Calciu in Plante

7/28/2019 Calciu in Plante

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THE FU NC TI ON OF CALCIUM IN PLANTS 1R . G . W Y N J O N E S ANB O . R . L U N T

D e p a r t m e n t o f B i o c h e m i s t r y & S o i l S c ie n ce ,U n i v e r s i t y C o l l e g e o f N o r t h W a l e s ,

B a n g o r , W a l e s ,a n d

D e p a r t m e n t o f B o t a n i c a l S c i en c e sU n i v e r s i t y o f C a l i fo r n i a

L o s A n g e l e s , C a l i fo r n i a , 9 0 0 2 4Introduction ................................................................................................................................................................................................ 07T h e calcium requirem ent of plants ................................................................................................................................ 408A nato m ic al and cyto lo gic al effe cts of calc iu m defic ie ncy ........................................................................ 410Gross defects ................................................................................................................................................................................... 10Cellular and subeellular effects ...................................................................................................................................... 411Physiological and biochemical evidence for the role of calcium .................................................412Cell wall ................................................................................................................................................................................................. 12Ion absorption and membranes ...................................................................................................................................... 414Ion absorption by whole cells ...................................................................................................................................... 414Mitoehondria ............................................................................................................................................................................... 416Membranes and mo del sys tems ................................................................................................................................ 417Nucleic ac ids and chromosomes ...................................................................................................................................... 418Enzyme activity .............................................................................................................................................................................. 18Concluding discussion .................................................................................................................................................................... 20Literature cited ....................................................................................................................................................................................... 21

I N T R O D U C T I O NT he p r ob l e m o f t he p r e c i s e b i oc he m i c a l f unc t i on o f s om e o f t he e s s e n t i a l

nu t r i e n t e l e m e n t s i n p l a n t m e t a bo l i s m , i nc l ud i ng t ha t o f c a l c i um , ha s p r ove dext remely in t rac tab le . Ca lc ium was recognized as an es sen t i a l e l ement in h igherp l a n t s du r i ng t he m i dd l e o f t he l as t c e n t u ry ( s e e T r u e 1922 ) a l t hough t he u s eof l imes tone and mar l in agr i cu l ture da tes back to pre -Chr i s t i an t imes (T i sda le& N e l s on 1966 ) . H ow e ve r , t he r o l e o f c a l c i um i n a g r i c u l t u r e a nd s o i l m a na ge -m e n t i nvo l ve s m a ny f a c t o r s , a num be r o f w h i c h a r e on l y i nd i r e c t l y r e l a t e d t ot he nu t r i t i ve va l ue o f t he e l e m e n t ( Cha pm a n 1966 ) .

The d i f f i cu l t i e s exper ienced in inves t iga t ing the ro le of ca lc ium may bei ll u st ra t ed by quo t i ng f r om L ud w i g J o s t ( s e e T r u e 1 922 ) , w ho w r o t e in 1907t ha t " . . . w e a r e bound t o a dm i t t ha t i ts f unc t i on ( i. e . c a l c i um ) ha s no t ye tbe e n d i s cove r e d ," a nd f r o m a r e c en t r e v i e w by N a s on a nd M c E l r oy ( 19 63 ) , w h oc onc lude d t ha t " . . . t h e ro l e o f c al c ium is no t e n t ir e l y c l e a r. " H ow e ve r , c on -s ide rab le ev idence , some of recen t o r ig in , has accumula ted about the poss ib le1. The work reported in this paper and the preparation of the manuscript were sup-ported b y National Science Foundation G ran t B5-1226 to Dr. O . R. Lunt.

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4 0 8 T H E B O T A N I C A L R E V IE Wf u n c t i o n s o f c a l c i u m a n d i t a p p e a r e d w o r t h w h i l e t o a t t e m p t t o i n t e g r a t e t h ec y t o l o g i c a l , p h y s i o l o g i c a l , a n d b i o c h e m i c a l d a t a . D u r i n g t h i s p r o c e s s w e h a v eb e e n d r a w n t o t h r e e m a i n a r e a s o f p l a n t m e t a b o l i s m , n a m e l y t h e c e l l w a l l ,m e m b r a n e s , a n d c h r o m o s o m e s . I n a d d i t i o n , t h e r e i s e v i d e n c e o f c a l c i u m i n v o l -v e m e n t i n t h e m a i n t e n a n c e o f t h e c o n f i g u r a t i o n o f s p e c i f i c e n z y m e s .

T H E C A L C I U M R E Q U I R E M E N T O F P L A N T ST h e a n a l y s e s o f a g r e a t v a r i e t y o f c r o p p l a n t s u n d e r m a n y e n v i r o n m e n t a l

c o n d i t i o n s i n d i c a te t h a t c a l c i u m l e v e l s o f s e v e r a l p e r c e n t o n a d r y w e i g h t b a s i sa r e c o m m o n l y f o u n d i n th e f o l i a g e o f l e g u m e s , t o m a t o , t o ba c c o, a n d o t h e rd i c o ty l e d o n s ( B e e s o n 1 9 4 1 ) ( T a b l e I ) . G r a s se s a n d c e re a ls g e n e r a l l y h a v es o m e w h a t l o w e r c o n c e n t ra t io n s o f t h e e le m e n t ; 0 . 2 - 0 . 5 p e r c e n t d r y w e i g h t( a p p r o x i m a t e l y 1 0 m m o l e s / k g , w e t w e i g h t ) is u s u a ll y r e p o r te d . I n b o t h m o n o -a n d d i c o t y l e d o n s t h e r o o t s o r d i n a r i l y c o n t a i n s i g n i f i c a n t l y l e s s c a l c i u m t h a n t h et o p s . I n k e e p i n g w i t h t h e h i g h v a l u e s o f t h e io n r e p o r t e d , t h e s o l u ti o n s u s e df o r h y d r o p o n i c c u lt u r e ( H o a g l a n d & A r n o n 1 9 5 0 ) a n d f o r c a ll u s o r r o o t -t ipc u l tu r e ( W h i t e 1 9 4 3 ) c o n t a in 2 0 0 - 3 0 0 p . p . m , c al c iu m s al ts o r a b o u t 2 - 3m M C a + + i o ns .

I n v i e w o f t h e s e o b s e r v a t i o n s i t i s n a t u r a l t h a t c a l c i u m s h o u l d b e r e g a r d e da s a m a c r o n u t r i e n t e l e m e n t . H o w e v e r , s e v e r al i n d i c a ti o n s h a v e a p p e a r e d r e c e n t ly

T A B L E ICALCIUM LEVELS IN PLANTS UNDER VARIOUS GROWTH CONDITIONSCA CONTENTPLANT ORGAN GROWTH CONDITION % DRY WT. REF.

Co rn m a tu re l eaves f ie ld 0 .34 1gr ain field 0.015 1leav es solution, 2 p.p.m. C a 0.01 2roots solution, 40 p.p.b. C a 0.005 3

W he a t s t r aw f ie ld 0 .29 1gra in f ield 0 .05 1

O a ts abov e g round f ie ld 0 .2 1por t ionA l fa l f a above g round f ie ld 2 -3 1por t ionP ota to tub er field 0.04- 1To bacc o l eaves f ie ld 3 -4 1

leave s solution, 200 p .p .m. Caand 50 p .p .m. M g 0 .4-solution, 200 p.p.m. Caand 50 p .p .m. M g 1 .1solution, 200 p.p.m. Ca

and 50 p .p .m. M g 0 .2 2leav es solution, 2 p.p.m. C a 0.08 2

r e m a i n d e r o fshootroots

Refe ren ces : 1 ) Beeson (1941) , 2 ) W al lace , Fro li ch , & Lu n t (1966) , 3 ) Jones & Lu n t(1968).


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FUNC TION OF CALCIUM IN PLANTS 4 0 9w hi c h s ugge s t t ha t t he c a l c i um r e qu i r e m e n t o f m a ny h i ghe r p l a n t s ha s be e nove r e s ti m a t e d . P ha ri s , B a r ne s , a nd N a y l o r ( 19 64 ) r e po r t e d a ve r y l ow r e qu ir e -m e n t i n Pinus taeda L . w he r e a s W a l l a c e , F r o l ic h , a nd L un t ( 19 66 ) w e r e a b l et o g r o w b o t h t o b a c c o (Nicotiana tabacum L O a M c o r n (Zea mays L . ) i n as o l u t i on c on t a i n i ng on l y a f e w pa r t s pe r m i l l i on o f C a, p r ov i de d o t he r po t e n -t i a l ly tox ic , a l though essen t ia l , ions such as Mg, Cu, Fe , Mn were kept a t al ow ba l a nc e d l e ve l . U nde r t he s e c ond i t i ons t he l e a f t i s s ue s o f c o r n c on t a i ne d0 . 01 - 2 pe r c e n t d r y w e i gh t o f c a l c i um , o r 0 . 1 pe r c e n t i n t oba c c o . T a l l w he a tgrass (Agropyron elongatum ( H o s t ) B e a u v . ) g r e w n o r m a l l y i n l o w c a lc iu mm e d i a a nd t he s hoo t s c on t a i ne d on l y 200 p . p . m . ( 0 . 02 pe r c e n t d r y w e i gh t )( L u n t & Jo n e s 1 9 6 8 ) .

