Planta (BEE.) 75, 10~22 (1967)
The Localization of Enzymes in the Cotyledons of P i s u m a r v e n s e L. during Germination A. IV[. FLrN~ a n d D. L. SMITH Botany Department, Queen's University, Belfast, Northern Ireland l~eceived December 24, 1966 S u m m a r y . Enzyme activity is not uniformly distributed through the cotyledon of P i s u m arvense. Initially the peripheral region, certain scattered cells of the
storage tissue and the procambium show a high level of activity of succinic dehydrogenase, cytochrome oxidase, acid phosphatase and esterase. Activity of acid phosphatase declines sharply after the first day of germination; activity of the other enzymes declines after about three days. In the storage tissue, where activity is lower initially, it declines after about five days and is ccrrelated with the disappearance of the reserves. The pattern of alkaline phosphat~se activity is similar except that activity is lower in the procambium but increases in the sieve-elements during differentiation of the phloem. 5-nucleotidase and glucose-6-phosphatase activity is low throughout the cotyledon but it also increases to a significant level in the sieve-elements. Activity of starch synthesizing enzymes is high in the parenchymatous bundle sheath, where they may be involved in the pathway from lipids to soluble carbohydrates.
Introduction The aim of the present s t u d y was to investigate changes i n the dist r i b u t i o n a n d a c t i v i t y of various enzymes i n the cotyledon of P i s u m a r v e n s e L. during g e r m i n a t i o n a n d to correlate these changes with histological a n d histochemical changes recorded previously (SMITK a n d FLIr162162 1967). The enzymes i n v e s t i g a t e d were: the respiratory enzymes succinic dehydrogenase a n d cytochrome oxidase; the h y d r o l y t i c enzymes acid phosphatase, alkaline phosphatase, 5-nucleotidase, glucose-6-phosphatase a n d esterase; a n d starch synthesizing enzymes, which m a y include starch s y n t h e t a s e a n d starch phosphorylase. Materials and Methods Seeds of P . arvense were soaked in distilled water overnight, after which they were planted in moistened vermiculite and kept in a greenhouse at about 200 C. Material was harvested 0.5, 1, 1.5, 2, 3, 5, 7, 9 and 12 days from the commencement of soaking. Fresh sections were used for all the localization procedures, the method of J~SEN (1955) being followed. A cavity was made in a block of Paraplast wax by means of a hot scalpel. A cotyledon was pressed into the cavity and the block immediately cooled in cold water. Sections were cut at 50 ~ on a rotary mierotome. Thinner sections of cotyledons proved unsatisfactory because many of the large storage parenchyma cells lost their contents during manipulation of the sections or during subsequent processing.
Localization of Enzymes in Cotyledons
11
For the histochemical localization of enzyme activity the following procedures were used: 1. Suceinie Dehydrogenase. The procedure used was the tetrazolium method of SELIGlYIA~and R~TTENBERG(JE~sE~, 1962). Controls were incubated in a medium without substrate. 2. Cytochrome Oxidase. The nadi reaction of GoMoRI (J]~Ns]~, 1962) was used. In the controls, sections were pretreated with alcohol or incubated in a reaction mixture containing 0.005 M sodium azide. 3. Esterases. The indigogenic method of HOLT (1958) was used. This procedure was also carried out with the following variations: a) Naphthol AS-D acetate was used as an alternative substrate (BURSTOI~E, 1962). b) The cotyledons were fixed in formol-calcium before sectioning (BURsTONE, 1962). c) The sections were first squashed to ensure that all the cells were damaged. d) Sections of several thicknesses, up to 100 [z, were used. 4. Acid Phosphatase. A modification of Go~oRfs lead sulphide method was used (J]~ss~, 1962). Controls were incubated in a medium without substrate or in one containing 0.001 M sodium fluoride. 5. Alkaline Phosphatase. A modification of the Go~o~I cobalt sulphide method (J]~NsE~, 1962) was used. Controls were the same as for acid phosphatase. 6. 5-Nueleotidase. The cobalt sulphide method of AvErtS (1961) was adopted. Controls were the same as for acid phosphatase. 7. Glucose-6-Phosphatase. The lead sulphide method of Av]~Rsand G n I ~ (1959) was used. Controls were the same as for acid phosphatase. 8. Starch Synthesizing Enzymes. The method used was that of DYAR (1950), which localizes enzymes catalyzing the synthesis of starch from glucose- 1-phosphate.
