Concentration of zinc in culture solution (mg/1)
Detail 0.05 0.1 0.125 0.15 0.175 0.2 0 . 4 Weight of seed per plant, oven dry
(gm) 0 .04 0.86 2 .5 4.0 3 .9 3 . 8 3 . 2 Number of seeds per plant 6 5 150 382 650 632 657 577 Number of mature burrs per plant 3 49 120 199 198 222 195 Number of seeds per mature burr 2. 2 3.1 3 2 3.3 3. 2 3 . 0 3.0 Weight of seed per mature burr (mg) 13 18 21 20 19 17 16 Mean weight per seed, oven dry
(mg) 6. 1 5 . 8 6. .6 6.2 6. 1 5.7 5.5 Percentage of inflorescences which
produced mature burrs 1. 8 23 37 33 37 39 37
α From Riceman and Jones (441 ).
content of inflorescence and seed as a proportion of the total zinc in t h e plants, especially over the period between 155 a n d 189 days w h e n inflorescence growth reached m a x i m u m development (cf. Fig. 2 2 in ref. 4 4 1 ) . T h e r e appeared to be a translocation of zinc from leaves to flowers at 189 days w h e n l u x u r y a m o u n t s w e r e provided (0.2 p p m ) . On t h e other h a n d n u m b e r s of leaves a n d leaf primordia decreased sharply over t h e same range.
2. E S S E N T I A L N U T R I E N T E L E M E N T S F O R P L A N T S 233 b. Anatomical and histological effects. Histological studies on zinc deficiency m a d e b y Reed ( 4 2 7 - 4 2 9 ) , Reed a n d D u f r e n o y ( 4 3 4 - 4 3 6 ) , Eltinge a n d Reed ( 1 3 2 ) , Carlton ( 6 4 ) , a n d M i l l i k a n (357) showed several changes in the cells. T h e palisade a n d " s p o n g y " mesophyll tissues i n leaves of tomato, peach, apricot, a n d w a l n u t w e r e a b n o r m a l l y compact a n d t h e usual intercellular spaces w e r e practically absent, a condition suggesting delayed differentiation. I n tomato the n u m b e r s of leaf palisade cells were decreased; t h e y w e r e three to four times the
FIG. 31. Effects of zinc nutrition on the growth of roots of tomato {Ly copersicon esculentum) plants. Root tips grown in hanging drops. (A) Control root tip, 24 hours; X37. (B) Exfoliated cell from (A) showing large starch grains; X360. (C) Exfoliated cell from (D) showing deposits of tannin in vacuole; X360. (D) Zinc-deficient root tip after 24 hours; X37. From Eltinge and Reed (132).
n o r m a l length a n d about double t h e n o r m a l width, i.e., twelve to sixteen times greater i n volume. Plastids w e r e decreased in size a n d n u m b e r s . T h e mesophyll cells tended to be atrophied. I n primordial leaves of orange, palisade cells r e m a i n e d undifferentiated a n d poly-gonal instead of becoming rhomboidal. Plastids r e m a i n e d primordial a n d undifferentiated. D u r i n g the expansion of zinc-deficient leaves t h e palisade cells enlarged a b n o r m a l l y , b u t occasionally cells divided trans-versely or longitudinally a n d irregular cell sizes resulted.
T h e root tips of zinc-deficient tomato plants (Fig. 31) showed a series of t h r e e to six swellings at 1 or 2 m m intervals behind t h e apex ( 1 3 2 ) ,
C D FIG. 32. Effects of zinc deficiency on growth of apricot (Prunus armeniaca),
orange (Citrus aurantium), and walnut tissues.