T h e n o r m a l d e v e l o p m e n t o f c o r n ro o t s re q u i re d o n l y 3 - 1 0 [zM C a i o n s int he e x t e r na l so l u t i ons i n the a bs e nc e o f o t he r i ons ( J o ne s & L un t 1 96 8 ) , a ndhe a l t hy r oo t s c on t a i ne d on l y 0 . 007 pe r c e n t C a d r y w e i gh t . H ow e ve r , t hepresen ce of toxic ions , e .g. , 0 .1 [zM cup r ic ions, in creased th e calc ium requ iredi n t he e x t e r na l s o l u t i on t o 10 0 - 2 00 l zM. T h e a b i l it y o f C a + + t o c oun t e r a c tthe adv er se e ff ec ts o f inc reas ing H + conce nt r a t ion on ro ot e long a t ion (Bi i r s t rom1952 , 1954 ) a nd i on a bs o r p t i on ( J a c obs on , M oor e , & H a nn a pe l 1960 , R a ins,S c hm i d , & E ps t e i n 1 964 ) i s w e l l doc um e n t e d . T h e a b i l i ty o f c al c ium t op r o t e c t a ga i n s t he a vy - m e t a l t ox i c i t y w a s obs e r ve d i n t he e a r l y s t ud i e s by T r ue( 1 9 1 4 ) , a n d m o r e r e c e n t l y w i t h Chlorella ( M c B r i e n & H a s s a l l 1 9 6 5 ) , y e a s t( C h e s t e r 1 9 6 5 ) , a n d a v ar ie t y o f b ac te r ia ( N i c h o l a s 1 9 6 3 ) . T h e o b s e rv a -t i ons c l e a r l y i m p l y t ha t t he h i gh l e ve l s o f c a l c i um u t i l i z e d i n p l a n t g r ow t hare r equi r ed pr imar i ly to amel iora te tox ic i t i e s and the ro le of ca lc ium underi de a l c ond i t i ons m a y a pp r oa c h t ha t o f a m i c r onu t r i e n t .

T h i s s i t ua t i on m or e ne a r l y a pp r oa c he s t ha t f ound i n l ow e r p l a n t s w he r en u m e r o u s e x a m p l e s e x i s t o f a v e r y lo w c a lc i um r e q u i re m e n t ( N i c h o l a s 1963,B o l l a r d & B u t l e r 1966). A n u m b e r o f r e p o r t s h a v e a p p e a r e d s u g g e s t i n g t h a tc a l c i um i s no t r e qu i r e d f o r t he g r ow t h o f s e ve r a l s pe c i e s o f ye a s t s a nd f ung i .T he l i t e r a t u r e i n t h i s f i e l d i s w e l l doc um e n t e d i n t he e xc e l l e n t r e v i e w byB o l l a r d a n d B u t l e r (1966) . I n f ung i i t a ppe a r s t ha t t he r e i s a h i ghe r c a l c i umr e qu i r e m e n t f o r s po r u l a t i on t ha n f o r ve ge t a t i ve g r ow t h . A c l o s e r e la t ionbe t w e e n c a l c i um a nd o t he r d i va l e n t c a t i ons a nd t he s t r uc t u r e a nd v i a b i l i t y o fs po r e s ha s a l so be e n r e po r t e d i n s e ve r a l ba c i ll i ( S l e pe c ky & F os t e r 19 59 ) . I ngenera l , a lgae r equi r e ca lc ium, a l though in Chlorella th i s r equi r ement i s t e s st ha n a pa r t pe r m i l l i on i n t he m e d i um . A s i n h i ghe r p l a n t s , i t m a y be a s s um e dt ha t t he obs e r ve d r e s pons e t o c a l c i um i s g r e a t l y i n f l ue nc e d by t he o t he r i onsi n t h e e x t e rn a l m e d i u m . O ' K e l l e y an d H e r n d o n ( 1 9 6 1 ) , i n d is c u ss in g th e c al-c i um r e qu i r e m e n t o f a lga e, s ugge s t e d t ha t i t m a y be r e l a te d t o t he q ua n t i t y o fpec t ic mate r ia l s ec re ted by the ce l l s .

A l t ho ugh t he r e qu i r e m e n t f o r c al cium ha s be e n w i de l y s tud i e d i n p l a n ts ,l i t t l e i s know n a bou t t he d i s t r i bu t i on o f t he i on w i t h i n t he c e l l . Ma ny t e c hn i c a ld i f f i cu l t i e s a r e involved in the use of so luble i so topes for r ad ioautography.Gie l ink , Sauer , and Ringoe t (1966) have r ecent ly r epor ted tha t f r eeze - subs t i tu -t ion us ing ace tone y ie lds sa t i s f ac tory au tographs in which the ca lc ium i s loca l -i z e d m a i n l y i n t he c e l l w a l l . F r e e z e - d r i e d pa r a f f i n - e m be dde d m a t e r i a l ha s be e n


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4 1 0 T H E B O T A N I C A L R E V I E Wu s e d b y L . J a c o b s o n ( p e r s o n a l c o m m u n i c at io n , 1 9 6 5 ) w h o f o u n d t h e i s o t o p em a i n l y i n t h e c e l l w a l l a n d t h e n u c l e u s . H o w e v e r , b o t h t h e s e t e c h n i q u e s m a yb e c r i t i c i z e d a n d a m u c h m o r e e l a b o r a t e a n d d e t a i l e d s t u d y i s r e q u i r e d t oe l u c i d a t e t h e p r o b l e m . T h e a c t u a l c y t o p l a s m i c a n d v a c u o l a r c o n c e n t r a t i o n o fc a l c i u m h a s b e e n r e p o r t e d i n Nitella b y S p a n sw i ck an d W i ll ia m s ( 1 9 6 5 ) . T h e yf o u n d 8 m M c a l c i u m i n t h e c y t o p l a s m a n d 1 2 m M i n t h e v a c u o l e a n d s u g g e s t e dt h a t t h e r e i s n o n e c e s s i t y f o r a c a l c i u m p u m p a t t h e p l a s m a l e m m a b e c a u s e t h el o w c y t o p l a s m i c c a l c i u m c o n t e n t c o u l d b e d u e t o l o w p e r m e a b i l i t y . M i l l i k a n a n dH a n g e r ( 1 9 6 5 ) h a v e re p o r t e d e x t en s iv e l y o n t h e in t er c el lu l ar m o v e m e n t o fCa 45 in su b te r r anea n c lover as in f luenced by ion leve l s and che la t ing agents andhave shown the d i f f e r en t ia l d i s t r ibu t ion of ca lc ium dur ing de f ic iency condi t ions .

A N A T O M I C A L A N D C Y T O L O G I C A L E FF EC T S O FC A L C I U M D E F I C IE N C Y

G R o s s D E F E C T SThe v i sua l e f f ec t s o f ca lc ium and o the r de f ic ienc ies in many c rop p lan t s mayb e f o u n d d e s c ri b e d b y W a l l a ce ( 1 9 6 1 ) a n d i n " D i a g n o s t ic C r it e ri a f o r P la n t sa n d S o i l s" ( C h a p m a n 1 9 6 6 ) . A g r e a t d e a l o f v a l u a b l e i n f o r m a t io n m a y a l so

