McNair, 1932). However, the informationpresented in Table II demonstrates that Ca oxalate crystals do not always form a crystalline structurecharacteristic of their hydrationform. It is probablethat the crystal chamberdictates the shape of the crystal, especially since the chamber has often been found to be formed before crystallizationoccurs. Since differentiationof a cell into a crystal idioblastis surely undergenetic control, the shape of the crystal formed may also be genetically determined. The functions assigned to Ca oxalate crystals in plants are varied and for the most part unproved. Certainlyone function does not necessarily exclude another, especially when one considers the great diversity in crystal shape and size and in idioblast morphology and location. The relationshipof Ca oxalate crystal formationto ionic balance and osmoregulation is an especially importantand interesting area which clearly needs furtherstudy. The similaritiesand differences between plant and animal Ca oxalate crystals are also intriguing.At the cellularand molecularlevels there may be some common relationshipswhich could help to explain certainbasic phenomenaoccurringin both Kingdoms. We believe that future studies at these levels, using tissue culture and protoplasttechniques, may provide a useful system to provide such understanding. ACKNOWLEDGMENTS

The authors acknowledge the support from several sources which helped to make this review possible. They are: Iowa State University GraduateCollege InitiationGrant (1977-1978; 405-21-07 to HTH); the Departmentof Botany and Iowa State University Library;and Ms. Jill Smartand Miss CarlaHolbrookfor typingthe manuscript.We also thank Drs. Lersten and Stewartfor criticallyreadingthe manuscriptand making numeroushelpful suggestions. LITERATURE CITED Albert, R., H. Konigshofer and H. Kinzel. 1980. Zur Osmoregulation einer physiologisch calciphoben und okologisch calcicolen Pflanze (Dianthus luminitzeri Wiesb.). Flora 169: 9-14.


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Calcium Oxalate Crystals in Plants Author(s)

Calcium Oxalate Crystals in Plants Author(s)

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