(A) Portion of a transverse section of an apricot leaf affected with zinc de-ficiency in which the perivascular cells were replete with masses of catechol. In the immediate vicinity of the bundle (x) the catechol masses formed a continuous phase enmeshing a number of vacuoles (t). Other cells contained spherical masses of catechol (c) enveloped by precipitation membranes and may be considered auto-complex coacervates. The plastids (pi) showed abnormalities. (B) Cells from the apical region of a vegetative shoot of an affected apricot tree. The catechol material in some vacuoles (v) condensed into globular masses (/); in others it formed an aggregate (c) enveloped by a precipitation membrane. Nucleus (n); mitochondria
(m). (C) Coacervated material demonstrated by the molybdenic reagent in hypo-plastic cells from root of an orange seedling which had grown seven months in a nutrient solution without zinc, (am) amyloplast; (c) coacervates exhibiting
vacuo-2 . E S S E N T I A L N U T R I E N T E L E M E N T S F O R P L A N T S vacuo-2 3 5
lated envelopes of phospholipid material; (m) mitochondria (frequently agglu-tinated); (n) elongated nucleus. (D) Portion of a transverse section of a walnut
(Juglans hindsii) leaflet, dwarfed and chlorotic by "little leaf." The hyper-trophied palisade cells had a definite polarization of contents, indicated by the posi-tion of vacuolar precipitates (t) at one, and nuclei (n) and plastids (pi) at the other, pole. The perivascular cells contained globulal masses (c) of catechol. Gum (g) and necrotic material (nec) occurred in intercellular spaces. From Reed and Dufrenoy (436).
a n d these w e r e associated w i t h t h e bulk of root h a i r production. Root hairs w e r e often crooked. T a n n i n s , calcium oxalate crystals, a n d oil globules appeared in root tip cells, b u t starch grains w e r e rare. All cells, including those in t h e meristematic zone, w e r e m u c h enlarged p r e m a -turely. I n tissues showing external symptoms, t h e meristematic cells w e r e scarcely recognizable as such. A r r a n g e m e n t w a s irregular w i t h n u m e r o u s intercellular spaces. M a t u r e cells w e r e isodiametric instead of columnar, a n d differentiation w a s suppressed. M e r i s t e m a t i c cells contained a b n o r m a l l y elongated a n d flattened vacuoles. Nucleoli w e r e smaller t h a n n o r m a l . Mitochondrial particles w e r e extra elongated a n d spirally distorted instead of being rod shaped. T h e effects of cell e n l a r g e m e n t a n d decreased differentiation in zinc-deficient tomato roots a n d stems are discussed b y Carlton (cf. Figs. 7 A a n d D a n d 8A a n d D of ref. 6 4 ) .
T h e most pronounced changes however, w e r e observed in contents of individual cells (Fig. 3 2 , Α - D ) . Plastids in green areas of mottled avocado leaves w e r e often n o r m a l w h e r e a s in the chlorotic areas chloro-plasts became agglutinated a n d segregated into groups at t h e i n n e r ends of the cells. Plastids became vacuolated a n d t a n n i n globules appeared. I n apricot plastids ( 4 3 6 ) , globules of lipid substances appeared in the stroma. T h i s w a s also observed in tomato chloroplasts. Plastids became vacuolated a n d t h e n s h r a n k to a fusiform outline; t h e y h a d increased capacity for staining w i t h acid fuchsin a n d h a d a decreased starch con-tent prior to disintegrating or dissolving b y a process resembling lysis.
I n m a i z e ( 4 2 7 ) t h e plastids w e r e m u c h fewer, clumped together, a n d vacuolated; t h e nucleus migrated to one end of the cell. Plastids devoid of starch w e r e m o r e prone to this behavior. It w a s suggested t h a t proc-esses of disintegration w e r e hastened b y stronger illumination. T h i s would be consistent w i t h the k n o w n effects of season on the incidence of zinc deficiency symptoms described below. Nuclei w e r e often m o r e resistant to b r e a k d o w n t h a n plastids. I n general, vacuolation a n d dis-integration of plastids w e r e t h e most consistent effects, but in m a n y plants phenolic compounds a n d oil droplets also accumulated. A t r o p h y of t h e vascular system occurred in tomato. T a n n i n s w e r e present in
apricot, peach, w a l n u t , avocado, orange (434, 4 3 5 ) , a n d b u c k w h e a t (436) a n d absent from sunflower, corn, a n d squash. T h i s distinction w a s related to t h e relative extent to w h i c h zinc deficiency caused
"little leaf" effects i n t h e tannin-containing group. Calcium oxalate crystals appeared i n tomato a n d orange leaves. Semipermeable prop-erties of cell m e m b r a n e s to toluidine blue a n d n e u t r a l r e d w e r e lost, a n d m a t e r i a l staining w i t h h e m a t o x y l i n collected in intercellular spaces of w a l n u t leaves ( 4 2 7 ) . This is consistent w i t h t h e observed exudations of cell contents from leaves of zinc-deficient plants.