b e f o u n d i n H e w i t t ' s e x c e l l e n t r e v i e w ( 1 9 6 3 ) . T h r e e p r i n c i p a l s y m p t o m s c o m -monly r esu l t f rom ca lc ium def ic iency , a l though o the r de fec t s have a l so beenr e p o r t e d . L o w c a l c i u m s t a t u s m a y r e s u l t i n a b l a c k e n i n g a n d c u r l i n g o f t h emarg ins of the ap ica l l eaves , u l t ima te ly l ead ing to acu te nec ros i s and the cessa-t io n o f g r o w t h . T h e p h y s i o l o g ic a l fo u n d a t i o n f o r t h is d e f o r m a t i o n m a y b ecom plex , h ow ever , a s it is c lose ly r e la ted to the mag nes iu m s ta tus an d occursm o r e r e a d i l y a t h i g h m a g n e s i u m l e v e l s ( H e w i t t 1 9 6 3 ; E . F r o I i c h , u n p u b l i s h e do b s e r v a t i o n s 1 9 6 6 ) . I t m a y a l s o b e n o t e d t h a t i n m a n y p l a n t s t h e s y m p t o m sof calc ium def ic iency c losely resemble t h o s e o f a l u m i n u m t o x i c i t y ( B o l l a r d &B u t l e r 1 9 6 6 ) . S in c e th e t y p i c a l e c o l o g ic a l c o n d i t i o n s i n w h i c h t h e s e ab n o r m a l i -t i e s w ou ld o ccur a r e so similar , i t m ay be d if fi cu l t to d i s t inguish b e tw een them .Poor roo t deve lopment i s a l so commonly as soc ia ted w i th ca lc ium def ic iency .H o w e v e r , t h e r e a r e v a r i o u s i n d i c a t i o n s t h a t t w o p h e n o m e n a , w h i c h m a y o r m a yn o t b e r e l a t e d , a r e i n v o l v e d . T h e r e i s e v i d e n c e o f a n i n t r a c e l l u l a r p h y s i o l o g i c a Ir equi r ement fo r ca lc ium, as we sha l l d i scuss . I n addi t ion , the re i s ev idence ofan ex te rna l r equi r em ent fo r d iva len t ca tions, u sua l ly ca lc ium, in the me diums u r r o u n d i n g t h e r o o ts . I n ea r ly s t u d ie s T r u e ( 1 9 1 4 ) f o u n d t h a t d i s ti l le d w a t e rw a s t o x i c t o p l a n t r o o t s a n d s u g g e s t e d t h a t c a l c i u m w a s r e q u i r e d i n t h e e x t e r n a lm e d i u m t o m a i n t a in t h e s e l ec t iv e p e r m e a b i l it y o f th e c e ll m e m b r a n e a n d p r e v e n tl e a k ag e . H a y n e s a n d R o b b i n s ( 1 9 4 8 ) s i m i la r ly c o n c l u d e d th a t ca lc iu m a n db o r o n m u s t b e p r e se n t i n t h e e x te r n al m e d i u m f o r r o o t d e v e lo p m e n t .

O u r o w n s t u d ie s ( J o n e s & L u n t 1 9 6 8 ) , a l th o u g h n o t in fu n d a m e n t a l d is -a g r e e m e n t w i t h t h e s e o b s e r v a t i o n s , s u g g e s t t h a t f u r t h e r i n v e s t i g a t i o n i s r e q u i r e d .C o r n r o o t s e x p o s e d to d i s ti l le d - d e i o n i z e d w a t e r ( m a x . 1 5 p . p . b . C a ) c e as e e l o n -g a t i n g w i t h i n h o u r s b u t s h o w o n l y l i t t l e a n d v e r y l o c a l i z e d d a m a g e . T h e r e i sn o e v i d e n c e o f a m a j o r b r e a k d o w n o f s e le c t iv e p e r m e a b i l i ty a f t e r 7 - 1 0 d a ys ,because l i t t l e po tas s ium i s released a n d t h e r a t e o f r u b i d i u m u p t a k e i s o n l y


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FUNC TION OF CALCIUM IN PLANTS 411

about hal f that found in normal roots . However , there i s a lmos t to ta l break-down of the cytoplasm of the cel ls in the root apex. Roots placed in ca. 1.0 jamalkal i earth ions elongate rapidly. The reason for the dramatic influence ofcalcium and related ions is not clear but i t may be a calcium requirement ofthe membrane s t ructure which may be more acute in dividing cel ls . As calciumis poo rly t ranslocated (B idd ulp h, Cory, & B idd ulp h 1 95 9), i t w ou ld also beexpected that the apex would be more severely affected.The resu l t s of work on the inf luence of ca lc ium on root developmentare also hampered by the close relat ionship between calcium status and thetoxic ity of o ther ions . As poin ted out by Edw ards (19 36 ) , the omission ofcalcium from an otherwise complete nutr ient solut ion exposes the roots toa h ighly toxic en vi ronm ent of potassium, magn es ium, and o ther m icronut r ien tions . A conf i rma t ion of th i s v iew is the recent demons t ra t ion (W al lace , Fro-l ich , & Lu nt 19 66) of the norm al grow th of tobacco and corn wi th one-f i f t ie ththe level of calcium in Hoagland's solut ion provided other ions are in balance.Thus i t i s probable that many early reports on the toxici ty of dis t i l led waterreflect the extre m e toxicity o f m an y ions, e.g., 0.1 tzM Cu ++, in the absence o fcalcium.The third defect associated with calcium deficiency is a decrease in fruitand seed form ation. H ew it t (1 96 3) describes the collapse of the ovule, wi thirregular necrosis of the embryonic cotyledons and shrinkage within the integ-um ents , as occu rring in broad-bean, pea, and Fren ch bean as a result o fcalcium deficiency. Likewise, calcium w as requ ired f or the diffe ren tiation ofspo rang i a and gemmae i n an Achlya species (Gr i f f in 1966) . An impor tan trole for calcium in the Jn v#ro germina t i on o f po l l en and t ube g rowth i si nd ica ted by t he work o f Kw ak (1965) and De Bruyn (196 6) . In v iew o fthis response to calcium and t ropic growth of pol len tubes of Antirrhdnummajus L. towards a calcium source (Mascarenhas & Machl is 1964), Mascaren-has (196 6) measured the d i s tr ibut ion of ca lc ium in the gynoecium but theJn vivo resul ts did not support the idea of a calcium-directed growth of thepol len tube.

Calcium deficiency readi ly induces "blossom-end rot" in tomato and maybe associated with "black-spot" in apples . The detai led anatomical changes inthe former are d i scussed by Spur t (1959) . The growth of the peanut f ru i t(Arachis hy[;ogaea L.) also exhibi ts a marked calcium requirement , whereasH ew it t (19 63 ) reports that grain fo rm ation is ofte n suppressed in cerealsduring deficiency.

CELLULA R AND SUBCELLULAREFF;.CTSIn the years before the Second W or ld W ar the cytological effects of calcium

deficiency received much at tent ion but confl ict ing resul ts were obtained by thevarious groups. W ith the adv ent of the electron microscope interest in thisproblem has renewed. In 1929 Sorokin and Sommer reported that the omissionof ca lc ium from a complete nut r ien t medium caused the rap id degenerat ion ofthe apical cells of Pisum sa t i v . m L . They found a decrease in cytoplasm andincreased vacuoIation of the meristematic cel ls . Abnormal mitot ic f igures and


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412 THE BOTANICA L REVIEW

p o l y p l o i d y w e r e o b s e r v e d . H o w e v e r , t h e a u t h o r s s pe ci fi ca lly d e n i e d a n y a d v e r s ee f f ec t s on the ce l l wa l l . They f e l t tha t the i r r e su l t s d id no t subs tan t ia te the con-c e p t o f c a l c i u m p e c t a t e a s a c e l l - w a l l b i n d i n g a g e n t . T h e s e r e s u l t s w e r esevere ly c ri ti cized by Ed w ards (1 93 6 ) bo th because o f h i s to log ica l a r ti f ac t sa n d b e c a u s e o f t h e t o x i c e f f e c t s o f t h e o t h e r i o n s i n t h e m e d i u m , a s p r e v i o u s l ym e n t i o n e d . B a m f o r d ( 1 9 3 1 ) r e g a rd e d t h e c h ro m o s o m a l d e fe c t s a s a t e rm i n a le f f ec t o f ca lc ium deficiency an d ion ic imba lance . H ow ev er , s ince th i s t imevar ious r epor t s have appeared ind ica t ing a c lose as soc ia t ion be tween ca lc iumd e fi ci en c y a n d c h r o m o s o m a l a b n o r m a l i t y ( S t e f f e n s e n 1 9 5 8 , H y d e & P a l i w a l1 9 5 8 ; se e H e w i t t 1 9 6 3 ) .

I n c o n t ra s t t o S o r o k in a n d S o m m e r ( 1 9 2 9 ) , R e a d ( 1 9 0 7 ) h a d f o u n di r r egula r i t i e s in the ce l l wa l l s o f ca lc ium-def ic ien t ce l l s and no ted an inab i l i tyt o f o r m a n e w c e l l p l a t e d u r i n g c e l l d i v i s i o n , w h i c h h e a t t r i b u t e d t o t h e a b s e n c eof calc ium.