Results 1. Succinic Dehydrogenase Succinic dehydrogenase is localized in small rod-like cytoplasmic particles less t h a n 1~ in length (Fig. 1). T h e y stain dark blue with the m e t h o d used. T h e y are presumed to be mitochondria. The enzyme is n o t u n i f o r m l y d i s t r i b u t e d t h r o u g h o u t the cotyledon; initially, there is a high level of a c t i v i t y in the hypodermis, the procamb i n m , the basal region a d j a c e n t to the petiole, a n d certain cells of the storage p a r e n c h y m a . The latter are of p a r t i c u l a r interest. T h e y are scattered t h r o u g h o u t the storage tissue (Fig. 2) b u t increase in frequency towards the periphery, often forming a n almost c o n t i n u o u s layer below the h y p o d e r m i s (Fig. 3). SMIT~ a n d FLIN~ (1967) showed t h a t certain cells of the cotyledon have a more opaque, g r a n u l a r cytoplasm a n d a relatively high cytoplasmic R N A c o n t e n t as shown b y azur B staining. These cells are a p p a r e n t l y identical with those h a v i n g a high level of a c t i v i t y of succinie dehydrogenase a n d of other enzymes as described below. There are two reasons for supposing this: a) their d i s t r i b u t i o n a n d frequency are comparable ; b) in both stained a n d u n s t a i n e d sections the cells are readily distinguishable u n d e r dark-field i l l u m i n a t i o n (SMIT~
12
A.M.
FLI~
and D. L. SMITR:
Figs. 1--3. Suceinic dehydrogenase, day 3. Fig. 1. Longitudinal section of a parenehyma cell of the bundle sheath. The enzyme activity is localized in cytoplasmic particles which are presumed to be mitochondria. • 1200. Fig. 2. Section of the central region of a cotyledon showing a high level of activity in only some of the storage cells. • 150. Fig. 3. Section of the peripheral region of the cotyledon showing high levels of activity in the epidermis, hypodermis and some of the storage cells. • 200
Localization of Enzymes in Cotyledons
13
and F L I ~ , 1967); in sections subjected to localization procedures only those cells so distinguishable showed heavy staining. Thus, initially, the activity of this enzyme is highest in the peripheral region of the cotyledon and falls off rapidly towards the centre, where it tends to be localized in scattered cells. The outer storage cells show some activity initially but there is a marked increase during the first two days of germination. After 3 to 5 days the peripheral cells lose much of their activity and there appears to be a degree of clumping of the mitochondria. After about 7 days these cells are no longer active. The activity in the storage cells remains high until the reserves disappear, when it falls off rapidly. I t finally disappears between the 9th and 12th days. During the differentiation of the vascular strands activity quickly disappears from the potential xylem but there is little or no change in the phloem or in the parenchyma of the bundle sheath. 2. Cytochrome Oxidase The nadi reaction for cytochrome oxidase localization stains sites of activity a bluish purple. The enzyme occurs in particles which are presumed to be mitochondria. The distribution of activity of this enzyme in the cotyledon is similar to t h a t of succiilic dehydrogenase in that initially the peripheral and basal regions, certain scattered cells of the storage tissue, and the procambium (Fig. 4) are much more active than the majority of the large storage cells. Enzyme activity in the latter increases during the first two days of germination. A striking feature of the localization of this enzyme is the reddish purple staining of globules of varying sizes. This staining is almost certainly not attributable to cytochrome oxidase activity since it occurs also in sections which have been incubated in a medium containing sodium azide. The large storage cells contain a few of these globules but the cells in the basal region are particularly rich in them. The cells near the periphery and the procambial cells contain m a n y smaller globules which~ however, are larger than mitochondria. A similar staining pattern is found in sections stained with Sudan I I I and it m a y therefore be concluded that these bodies are lipids. According to Jn~sE~ (1962) the staining of lipids in the nadi reaction is probably attributable to the non-enzymatic formation of indophenol blue from the nadi reagent, catalyzed by fat peroxides. 3. Esterases The sites of esterase activity stain a deep bluish green with the indigogenic method. The localization is very precise. The distribution
14
A.M. FLI~I~ and D. L. SMIT~:
of the enzyme appears to be mainly cytoplasmic and non-particulate. However. in some cells near the outside of the cotyledon the nuclei or the periphery of the nuclei may stain more intensely than the surrounding cytoplasm. I t is possible that this may be due to aggregation of small stained particles round the nucleus rather than to actual nuclear staining.