I n meristematic buds of zinc-deficient apricot t h e cells showed in-creased contents of t a n n i n of two sorts, differentiated b y staining capac-ity for m e t h y l green. Vacuolation w a s p r e m a t u r e a n d cell contents w e r e often "polarized" to one end. Nuclei carried several small spher-ical bodies. Cell division appeared to b e inhibited a n d cell expansion occurred p r e m a t u r e l y . Plastid differentiation w a s delayed. Polarization of cell contents, with arrested development of plastids, w a s character-istic i n t h e w a l n u t ( 4 2 9 ) . Reed a n d D u f r e n o y (436) showed t h a t w h e r e t a n n i n s accumulated on spherical droplets or complex coacervates t h e y comprised catechol t a n n i n s surrounded b y a phospholipid m e m b r a n e . T h e s e appeared to h a v e been produced v e r y rapidly, as judged b y the occasional inclusion i n t h e m of t h e cell fluids (Fig. 3 2 ) .
Histological changes i n flax, lucerne, a n d s u b t e r r a n e a n clover have been studied b y M i l l i k a n ( 3 5 5 - 3 5 7 ) . I n flax two types of necrosis occur. Bronzing of t h e leaves w a s due to collapse of small groups of cells of t h e u p p e r epidermis. U n d e r l y i n g cells sometimes became necrotic later. Necrosis of t h e leaf stalk a n d leaf base w a s associated w i t h col-lapse a n d necrosis of p a r e n c h y m a cells of t h e petiole a n d preceded a n y ab-n o r m a l i t y i ab-n t h e epidermis. T h e vascular tissues w e r e t h e least affected.
Bronzing of stem tips w a s related to necrosis of t h e cortex p a r e n c h y m a followed b y epidermal necrosis a n d , last of all, necrosis i n t h e vascular tissues. Dieback of t h e stem apex w a s associated w i t h necrosis of cells just behind t h e apical dome, a n d it occurred i n d e p e n d e n t l y of t h e necro-sis i n leaves or petioles. T a n n i n s w e r e detected i n cells of zinc-deficient tissues.
"Little leaf" effects i n s u b t e r r a n e a n clover w e r e associated w i t h thick-ening of t h e leaves. T h i s w a s d u e to proliferation of palisade cells.
T h e s e either divided transversely to produce several layers of irregular, b u t still r o u g h l y columnar, cells, or, i n regions w h e r e n o t thickened, t h e cells became rhomboidal a n d w e r e not differentiated from t h e lower meso-p h y l l cells. Cells of t h e u meso-p meso-p e r emeso-pidermis meso-produced irregular meso-projections or collapsed completely into necrotic areas. Plastids b e c a m e elongated a n d lysis caused their disappearance. T h i s w a s associated w i t h t h e
2 . E S S E N T I A L N U T R I E N T E L E M E N T S F O R P L A N T S 2 3 7
irregular chlorosis a n d red-purple tinting of foliage. Cell necrosis i n t h e
"little leaf" tissues occurred first i n t h e mesophyll. T h e first-formed leaves of zinc-deficient plants showed somewhat different behavior.
Epidermal, palisade, a n d mesophyll cells r e m a i n e d n o r m a l i n shape, but m a n y cells w e r e devoid of plastids. Cells of all tissues, especially those of the palisade, contained spherical coacervates w i t h a phospho-lipid m e m b r a n e . T h e s e preceded necrosis of the cells. T h e oldest leaflets of n o r m a l size on zinc-deficient plants showed necrosis in u n d e r l y i n g tissues. M a n y of the effects described for s u b t e r r a n e a n clover w e r e anal-ogous to those observed in citrus, tomato, a n d m a i z e noted above. I n lucerne t h e palisade cell contents disintegrated a n d the cells subdivided.
Bronze necrotic spots developed in t h e u p p e r epidermis.