T h e r e c e n t e le c t ro n m i c r o s c o p ic s t u d ie s b y M a r i n o s ( 1 9 6 2 ) o n b a r l e y s t ema p e x r e v e a l e d e x te n s i v e m e m b r a n e a n d m i t o c h o n d r i a l d i s i n te g r a t io n i n d ef i-c i e n t c e ll s. F l o r e l l ( 1 9 5 6 ) p r e v i o u s l y r e p o r t e d f e w e r m i t o c h o n d r i a i n w h e a tr o o t s l o w i n c a l c i u m w h e r e a s c o m p a r a b l e m e m b r a n e a n d c y t o p l a s m i c b r e a k d o w nh a s a ls o b e e n o b s e r v e d i n b a r le y r o o t s ( M a r s c h n e r & G u n t h e r 1 9 6 4 , M a r s c h -n e r , H a n d l e y , & O v e r s t r e e t 1 9 6 6 ) . T h e l a t t e r w o r k a l s o i l l u s t r a t e d c y t o l o g i c a l l yt h e a b i li t y o f C a + + t o p r o t e c t a g a i n s t H + t o x i c it y w h i c h h a d p r e v i o u s l y b e e no b s e r v e d f r o m l e a k a g e a n d g r o w t h m e a s u r e m e n t s . I n o u r w o r k ( G a l e y , J o n e s , &L u n t 1 9 6 8 ) w e h a v e a ls o o b s e r v e d t h e su b s t a n t ia l b r e a k - u p o f th e e n d o p l a s m i cr e t ic u l u m a n d o t h e r c y t o p l a s m i c m e m b r a n e s i n t h e c e ll s o f c o r n r o o t a p ic ese x p o s e d t o a d i v a l e n t - i o n - f r e e e n v i r o n m e n t . I t w o u l d a p p e a r t h e r e f o r e t h a tcy top lasmic s t ruc ture i s adver se ly a f f ec ted by ca lc ium def ic iency a l though th i sp r o c e s s i s g r e a t l y w o r s e n e d b y f u r t h e r i o n i c i m b a l a n c e ( S o r o k i n & S o m m e r1 9 2 9 , M a r s c h n er , H a n d l e y , & O v e r st re e t 1 9 6 6 ) .

T H E P H Y S I O L O G I C A L A N D B I O C H E M I C A L E V I D E N C EF O R T H E R O L E O F C A L C I U M

T h e e v i d e n c e r e g a r d i n g t h e p h y s i o l o g i c a l f u n c t i o n o f c a l c i u m f a l l s l a r g e l yi n t o f o u r c a t e g o r ie s : t h e c e ll w a l l , m e m b r a n e s , c h r o m o s o m e s , a n d e n z y m eac t iva t ion . As in any compar tmenta l iza t ion of th i s type , i t i s a s impl i f i ca t ionwhich tends to b lur ce r ta in r e la t ionsh ips and i s a t bes t r a the r a rb i t r a ry . How-e v e r , w e h o p e t h a t i n a d o p t i n g t h i s l a y o u t t h e f i e l d w i l l b e e n l i g h t e n e d r a t h e rt h a n o b s c u r e d .

T H E C E L L W A L LT h e h y p o t h e s i s o f c a l c i u m p e c t a t e a c t i n g a s a c e m e n t i n g a g e n t i n t h e c e l lw a l l o f p l a n t c e l l s w a s o r i g i n a t e d d u r i n g t h e l a s t c e n t u r y ( s e e T r u e 1 9 2 2 ) a n dh a s f o u n d g e n e r a l a c c e p t a n c e i n p l a n t p h y s i o l o g y te x ts . H o w e v e r , t h e a t t e m p tb y B e n n e t - C l a r k e ( 1 9 5 6 ) t o r e l a te th e p r o p o s e d r o l e o f ca lc iu m a s a n i o n i cbr id ge to auxin ac t ion has been sub jec ted to cons ide rab le c r it ic ism. I t was


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proposed that IAA" s t imulated cel l e longat ion by complexing calc ium in amanner comparable to EDTA and thus increasing the plast ici ty of the cel lwall . How ever , nei ther Cle land (19 60) nor Bur l ing and Jackson (19 65)detected any re lease or red is t ribut ion of Ca ++ du r ing IAA -s t imulated gro wthof coleoptiles, as would be expected from the calcium-bridge hypothesis.Considerable evidence has accumulated that the formation of calcium pectateincreased th e r igidi ty of the cell w al l (Ta gaw a & Bon ner 1957, Rasmussen1966, Cormack 1965). The process of calcification increases the resistanceof t issue to infect ion probably by increasing the resis tance of the wal ls topolygalacturonidase (Bateman & Lumsden 1965) . Fur ther ev idence for aro le of ca lc ium in the ce l l wal l may be deduced f rom the use of EDTA andEDTA with protein or l ipid-dissolving agents to increase t issue macerat ionand to produce i so la ted cel l s (Ginzberg 1958) . EDTA t reatment a l so causesthe removal of Ca ++ a nd pect in and increases cel l plastici ty (Ta ylor &W a i n 1 9 6 6 ) .

A more complex relat ionship between cel l r igidi ty, elongat ion, and calciumis indicated by the detai led and extensive s tudies of Burstr6m (1952, 1954,1957). He concluded that root cel l growth occurs in two stages: a) an increasein plast ici ty an d elast icity of the cel l wal l , an d b ) the biosynthesis and layingdown of new cel l -wal l material . The fi rs t s tage is enhanced by auxin butantagonized by calcium, whereas the relat ionship is reversed in the secondstage. Burstr6m evolved this postulate to explain the differences between hisobservation of Ca+ +-induced elon gat ion o f root cells and the effect of Ca ++,noted by Cormack (19 49 ) , of hard ening and produ cing shor ter ce ll s. Burs tr6m'sproposal for the role of IAA in root t issue differs somewhat from the recentwork o f Mor r6 and Bonne t (1965) , who f i nd t ha t IAA inh ib i t ed roo t g rowthby shor ten ing the per iod of ce ll e longat ion .Li t t le change in pect in and hemicel lulose contents of Ca-deficient roots wasfound (Burs t r6m 1958) , a l though a 20 per cent decrease in ce l lu lose contentoccurred. In comparable studies on coleoptile cell-wall constituents, Ra y an dBake r (1 96 5) fo un d that the synthesis of matrix polysaccharides exce pt ~-cel-lu lose was induced in co leopt i le segments by IAA when e longat ion i s inh ib i tedby Ca + +. I n the absence of Ca + + a n increased synthesis of ~ -cellulose was alsoobserved. The comparison of these results is complicated by opposite effectsof ions on root an d coleopti le e longat ion . The form er i s induced by Ca ++whereas the lat ter is s t imulated by monovalent cat ions but not by divalent ions(P urves 1966) .

It is difficult to determine whether these interactions are related to a directinfluence on th e cell wal l or to a more subt le al terat ion in metabolism. IA A atlow concent ra tion has been show n by Sark issian and M cD aniel (196 6) toincrease the respirat ion and the P:O rat io of maize-scutel lum mitochondria2. Th e following abbreviations are us ed : ED TA , ethyle ne diaminetetraacetic ac id;IAA , indoleacetic acid; R N A , ribonucleic acid; sR N A , soluble RN A; RN Aase,ribonuclease; D N A , deoxyribonucleic acid; N A D PH , reduced nicotinamide ade-ninedinucleotide phosphate; AT P, adenosine triphosphate; A TP ase , adenosine tri-phosphatase; K,, M ichaelis constant.


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whereas H anson, M alhot ra , and S toner (19 65) have demon st ra ted that ca lc iumrechannels energy f rom A TP form at ion to ion accumulat ion . Th us bo th com-pounds may substant ial ly al ter the metabol ic balance within the cel l . I t isin teres t ing to note that La mp ort ( 196 5) has proposed a hypothes i s of ce l l-walle longat ion based on the concept of IA A increasing the NA D P H level andtherefore s t imulat ing the reduct ion of su l fhydry l bonds of the ce ll -wal l pro te in .H e suggest s that IA A causes an increase in A TP levels which in turn channe lg lucose-6-phosphate in to the NA D PH -ge ner at ing pentose phospha te cycle.Thus these metabolic interactions must aIso be considered as well as cell-waildeposi t ions when interpret ing the varying effects of calcium on cel l elongat ion.The resul ts suggest that the deposi t ion of calcium pectate in the cel l wal lincreases the r igidi ty of the s t ructure. However, i t i s not clear how vi tal thisprocess is to the growth of the plant in the absence of s t ress , as such low levelsof calcium have been found in seemingly heal thy t issue, part icularly roots .The complex interact ions thereto described cannot be resolved into a unifiedhypothesis , al though i t is possible that they relate more to the interact ion ofcalc ium wi th membranes than wi th the ce l l wal l .