Fig. 4. Cytochrome oxidase, day 1. Longitudinal section of the procambium, where activity is much higher than in the surrounding storage cells. X 75 Fig. 5. Esterase, day 5. Section of the central region of the cotyledon, showing a high level of activity in some of the storage cells. • 150 The distribution of the enzyme through the cotyledon is similar to that of the respiratory enzymes (Fig. 5), though there is a more striking difference between cells of high activity and those of low activity. Only very slight staining is detectable in the bulk of the storage cells. I n view of the peculiar distribution of activity of this and other enzymes in the storage tissue it is essential to consider the possibility that the oeeurrenee of high levels of activity in only some of the cells is an artifact. The possibility exists that the pattern of enzyme distribution in this tissue could be interpreted as the result of variation in uptake of the substrates by the cells or as a wound response. The following
Localization of Enzymes in Cotyledons
15
evidence, which is based on a more detailed investigation of esterase activity, is opposed to this view: a) The pattern of enzyme activity is similar with both the substrates used. b) The pattern is similar in fixed and unfixed sections. c) The pattern is similar in sections which had been squashed before treatment. This tends to eliminate the possibility t h a t only undamaged cells are showing high activity. d) The proportion of cells showing high activity is more or less constant in sections of different thickness. H a d the effect been a wound response the proportion should have been lower in thicker sections, since the proportion of cut cells would have been reduced. This tends to eliminate the possibility that only damaged cells are showing high activity. e) Although it is difficult to determine conclusively whether a particular cell is intact or has been cut slightly during sectioning, both stained and unstained cells which appear to be intact can be found in sections. The intensity of staining in the peripheral cells decreases rapidly after 5 to 7 days. As the reserves of the storage cells become exhausted their staining intensity also decreases. Activity is still present in the centre of the cotyledon on the 9th day but is undetcctablc by the 12th day. In the vascular strands much of the activity is lost during the first 2 days, when the mature conducting elements are undergoing differentiation. The parenchyma of the bundle sheath remains highly active until a late stage and some activity is still present on the 12th day.
4. Acid Phosphatase Acid phosphatase was found to be localized in cytoplasmic particles (Fig. 6). These appeared to be considerably larger than the particles staining with the localization methods for the respiratory enzymes and presumed to be mitochondria. I t is possible, though unlikely, that these larger particles m a y be aggregates of smaller bodies formed during the processing of the sections. The peripheral cells and scattered cells of the storage tissue appear to be more active than the majority of the storage cells, but not markedly so. The activity is only apparent during the first 2 days of germination, and after the first day the particles are difficult to see. Initially, activity is fairly high in the vascular strands. I t disappears quickly from the differentiating xylem and possibly from the phloem but remains
16
A.M. F L I ~ and D. L. SMIT~:
Fig. 6. Acid phosphatase, day 0.5. Section of the outer storage tissue, showing the particulate distribution of the enzyme. • 300 Fig. 7. Alkaline phosphatase, day 2. Longitudinal section of ]?art of a vascular strand, showing the localization of enzyme activity in the sieve-elements of the phloem. • 580 Fig. 8. Glucose-6-phosphatase, day 3. Longitudinal section of a vascular strand, showing a high level of activity in the sieve-elements. • 580
Localization of E n z y m e s in C o t y l e d o n s
17
detectable for a few days in the parenchyma of the strands as weaklystaining particles.