T h e abnormalities in ovule development of peas w e r e observed b y Reed ( 4 3 0 , 4 3 1 ) to occur at zinc levels above those adequate for n o r m a l vegetative growth. Absence of micropile or i n t e g u m e n t or of e m b r y o sac nuclei w e r e observed. P r e m a t u r e vacuolation was common, a n d lysis of cell contents also occurred. Ovules tended to be anatropous instead of campylotropous. T h e effects of zinc on the flower a n d seed production in s u b t e r r a n e a n clover h a v e already been noted ( 4 4 1 ) .
c. Relation to auxin activity and light. T h e zinc status of some plants probably affects t h e activity of auxins, in particular t h a t of indoleacetic acid. T h e action m a y involve two i n d e p e n d e n t effects.
Skoog ( 4 8 1 ) found t h a t zinc deficiency greatly decreased the a m o u n t s of indoleacetic acid obtained b y diffusion, or ether extractions, from stem apices or leaves of tomato. T h e a m o u n t s obtained from deficient tissues w e r e between one-tenth a n d one-fiftieth of the n o r m a l . T h e effects of zinc deficiency on a u x i n content preceded those on g r o w t h or t h e visual symptoms, in contrast to effects of copper or m a n g a n e s e deficiencies, w h e r e decreased a u x i n occurred o n l y in advanced stages of deficiency.
A u x i n concentrations increased 2 days after restoration of t h e zinc supply, b u t r e n e w e d growth was not a p p a r e n t until several days later.
A u x i n was inactivated m o r e r a p i d l y in contact w i t h zinc-deficient t h a n w i t h n o r m a l tissues. Blue light, w h i c h n o r m a l l y inhibits a u x i n responses, h a d a greater proportional effect o n zinc-deficient t h a n on n o r m a l tissues in this respect. Red light, w h i c h favors elongation, u n d e r a u x i n action permitted greater elongation of zinc-deficient tissues t h a n occurred w i t h n o r m a l light. A u x i n inactivation b y zinc-deficient tis-sues was decreased b y red as compared w i t h n o r m a l light, a n d in this respect red light decreased t h e effects of zinc deficiency. T h e s e relation-ships m a y h a v e a bearing on t h e increased zinc r e q u i r e m e n t s observed in long days a n d seasons of m a x i m u m light intensity. It was also found t h a t peroxidase activity, as revealed b y oxidation of benzidine in the
presence of h y d r o g e n peroxide, w a s greater in zinc-deficient t h a n in n o r m a l tissues. Skoog reported w o r k b y Bean w h i c h h a d shown t h a t blue light increased t h e peroxidase activity of tissues a n d also accel-erated t h e breakdown of auxin. Increased peroxidase activity m a y oc-cur i n other plants w h e n t h e y a r e deficient i n zinc (209, 3 7 1 ) . It w a s concluded b y Skoog (481) t h a t zinc is responsible for m a i n t a i n i n g a u x i n in a n active state, b u t not for t h e synthesis of auxin. It seems possible, however, t h a t a u x i n content decreased i n zinc-deficient tis-sues because inactivation was m o r e rapid, owing to increased auxin-oxidase or perauxin-oxidase activity as elucidated b y W a y g o o d a n d his col-laborators (313, 570, 571) a n d b y K e n t e n ( 2 6 4 ) . T h e frequent obser-vation t h a t t a n n i n s accumulate i n zinc-deficient tissues would also support t h e idea of increased oxidase activity.
T h e effects of light a n d season on zinc deficiency h a v e been noted b y several observers w i t h some contradictory conclusions. H o a g l a n d (228) reported t h a t visible symptoms of zinc deficiency a r e restricted to t h e s u m m e r i n California a n d are not observed i n t h e winter. Ferres ( 1 3 9 ) , however, found t h a t zinc deficiency in s u b t e r r a n e a n clover in South Australia is most severe d u r i n g t h e short-day period. O z a n n e (397) concluded t h a t both d a y length a n d light intensity are involved a n d t h a t light intensity effects m a y be the m o r e important. M e d i a n light inten-sities of 1400 foot-candles caused m a x i m u m immobilization of zinc in roots, a n d this intensity was similar to t h a t to w h i c h zinc-deficient clover w a s exposed i n t h e e x p e r i m e n t b y Ferres ( 1 3 9 ) .