ION ABSORVTION AND MEMBRANESa) Ion absorp t ion by whole ce l l s

As previously discussed, there is evidence from both l ight- and electron-microscopy that calcium deficiency causes a breakdown of membranous s t ruc-tures and the decreased synthesis of mitochondria. A further substant ial bodyof ev idence re la t ing calc ium to me mb rane in tegr i ty comes f rom w ork on ionabsorpt ion. U nd er certain con di t ions calcium enhances the up take of potassium byexcised roots and other t issue--the Viers effect (1944). In a comprehensivestud y Jacobson a nd his collaborators (1960 , 1961a, b) show ed that the influenceof Ca ++ on the up take o f m onov atent ions i s depend ent on the p H . K + absorp-t ion is rapidly inh ibi ted b y increasing H + and calcium is able to co unteractthis effect . The rapid loss of ions from excised roots at low pH is also mini-mized by Ca ++ ions. Ho weve r , a t m i ld ly a lkal ine or neut ra l pH , ca lc ium ei therhas no effect on potassium upta ke or is s l ight ly inhibi tory. At ne utral pH calciumenables roots pre loaded w i th K + to re ta in the ion w hich wo uld o therw ise beleached out (M arschner , Ha ndley , & Overs t ree t 1966) . Eps te in (1 961 ) con-cludes f rom exper iments on the compet i t ion between Na and K that ca lc ium i srequ ired at 1 mM fo r the m aintena nce of the selectivity of the absorp t ion process.La ter resul ts (Ra ins, Schmid, & Epstein 1964) show ed that influence of Ca onRb uptake was h ighly dependent on pH. These resu l t s imply that ca lc ium de-creases the efflux of ions, particularly in a toxic ionic environment, and is essen-t ia l for the in tegr i ty of the bou ndary membranes , poss ib ly the p lasmalem ma.

How ever , W aisel (196 2) ma in ta ins that ca lc ium increases the ra te of passivedi f fus ion of the potass ium across the p lasmalem ma. Ha ndle y and h is co lleagues(1965), in an at tempt to dissociate the cytoplasm and vacuole, s tudied the in-f luence of Ca on K and Na uptake and release by the f i rs t unvacuolated (0-1.8ram . ) and second vacuola ted (1 .8-3 .8 ram . ) segments of corn root . They sug-gest that K absorpt ion in to the cytoplasm is passive an d tha t calcium inh ibi ts this


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d i f f u s i on by de c r e a s i ng t he po r e s i z e . T he a c t i ve up t a ke i n t o t he t onop l a s t , how -ever , is s t imulated by calc ium.T he s e i n t e r p r e t a t i ons d i f f e r f unda m e n t a l l y i n t he c onc e p t o f t he p l a c e a nd

m e c h a n i s m o f i on a bs o r p t i on . E ps t e in , R a in s , a nd E l z a m ( 19 63 ) de m ons t r a t e d ab i pha s i c a bs o r p t i on i s o t he r m o f po t a s s i um by b a r l e y r o o t s . T h e t w o p h a s e s h a v ebe e n a t t r ibu t e d by T o r i i a n d L a t ie s ( 1 96 6 ) t o a l ow K s s ys te m ope r a t i ng a t t hep l a s m a l em m a a n d a h i g h K , s y s t em o p e r a ti n g a t t h e to n o p l a st . A t t h e h i g h e rc onc e n t r a t i on t he p l a s m a l e m m a s ys t e m i s s a t u r a t e d a nd d i f f u s i on be c om e s i n -c r e a s i ng l y i m por t a n t . A f u r t he r m od i f i c a t i on o f t he s e i de a s ha s a ppe a r e d r e c e n t l y( P i t m a n & S a d d le r 1 9 6 7 ) . E v i d e n c e is p ro d u c e d t o s h o w t h a t th e l o w K , s y s te ma t t h e p l a s m a l e m m a i s a s o d i u m / p o t a s s i u m e x c h a n g e p u m p . I n s e v e r a l a n i m a lt i s sues , e .g . , r ed b lood ce l l s , ne rve f ibe r s , the up take of po tass ium i s accompaniedby a m e t a bo l i c a l l y m e d i a t e d m ove m e n t o f s od i um a c r os s t he m e m br a ne a ga i n s tt he e l e c t roc he m i c a l g r a d i e n t . T h e m ove m e n t i s m e d i a t e d by a K + a nd N a + -s t im u l a t e d A T P a s e e n z y m e w h i c h is u s u al ly i n h i b i te d b y C a + + ( S k o u 1 9 6 5 ) .T h i s t y p e o f s o d i u m e x c l u s i o n p u m p h a s n o t p r e v i o u s l y b e e n r e p o r t e d a sope r a t i ng i n r oo t i on a c c um ul a t i on ( s e e S kou 1965 ) . I n a dd i t i on , a n A T P a s ea c t iv i ty has r e c e n tl y be e n r e po r t e d i n p l a n t c e l l w a l l s w h i c h i s s t i m u l a t e d by K +a n d is d e p e n d e n t o n C a + + o r M g + + ( D o d d s & E llis 1 9 6 6 ) . T h e o b s e rv a t io n scor r e la te ve ry we l l and y ie ld the a t t r ac t ive hy pothe s i s tha t th e in f luence of cal -c i um on po t a s s i um up t a ke i s due t o i t s r o l e a s a n a c t i va t o r o f A T P a s e . A pos s i b l ee xp l a na t i on f o r the a nom a l ous r e su l ts ob t a i ne d by H a nd l e y , M e t w a l l y , a nd O ve r -s t re e t ( 1 96 5 ) m a y l i e i n t he re l a ti ve ly h i gh c onc e n t r a ti on o f po t a s si um , 1 m M ,t he y e m p l oy e d . T he l ow K ~ s ys te m o f E ps t e in a nd L a t ie s i s s a t u r a te d a t one - fi f tht o one - t e n t h o f t h i s c onc e n t ra t ion a nd m a y no t be obs e r ve d und e r t he c on d i t i onse m p l o y e d .

A n a l t e r na t i ve a pp r oa c h t o t he r o l e o f c a l c i um i n m e m br a ne s a nd i on a bs o r p -t i o n h a s b e e n t h e u s e o f E D T A t o r e m o v e a c o n s i d e r a b l e p r o p o r t i o n o f t h et is s ue c al ci um . H a ns o n ( 19 60 ) f ou nd t ha t t r e a t m e n t o f e xci se d r oo t s w i t hE D T A , R N A a s e , o r K C I c a us e d de c r e a s e d r e s p i r a t i on a nd t he l o s s o f nuc l e o t i de st o t he s o l u t i on . C a l c i um a nd m a gne s i um p r o t e c t e d t he c e l l s f r om t he da m a ge .I n l a te r w o r k ( F o o t e & H a n s o n 1 9 6 4 ) t h e se tr e at m e n t s w e r e f o u n d t o c a u se a nin i t i a l inc rease in po tass ium uptake which was a t t r ibu ted to a r ap id f lux ofpotass ium in to the vaca ted exchange s i t e s of the cy toplasm. The loss of ca lc iuma n d d e g r a d a t i o n o f R N A w a s t h o u g h t t o i n c r e a s e t h e m e m b r a n e p e r m e a b i l i t y .T a n a da ( 195 5 , 195 6 ) ha d p r e v i ous l y s how n the a dve r s e e ff ec ts o f u l tr a v i o l e tl i gh t i r r a d i a t i on a nd R N A a s e on a bs o r p t i on . O n t h i s ba s i s i t w a s s ugge s t e d t ha ta C a - r i bonuc l e op r o t e i n c om pl e x i s i nvo l ve d i n po t a s s i um a bs o r p t i on .

T h e r e m o v a l o f c a lc iu m b y E D T A a ls o p r e v e n t e d c h l o r id e a n d p h o s p h a t ea bs o r p t ion . T he i m pa i r e d up t a ke o f phos ph a t e , n i t ra t e , c h l o ri de , a nd b r om i deha s p r e v i ous l y be e n de m ons t r a t e d i n c a l c i um - de f i c i e n t t i s s ue ( F oo t e & H a ns on1 96 4 , H o o y m a n s 1 9 6 4 , P i t m a n 1 9 6 5 , H y d e 1 9 6 6 , L e g g et , G a l l o w a y , & G a u c h1965) . T he i nc r e a s e d l e a k i ne s s o f be e t r oo t d i s k s e xpos e d t o E D T A w a s r e -p o r t e d b y V a n S t e v en i n c k ( 1 9 6 5 ) . H o w e v e r , so m e w h a t d i ff e re n t re s u lt s w e r eo b t ai n e d b y H i r a ta a n d M i t su i ( 1 9 6 5 ) , w h o f o u n d t h a t b o t h E D T A a n d M g -E D T A t r e a t m e n t i nduc e d t he l o s s o f c a l c i um a nd c a us e d de c r e a s e d po t a s s i um


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absorpt ion . O nly ED TA and no t M g-E DT A caused nucleot ide leakage; thusno d i rect re la t ion between the inf luence of CA on K uptake and RNA metab-ol ism was postulated. The difference between the resul ts of the Japaneseworkers and those of Hanson and h i s group are probably due to the speci f iccondi t ions employed, as Foote and H anso n noted that longer exposure to ED TAdecreased potassium uptake. Possibly a tenuous balance exists between destruc-t ion of cytoplasm and increasing the number of exchange s i tes .b) Mitochondria