5. Alkaline Phosphatase The localization of alkaline phosphatase and 5-nucleotidase is made more difficult by the non-enzymatic formation of cobalt sulphide within the cell walls. The nucleoli of some cells also stain deeply but this too appears to be non-enzymatic since it also occurs in the controls. However, the non-particulate, cytoplasmic localization of these enzymes appears to be genuine. Initially, the activity of alkaline phosphatase is at its highest in the peripheral region of the cotyledon. Activity in this region increases during the first 2 or 3 days of germination and subsequently a wave of activity progresses from the periphery towards the centre of the cotyledon. Activity in the outer storage parenchyma reaches its peak between days 3 and 5 and in the inner storage parenchyma between days 5 and 7. Thereafter, the cytoplasmic staining decreases as the reserves become depleted. Activity is also initially present, but only at a low level, in the procambium. As differentiation proceeds the phloem elements become conspicuously active (Fig. 7) but all activity is lost from the xylem. Some activity persists in the bundle sheath. 6. 5-Nucleotidase The activity of 5-nueleotidase is very low in all the cells of the cotyledon. Little change can be detected in the peripheral or storage regions during germination. A low level of activity is present initially in the procambium but during and after differentiation a rather higher level is detectable in the sieve elements of the phloem. 7. Glucose-6-Phosphatase This enzyme, like alkaline phosphatase and 5-nucleotidase, has a non-particulate, cytoplasmic distribution. No activity is detectable in the peripheral or storage regions of the cotyledon. The localization procedure used results in slight staining of the cell walls but as this occurs also in the controls it cannot be attributed to enzyme activity. Such wall staining would probably make difficult the detection of low levels of activity in the cytoplasm. Some activity is detectable in the procambium during the first day of germination and by the second day the sieve tubes of the phloem show a high level of activity (Fig. 8). This activity persists until about the seventh day, after which it declines and is nndetectable by the ninth. 2a Planta (Berl.), Bd. 75
18
A.M. FLINN and D. L. SM~Tg:
8. Starch Synthesizing Enzymes The method used here was t h a t devized by DYAR (1950) for the localization of phosphorylase. The tissue is supplied with glucose-lphosphate and subsequently stained with iodine-potassium iodide solution. New starch, formed during the localization procedure, has few branched chains and stains a reddish brown eolour. The specificity of this method for the localization of phosphory]ase is questionable since it has now been established t h a t in vivo starch is synthesised from uridine diphosphate glucose and adenosine diphosphate glucose by the action of starch synthetase (NOI~DIN and K m ~ w o o D , 1965). I t is likely t h a t this enzyme would also be localized b y the procedure used here, since adenosine diphosphate glucose is probably synthesized in vivo from glucose- 1-phosphate and ATP (ESPADA, 1962). The starch synthesizing enzymes are not demonstrably active in the majority of the cotyledon cells. However, the cells at the periphery of the procambium show a high aetiFig. 9. Starch synthesizing enzymes, v i t y and this persists in the parday 3. Section of a vascular strand, enchyma of the bundle sheath after showing enzyme activity in the paren- differentiation of the vascular strands chyma of the bundle sheath. The new, (Fig. 9). The hypodermis and some of small starch grains, formed during the localization procedure, appear paler the underlying storage cells appear than the much larger old grains. • 500 to have a very slight activity. Discussion Although the cells of the storage parenchyma of the cotyledon of Pisum appear morphologically similar under the light microscope recent work has shown t h a t they are not homogenous at the ultrastructural and physiological levels. I n P. sativum, BAI~ and MEI~CS,Ir (1966, 1) have shown t h a t in certain cells the protoplast breaks down during maturation and consequently the storage products, fragmented plastids and mitochondria are dispersed through a vesiculated cytoplasm. I n P. arvense, as described above, certain cells of the storage tissue have a higher cytoplasmic R N A content and a higher level of enzyme activity than the other cells. Although there is no direct evidence for it, it seems
Localization of Enzymes in Cotyledons
19
likely t h a t these cells are similar to the ones described by BAI~ and MERCE}~ (1966, I). In view of the high IINA content and high levels of enzyme activity it appears t h a t the cells must be specialized as sites of enzyme synthesis. I t is possible that they are concerned with the initiation of breakdown of the storage reserves in the cotyledon. Some of the enzymes, such as acid phosphatase and the esterases, must be synthesised during the maturation of the embryo since their activity is high from the beginning of germination. As in the case of the scattered cells of the storage tissue, other cells which have been shown to have a high level of cytoplasmic RNA (SMITIt and FLI~N, 1967) have now been shown to have a high level of enzyme activity. Thus the general pattern of enzyme activity in the cotyledon