Zinc m a y , however, directly affect a u x i n production, according to T s u i ( 5 3 1 ) , w h o concluded t h a t zinc w a s r e q u i r e d for synthesis, in tomato, of t r y p t o p h a n , w h i c h is a precursor of auxin. It m a y b e rel-evant t h a t N a s o n (370) found t h a t t h e production of a n e n z y m e in Escherichia coli, w h i c h converted indole a n d serine to t r y p t o p h a n , was dependent u p o n a supply of zinc. Z i n c m a y therefore h a v e two inde-p e n d e n t effects on a u x i n inde-production. Carlton (64) observed effects resembling t r e a t m e n t with auxins i n zinc-deficient tomato roots. Zinc also appears to increase t h e action of gibberellin on bean internodes, according to D a n c e r ( 9 0 ) .
d. Relation to nitrogen and phosphorus metabolism and enzyme ac-tivity. Z i n c deficiency caused great increases in amide a n d total a m i n o nitrogen compounds in tomato ( 4 1 6 ) . G l u t a m i n e was increased seven-fold a n d became t h e major soluble nitrogen compound. Asparagine w a s increased n e a r l y fiftyfold a n d was second in importance to glutamine. T h e r e w e r e also proportionally large increases in the levels of aspartic acid, lysine, histidine, arginine, serine, threonine, alanine, p h e n y l a l a n i n e , valine, leucine, a n d proline. However, some decreases
2 . E S S E N T I A L N U T R I E N T E L E M E N T S F O R P L A N T S 2 3 9
i n t h e concentrations of substances w e r e attributed to citrulline a n d e t h a n o l a m i n e ( T a b l e X V I I I ) . Earlier observations b y Steinberg ( 5 0 3 , 5 0 5 , 5 0 8 ) showed t h a t zinc deficiency i n c o m m o n w i t h most m i n e r a l deficiencies caused increases i n t h e total soluble nitrogen of tobacco plants. L a t e r data on a m i n o acids, from analyses b y Z a c h a r i a s a n d deficiency is striking; this suggests t h a t some well-known effects of zinc deficiency m a y be the result of the accumulation of specific a m i n o correla-tion between protein nitrogen a n d zinc retencorrela-tion.
Reed ( 4 3 2 ) observed t h a t zinc-deficient tomato h a d a h i g h e r con-centration of inorganic phosphate t h a n n o r m a l plants a n d also a high acid phosphatase activity, shown histochemically in vivo. This has also been confirmed i n leaf extracts, t h e data being expressed on a protein basis, b y H e w i t t a n d T a t h a m ( 2 2 5 ) for tomato g r o w n w i t h different sources of nitrogen. T h e effect is not specific, however, as it occurred also w i t h deficiencies of phosphorus, boron, a n d m o l y b -d e n u m a n -d of copper w h e n nitrogen w a s given as nitrite ( T a b l e X V ) . T h e association of zinc s u p p l y w i t h aldolase activity in clover ( 4 1 9 , 4 2 0 ) m i g h t account for effects of zinc on inorganic phosphorus status.
T h u s decreased aldolase would decrease t h e production of glyceralde-hyde-3-phosphate, a n d this i n t u r n could cause decreased phosphoryla-tion of adenosine diphosphate d u r i n g triosephosphate dehydrogenase action. Reed ( 4 3 2 ) reported decreased dehydrogenase activity i n zinc-deficient tomato tissues. Z i n c h a s also been identified as a probable constituent of several diphosphopyridine nucleotidependent de-hydrogenases i n yeast a n d a n i m a l tissues, a n d it m a y be similarly in-volved i n plants. Loustalot et al. ( 3 0 5 ) found t h a t zinc deficiency de-creased photosynthetic carbon dioxide u p t a k e b y t u n g trees. T h i s would be consistent w i t h other effects described above.
Zinc-deficient tomato plants ( 3 7 1 ) m a y h a v e g r e a t l y increased polyphenol oxidase a n d peroxidase activities, a n d these changes m i g h t
T A B L E X V
EFFECTS OF MICRONUTRIENT DEFICIENCIES ON ACID PHOSPHATASE ACTIVITY IN