The major problem in the use of excised roots or s torage t issue disks ins tudying ion absorp t ion is the complexi ty of the ce ll s and t is sue employed. O nem etho d of s im pl i fy ing the problem has been the use of i so la ted mi tochon dr ia .The work o f Lehn inger (1 967) and o the rs demons t ra ted t ha t an imal mitochon-dria can accumulate large quan t i ties of Ca ++, M g ++, and inorgan ic phosp hatewhen an energy source is provided, and under certain condi t ions can also takeup K + ( H arris , Cockrel l, & Pressman 196 6). H an son and his colleagues in aseries of papers (H odg es & Hans on 1965, Truelove & Ha nson 1966, Kenef ick &H anso n 1966) have sho wn the m utual ly depende nt absorp tion of Ca + +, Mg + +,and inorganic phosphate by mi tochondr ia f rom corn seedl ings . The process re-qui res energy which may be furn i shed by a Krebs-cyde in termedia te such assuccinate , ATP, or the potent ia l energy of the cont racted mi tochondr ion . I t hasbeen suggested that a high ene rgy pho spho rylated interme diate is involved inthe upta ke of the d ivalent ion, al thou gh the idea of charge segregation, asadvocated by M i tchel l (19 66 ) , i s no t excluded. M i tochondr ia f rom red b eeta l so accumulate Ca and inorganic phosphate and the inhib i t ion of th i s uptakeby monovalent ions was interpreted as evidence for the chemo-osmotic theory(Mi l lard , Wiskich , & Rober t son 1965) . The main product of the accumulat ionis the precip i ta t ion of ca lc ium phosphate wi th in the mi tochondr ion . However ,G run w ald (19 66 ) has also fo un d a less soluble Ca45-1abeled fract ion associatedwi th R N A which m ay be re la ted to the car rier complex .

In addi t ion to mi tochondr ia , the magnes ium- and l ight -dependent uptake ofcalcium and phosphate has been demonstrated in isolated spinach chloroplastsw hen cycl ic e lec tron t ranspor t was coupled to phen azine metho sul fa te (N obe l &Packer 1964, 1965).The ques t ion ar i ses whether the process of ca lc ium and phosphate uptakeby m itoch ond ria an d chloroplasts bears any relat ion to the mechanisms of assim-ilation of ions by intact cells. There are conflicting suggestions whether calciumuptake into roots is either active or passive. However, there is considerable evi-dence of calcium deficiency diminishing phosphate uptake. In view of the closerelat ion betw een ion absorpt ion and respiration, i t appears desirable in ma nyins tances to s tudy the s i tuat ion in m i tochondr ia , a l though the case for analogousmechanisms in both organelles and cells cannot be pressed at this t ime.

As wel l as the use of mi tochondr ia as a model sys tem for examining ionabsorption, the re is evidence of calcium influ enc ing respiration. T he o ld obser-vat ion o f the Ca + + ion unco upl ing oxidat ion pho sphoryla t ion ma y now be ex-p la ined in t erms of a red i rect ion of energy f rom ATP format ion to ca lc ium


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FUNC TION OF CALCIUM IN PLANTS 4 1 7p h o s p h a t e a c c u m u l a ti o n . T h e s t im u l a ti o n o f r e s p i r a ti o n b y t h e l o w l e v e l o f C a + +n o t e d i n a n i m a l s h a s n o t b e e n f o u n d i n p l a n t m i t o c h o n d r i a .

I t a p p e a r s l i k e l y t h e c a l c i u m i s i n v o l v e d i n t h e s t r u c t u r a l o r g a n i z a t i o n o fm i t o c h o n d r i a b e c a u s e o f t h e c y t o lo g i c a l e v i d e n c e p r e v i o u s l y d i s c u s se d a n d aRuss ian repor t specif ical ly descr ibing the swel l ing of cr is tae in calc ium-def ic ientp l a n ts ( B u s h u e v a & S e m i k h a t o v a 1965).c) Mem branes and M odel Systems

I n v i e w o f t h e e v i d e n c e l i n k i n g c a l c i u m w i t h t h e s t a b i l i t y o f m e m b r a n o u ss tructures , w e sha l l con s ide r b r ie f ly the cu r r en t theor ies o f m em bra ne s t ruc turea n d p o s s i b l e r o le s t h a t c a l c iu m m a y p l a y .

U n t i l r e c e n t l y , t h i n k i n g a b o u t m e m b r a n e s t r u c t u r e a n d f u n c t i o n h a s b e e nd o m i n a t e d b y t h e b i m o l e c u l a r le a fl et c o n c e p t ( D a n i e l l i & D a v s o n 1 9 3 5 ) , w h i c hw a s d e v e l o p e d i n to t h e u n i t m e m b r a n e t h e o ry b y R o b e r t so n ( 1 9 5 9 ) . T h i s c o n -c e p t , w h i c h w a s a p p l i e d t o a l l m e m b r a n e s , p r o p o s e d a b i m o l e c u l a r l e a f l e t o fp h o s p h o l i p i d w h o s e n o n - p o l a r t a i l s w e r e d i r e c t e d i n w a r d p e r p e n d i c u l a r t o t h ep l a n e o f t h e m e m b r a n e . T h e p o l a r p h o s p h o l i p i d g r o u p s c o m p r i s e t h e e x t e r n a ls u r f a c e w h i c h w a s c o v e r e d b y a l a y e r o f p r o t e i n . T h i s t h e o r y h a s r e c e n t l y b e e nc ritic ally e x a m i n e d b y K o r n ( 1 9 6 6 ) , K a v a n a u ( 1 9 6 6 ) , a n d o t h e rs w h o h a v ef o u n d i t i n a d e q u a t e i n m a n y r e s p e c t s . A m o r e d y n a m i c c o n c e p t o f m e m b r a n es t r u c t u r e i s n o w a d v a n c e d a n d t h e i d e a o f a u n i v e r s a l u n i t m e m b r a n e i s s p e -c i f i c a l l y d e n i e d . I n s e v e r a l o f t h e s e s u g g e s t i o n s , t h e m e m b r a n e i s c o m p o s e d o fl i p o - p r o t e i n o r p r o t e i n g l o b u l e s e m b e d d e d i n a p r o t e i n m a t r i x o r s a n d w i c h e db e t w e e n p r o t e i n l a y e r s . T h e s e c o n c e p t s a r e s u p p o r t e d b y t h e e l e c t r o n - m i c r o s c o p i ce v i d e n c e o f S j/S s tr an d , C e d e r g r e n , a n d K a r l ss o n ( 1 9 6 4 ) a n d b y f r e e z e -e t c h i n gr ep li ca s o b s e r v e d b y B r a n t o n ( 1 9 6 6 ) . K a v a n a u i n p a r t ic u l a r s tr es se s t h e d y n a m i cr e q u i re m e n t o f t h e v a ri o u s fu n c t io n s p e r f o r m e d b y m e m b r a n e s a n d p r o p o s e st h a t t h e l i p i d " s p h e r o c y l i n d e r " o f t h e l e a f l e t i s c a p a b l e o f a l t e r n a t i n g b e t w e e na c y l i n d r i c a l a n d a d i s k f o r m . A s i m i l a r " c o r p u s c u l a r " t h e o r y o f t h e s t r u c t u r eo f b i o lo g i c al m e m b r a n e s h a s b e e n p r o p o s e d b y B e n s o n ( 1 9 6 6 ) .

A prec i se incorpora t ion of ca lc ium in to these s t ruc tures i s d i f f i cu l t a t p r esen t ,a l t h o u g h t h e r e i s c o n s i d e r a b l e e v i d e n c e o f c al ci um i n t e ra c t in g w i t h p h o s p h o l i p i d sa n d n e u t r a l l i p i d s in vi tro. S h a h a n d S c h u l m a n ( 1 9 6 7 ) d e m o n s t r a t e d t h a t t h eb i n d i n g o f C a + + t o m o n o l a y e r s o f l e c it h i n a n d s p h i n g o m y e l i n i nc re as es t h e s u r-f a c e p o t e n t i a l . T h e b i n d i n g i s g r e a t e r w i t h d i p a l m i t o y l l e c i th i n t h a n w i t h s p h i n -g o m y e l i n - - t h i s t h e y a t t r i b u t e t o s t e r i c i n t e r f e r e n c e f r o m t h e 3 - C h y d r o x y l o f t h el a t t e r . C a l c i u m h a s b e e n f o u n d t o r e d u c e t h e s u r f a c e p r e s s u r e o f p h o s p h o l i p i dm o n o l a y e r s a s c o m p a r e d w i t h K a n d N a . T h e p r e s e n c e o f 1 0 m M K a n d N ad o e s n o t a ff e c t t h is b i n d i n g b u t t h e C a is d i s p la c e d b y 1 0 0 m M o f t h e m o n o -v a l e n t i o n s ( R o j a s & T o b i a s 1 9 6 5 ) .T h e b i n d i n g o f C a + + w i t h p h o s p h a t i d y l s e ri n e ha s b e e n d e m o n s t ra t e d e v e ni n t h e p r e se n c e o f 1 4 5 m M N a o r K . B a n g h a m a n d P a p a h a d j o p o u l o s ( 1 9 6 6 )f o u n d a n a l t e r a ti o n i n t h e b i n d i n g o f t h e C a + + a t 1 m M f r o m a 1 : 1 r a ti o t o aC a + + n -I c o m p l e x a t h i g h e r c o n c e n t r at i o n . I n a s t u d y o f t h e p e r m e a b i l i t y o f p h o s -pha t idy l s e r ine l iqu id c rys ta l s to monova len t ions , ca lc ium a t above 1 mM was


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418 THE BOTANICALREVIEW

f ound t o s ubs t a n t i a l l y i nc r e a s e t he d i f f u s i on o f t he m onova l e n t i ons . Mg ha da s imi la r e f f ec t to C a but a t a h ighe r co ncent r a t ion .

A l t hough t he s e m ode l s y s t e m s do l i t t l e t o c l a r i f y t he p r opos e d r o l e o f c a l -c i um i n t he p l a s m a l e m m a , t he y a t l e a s t s how t he i n f l ue nc e o f c a l c i um on t hephys i c a l - c he m i c a l p r ope r t i e s o f t he p r oba b l e c ons t i t ue n t s o f t he m e m br a ne s .

NUCLEIC ACIDS AND CHROMOSOMESA n i m por t a n t f unc t i on o f c a l c i um i n m a i n t a i n i ng t he s t r uc t u r e o f c h r om o-

s o m e s m a y b e i m p l i e d f r o m f i n d in g s o f S o ro k i n a n d S o m m e r ( 1 9 2 9 ) . T h ea b i l i t y o f D N A t o f o r m c om pl e xe s w i t h c a l c i um m a y a l s o a c c oun t f o r t he c y t o -l o g ic a l o b s e rv a t io n s o f S t ef fe n s en ( 1 9 5 8 ) , H y d e a n d P a li v a l ( 1 9 5 8 ) , a n d o t h e r s( s e e H e w i t t 1 96 3 ) , w h o f ou nd a c l o s e a ss oc ia ti on be t w e e n c h r om os om a l abe r ra -t i on a nd c a l c i um de f i c i e nc y . T r e a t m e n t o f c h r om os om e s w i t h E D T A i n a l owi on i c s t r e ng t h m e d i um o r c i t r a t e i n w a t e r c a us e s d i s pe r s i on pos s i b l y due t o t hed i s s o l u t i on o f i on i c b r i dge s .

T h e f o r m a t i o n o f c o m p o u n d s b e t w e e n D N A a n d m e t a l i o n s i n c l u d i n g c a l -c i u m h a s b e e n d e s cr ib e d b y K i r b y ( 1 9 5 7 ) a n d m o r e r ec e n tl y b y C h e n g ( 1 9 6 5 ) .T he r e i s a l s o a m p l e e v i de nc e o f t he a b i l i t y o f c a l c i um t o f o r m a s pe c i f i c c om -p l ex w i t h A T P ( E p st e in & W h i t t a m 1 9 6 6 ) . A l le n d e e t a l . ( 1 9 6 5 ) h a v e re -c e n t l y p r opos e d t ha t c a l c i um o r a no t he r d i va l e n t c a t i on i s i nvo l ve d i n t he f o r m a -t i on o f t h r e ony l - s R N A . T he e v i de nc e doe s no t t e l l u s w he t he r t he i on c on f e r sa spec i f ic conf igura t ion on the sRNA or on the enzyme.

A s m e n t i one d , t he e xpos u r e o f r oo t s t o E D T A c a us e s t he r e l e a s e o f nuc l e o -t i de s . F u r t he r m or e , t he r e a r e i nd i c a t i ons f r om t he m i t oc hondr i a l obs e r va t i on o fG r u n w a l d ( 1 9 6 6 ) a n d fr o m F o o t e a n d H a n s o n 's ( 1 9 6 4 ) w o r k th a t c a lc iu ma nd nuc l e i c a c i d s a r e i nvo l ve d i n m e m br a ne t r a ns po r t . I t i s i n t e r e s t i ng t ha tt he p r o t op l a s t s p r e pa r e d f r om A v e n a c o l eo p t il e s ( R u e s i n k & T h i m a n n 1 9 6 5 ) ,a l though s tab le to va r ious pro te inases and l ipases , a r e immedia te ly lysed byR N A a s e . I n v i e w o f t he s e obs e r va t i ons i t i s no t i nc onc e i va b l e t ha t t he f unc t i onso f c a l c i um i n nuc l e i c a c i d m e t a bo l i s m a nd i n m e m br a ne pe r m e a b i l i t y a r e s i m i l a r .

ENZYME ACTIVITYT he va r i ous r e po r t s o f c a l c i um i nvo l ve m e n t i n e nz ym e a c t i v i t y a r e no t e d

i n T a b l e I I , a l t hough w e c a nno t c l a i m t ha t t h i s t a bu l a t i on i s e xha us t i ve . P e r -ha ps t he m os t i n t e r e s t i ng e nz ym e s ys t e m s i n w h i c h c a l c i um i s w e l l know n t o bei nvo l ve d a r e a - a m y l a s e a nd A T P a s e . H ow e ve r , i n a n i m a l s t he i n t e r a c t i ons o ft h e i o n w i t h tr y p s in o g e n ( S m i t h 1 9 5 1 ) a n d i n p r o t h r o m b i n f o r m a t i o n ( C o l e ,K o p p e l , & O l w i n 1 9 6 4 ) a r e w e l l d o c u m e n t e d .

A va r i e t y o f A T P a s e s ha ve be e n de s c r i be d f r om a n i m a l a nd p l a n t s ou r c e sw h i c h ha ve ve r y d i f f e r e n t r e s pons e s t o c a l c i um . T he c l a s s i c a l m a m m a l i a n e nz ym ei s a c t iva t e d by N a p l u s K a nd i nh i b i t e d by c a l ci um ( s e e S kou 1 96 5 ) , w h e r e a st he p l a n t c e l l - w a l l e nz ym e r e qu i r e s C a o r Mg f o r a c t i v i t y ( D odds & E l l i s 1966 ) .C a l c i um - o r m a gne s i um - s t i m u l a t e d A T P a s e s ha ve a l s o be e n de s c r i be d i n s yna p t i cve s ic l e s o f ra t b r a i n ( G e r m a i n & P r ou l x 19 65 ) a nd i n m yo f ib r i ls ( M a r yya m a &


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FUNCTION OF CALCIUMIN PLANTS

T A B L E I ISOME ENZYMATIC ACTIVITIES INF LUEN CED BY CA + +

4 1 9

INFLUENCE.ENZYME SOURCE REF.OF CA ++

or-amylase barley aleurones q- C hr is p ee ls& Varner 1967Baci l lus sub l i l is + Imanishi 1966Cohn emend.PrazmowskiB. subt i l is + Ta kag i & Isemura 1965

AT Pase plant cell walls + Dodds & Ellis 1966d r a c h i s h y p o g a e a - - Brown et al. 1965rat brain "-b G erm ain & Prou lx 1965many anim al sources -- Skou 1965phospholipase cabbage leaf + Davidson & Long 1958carrot q- Einset & Cl ark e 1958

esterase hog liver + K eay & Crook 1965p yru va te k i n a s e m a m m a l i a n - - M i ld va n & C o h n 1965threonyl-,RNA Escherichia coi l q- Allende et a l . 1965synthetase (Migula) Castellani &Chalmerspo lyga la c tu r on ic - Er~winia carotovora q- Starr & M oran 1962(Jones) Holland.

transeleminase B . p o l y m y x a(Prazmowski) Migulaglucose-6-phosphate B. subt i l is q- M arquet 1964dehydrogenasearginine kinase }lecithinasepectin polygalact-uronidase various sourcesadenyl kinaseapyrase

n - McE lroy & Nason 1954

I sh ika wa - Ka t suk i 196 6) . How e ve r , t he e nzyme f r om A r a c h i s h y p o g a e a is in-h ib i te d by C a + + ( B r ow n e t a l . 1 9 6 5 ) .C a lcium i s f oun d a s a c ompon e n t o f a - a my la se f r om a va r ie ty o f a n ima l

and bac te r ia l sources and i s probably requi red for the s tab i l iza t ion of the en-zymica l ly ac t ive conf igura t ion ( Im anish i 1966, Tak agi & Isem ura 1 96 5) . Re-cent ly Chr ispee ls and V arner (1 96 7) , in the i r wo rk on g ibbere l l ic -ac id-enhancedsynthesis o f ~-amylase in bar ley a leurones, hav e show n t hat 20 m M C a ++ isr e qu i r ed f o r t he a c t iv i ty o f t he e nz yme in h igh e r p l an t s.The sugge s t e d invo lve me n t o f c a l c ium in the ma in te na nc e o f me mbr a nef unc t ion a nd c y top la smic o r ga n iz a t ion wou ld o f c ou r se p r o f ound ly in f lue nc e agrea t va r ie ty of enzymic ac tivi ties . I t has a lso been sugges ted by H ew it t (1 96 3)tha t an appropr ia te ra t io of K to Ca i s r equi red for normal me tabol i sm. Am or e e l a bo r at e hypo the s is ha s be e n adva nc e d by D e K oc k ( 19 64 ) wh o imp l i -


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420 THE BOTANICAL REVIEW

cates the rat io of K/Ca and P in the control of the ci t r ic acid cycle and in deter-m inin g the level of ci trate, m alate, and oxalate.

C O N C L U D I N G D I S C U S S I O NThe proposed role of calcium in protect ing t issue, part icularly roots , againsttoxic ionic env ironm ents raises some interest ing quest ions. Even w ithin com mer-cial ly important plants and certainly within the higher plants , there exists a vastrange of ionic habi tats in which plant roots develop. A strain of A g r o s t i s t e n u i s

Sibth . w as fou nd w el l adapted to grow th in a soi l conta in ing 1 per cent l ead and0.03 per cent zinc but low in calcium and phosphate. However, i t did not growwell in a garden soi l (Bradshaw 1952). Remarkable differences in resis tance tocopper, nickel, lead, and zinc have been detected in A g r o s t i s species and thesecould be re la ted to the or ig inal ecological habi ta t (Gregory & Bradshaw 1965) .Th e results obtained by W ilkin s (19 60 ) on the lead tolerance of species ofFestuca ovina L . imply a genet ica l ly determined " to lerance factor . " A compa-rable pro blem i s presented by the adapta t ion of certain p lan ts to a h igh H + andA1 +++ ion e nviro nm ent and others to calcifuge condi t ions. Clarkson (19 65 )studied the growth response and calcium uptake by five A g r o s t i s species andfound that the ca lc iphobe species , A. se tacea Curt . , responded maximal ly to0.125 mM Ca ++ whereas the calcicole species show ed fur the r response even ov er1 mM . Th ese results we re taken to indicate that the calcium uptak e m echan ismof th e calciphobe h ad a low er Ks.Since calcium is thought to play an important role in the s tabi l izat ion ofthe p lasmalem ma un der adverse condi t ions , th i s poses the ques t ion of the mech-anism by which these p lants adapt to toxic envi ronments . Unfor tunate ly , ourknowledge of the composi t ion of p lan t membranes i s very sparse (Thompson1965) and to date there has been no at tempt to correlate ecological adaptat ionwi th membrane composi t ion and s t ructure . However , th i s would appear impor-tan t for both sc ien t i f ic and agronomic reasons . In v iew of our profound igno-rance of th e chemical s t ructure of the p lasma lemm a one can only speculate abou tthe role of calcium in the structure.The role of calcium in cel l membranes is most l ikely related to i ts binding tophosphol ip ids , in v iew of the in v i tro interactions, alth ou gh there is l i t t le directin v ivo evidence. As i t has been shown in a-amylase and lact ic dehydrogenasethat calcium can al ter the al losteric s t ructure of protein, this type of interact ioncannot be discounted. An assessment of the role of the proposed Ca-ribonucleo-protein in ion uptake is diff icul t . The hypothesis is based on the s imilar effectsof EDTA, RNAase, and u l t rav io le t l igh t i r rad ia t ion to which the sens i t iv i ty ofprotoplasts to RNAase, the associat ion of Ca45 wi th mi tochondr ia , and thechemical complexes o f Ca w ith nucleic acid m ay be add ed as add i t ional evidence.The use of EDTA causes such drast ic effects that some doubt is cast on theval id i ty of deduct ions ma de f rom the resu lt . Fur therm ore , Foote and H ans on(19 64 ) , a l thoug h expla in ing the rap id in i ti a l in flux of K + as due to increasedperm eabi l i ty and an ex cess of vacated cytoplasmic excha nge sites, did no t f ind acom parable influx of Ca ++ . H ow ever, one eagerly awaits chemical evidence forthe presence or absence of R N A in me m bran e fractions.


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F U N C T I O N O F C A LC IU M I N P L A N T S 4 2 1The proposed funct ion of ca lc ium in membranes i s not only re la ted to thep l asmalemma bu t may a l so accoun t fo r t he b reakdown o f mi tochondr i a andendoplasmic ret iculum and cytoplasmic dis integrat ion observed in calcium-

deficient t issue. The beneficial effect of calcium on cytoplasm was cited over 40years ago by Tru e (19 14 ) an d has s ince appe ared in several guises . Ho w ever,we are s t i l l some way f rom a b iochemical or molecular unders tanding of th i ss tatement . I t may be interest ing to note a recent approach to this problem byCerbon (1965). The 1.3 p.p.m, nuclear magnet ic resonance s ignal relat ive to ate t ramethyl -s i lane s tandard w as fo un d to be re la ted in Nocardia asteroides(Ep ping er) Blanchard to ce l lu lar l ip il s. G row ing cell s and those exposed toisotonic media gave no signal, but one was observed in cells exposed to hypo-or hyper tonic condi t ions . The presence of Ca or Mg induced the s ignal underisotonic conditions, ind icatin g an a lteration in the l ipid phase.In addi t ion to the influence of calcium on organizat ion and possibly indis-t inguishable from i t , the ion may be required specif ical ly for the act ivat ion ofcertain enzymes. Possibly the most interest ing of these is the ATPase found incell-wall fractions. I t is tem ptin g to cons ider that the V iers effects m ay be ex-p la ined by the ac t ivation of the A TPase by Ca or M g.

D esp ite the lon gev ity of the calcium-pectate theo ry, certain difficulties re-main . The m ajor problem i s the ex t remely low calc ium content (Tab le I ) nowfou nd in he al thy roots . P re l iminary studies have taken p lace on the d i s t r ibut ionof ca lc ium wi th in ce l l s and perhaps fur ther inves t igat ion wi l l p rovide more de-ta i led inform at ion .

Although there is evidence point ing to a calcium involvement in three areasof metaboli sm, no sa t i s fy ing explanat ion can be advanced for the impor tance ofCa ++ and o ther d ivalent ions in root e longat ion .I t has been the purpose of th is arti cle to br ing together inform at ion re la t ingto the funct ion of calcium scat tered throughout the biological and biochemicall i terature. In this s tudy the evidence for the possible role of calcium in mem-

branes, chromosomes, cell walls, and in the activation of certain enzymes hasbeen most compel l ing and these topics would appear to be profi table l ines forfu ture s tudy.L I T E R A T U R E C I T E D

ALLENDE, J . E . , G. MORA , M . GATICA, & C. C. ALLENDE. 1965. T h e role of m etal ionsin t h e f o r m a t io n o f t h r e o n y l - s o lu b l e RNA f r o m th r e o n y l a d e n y l a t e - e n z y m e c o m -p lex . Jour . Bio l . Chem . 240 : PC 3229-3232 .BAMFORD~ R. 1931 . Ch anges in roo t t ips o f wh ea t and corn g row n in nu t r ien t so lu t ionsdef ic ien t in ca lc ium . Bu l l . To r re y Bot . Club 58 : 149-178 .BANGHA M, A . D ., & D . PAPAHADJOPOULO$.1966 . Biop hys ica l p rop er t ie s o f pho spho l ip ids .I . I n t e r a c t i o n o f p h o s p h a t i d y l s e ri n e m o n o la y e r s w i th m e t a l i on s. B io c h im .Biophys. Acta 126: 181-184.BATEMAN, D. F ., & R. D. LUMSDEN. 1965 . Re la t ion o f ca lc iu m con ten t and na tu re o f th epec t ic subs tances in bean hypoco ty ls o f d i f fe ren t ages to suscep t ib i l i ty to aniso la te o f Rhizoctonia solani. Phytopa tho logy 55 : 734-738 .BEESON, K. C. 1941 . M ine ra l compo s i t ion o f c rops. U.S . Dep . A gr . M isc . Pub l . 369 , pp .93-117.BENNET-CLARKE, J . A . 19 56 . Sa l t a ccum ula t ion and the m ode o f ac t ion o f aux in . A p re -


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4 2 2 T HE BOT ANICAL RE VIE Wl im in a r y h y p o th e s i s . In : " T h e C h e m i s t ry a n d M o d e o f A c t i o n o f P l a n t G r o w t hSubs tances ," ed 9 b y R . L . W a in & F . W ig h tm a n , Bu t t e r ~ o r th S c i e n ti f ic P u b l . ,London, pp. 284-291.

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426 THE BOTANICAL REVIEWSKOU, J . C . 1965 9 E n z y m a t i c b a s i s f o r a c t i v e t r a n s p o r t o f N a + a n d K + a c r o s s c e l lm e m b r a n e 9 P h y s i o l 9 R e v

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