coincides with the pattern of high levels of RNA. The region showing the highest activity initially is the peripheral region, comprising the epidermis, hypodermis and underlying storage cells. I t has previously been shown (SMITH and FLINN,1967) t h a t this is also the region in which breakdown of the reserve starch and protein is initiated. The decrease in lgNA content of the cells coincides with the disappearance of the reserves during the second or third day of germination; enzyme activity in this region persists up to the fifth day and then declines rapidly. Although the majority of enzymes conform to the general tissue activity pattern already indicated, the different enzymes show interesting variations. Cytochrome oxidase and succinic dehydrogenase are enzymes generally associated with mitochondrial respiration. Both enzymes increase in activity in the storage cells during the first 3 days. Thus the m a x i m u m activity is reached as the reserves visibly begin to disappear. A fall in the activity of these enzymes is apparent in those cells which have lost all their reserves. Significantly, the cells of the phloem and the bundle sheath are the last to show a fall off in activity. BAIN and gm~CEl~ (1966, 2) have shown t h a t in P . sativum the mitochondria assume their normal functional morphology during the first few days of germination. Thus, the increase in activity of these enzymes is not only a function of synthesis but of organelle completion at the ultrastructura] level. The phosphatases - - acid phosphatase, alkaline phosphatase, 5-nueleotidase and glucose-6-phosphatase - - are hydrolytic enzymes under physiological conditions. Acid phosphatase was found to be localized in cytoplasmic particles apparently bigger than mitochondria. These disappeared rapidly in most cells, the exception being some of the cells of the vascular strands. The association of the enzyme with cytoplasmic particles is in agreement with previous observations (AVERS and KI~O, 2*
20
A.M. F L ~ and D. L. SMIT~:
1960; AvErs, 1961). GA~A~ (1965) has demonstrated t h a t a considerable period of incubation is necessary to demonstrate the presence of such particles in frozen sections of root tips of V i c i a . This implies t h a t the enzyme is present in an inactive form. I t therefore seems likely that the acid phosphatase in this form is present within a membrane. GAI~A~ and MArL~ (1966) produced evidence for the concept that self digestion of the cell contents occurs b y the release of hydrolytic enzymes, in particular acid phosphatase, from lysosomes. They showed t h a t this happens in the developing protoxylem elements in root tips of V i c i a . I t seems reasonable to conclude, on this evidence, t h a t in cotyledons of P . arvense the acid phosphatase is localized initially in lysosome-like particles and is then released during the early stages of germination to aid in some way in the mobilization of the storage reserves. I t must be borne in mind, however, that no organelles comparable to the lysosomes of animal cells have yet been identified in plant cells. The scheme suggested here is different from the one proposed b y P o v x (1963), where the protein bodies of T r i t i c u m embryos contain acid phosphatase in a form which becomes activated only at germination. I n P . arvense no evidence of acid phosphatase activity within the protein bodies was obtained. Alkaline phosphatase, 5-nueleotidase and glueose-6-phosphatase do not have a particulate distribution. There is a significant increase in activity of alkaline phosphatase throughout the cotyledon during the first few days of germination. The low activity of 5-nueleotidase in the cotyledons is perhaps surprising since there is probably a considerable turnover of I~NA in the storage tissue, related to enzyme synthesis. Of special interest is the increase in activity of all three enzymes in the sieve-elements during the differentiation of the phloem. As a high level of activity is retained until the storage reserves are exhausted it indicates a probable involvement of the enzymes in the transport of materials through the phloem. On evidence from other sources, it has been suggested t h a t phosphatase activity in phloem is associated with passage of sugars into the sieve-elements and it is possible also t h a t the processes of phosphorylation and dephosphorylation m a y be involved in the subsequent transport of sugars through the sieve-plates (Kt;~SA~OV, 1963). The localization procedure used for esterases probably reveals a range of these enzymes. The initial distribution is predominantly in the peripheral region and in the procambium, subsequently in the bundle sheath. These regions have a high lipid content and it is probable t h a t one of the functions of the csterases is to break down the lipids to their constituents. They might also be involved in the destruction of organelle membranes, including those of the protein bodies.
Localization of Enzymes in Cotyledons
21
Of the enzymes which are probably localized by the starch method of DYA~ (1950) starch synthetase is probably the one responsible for starch synthesis in vivo. I n vitro, phosphorylase can catalyze the synthesis of starch from glucose-l-phosphate but in vivo it is now considered to be an agent only of degradation (NoRDIN and KncKwoo]), 1965). Unlike most of the other enzymes investigated, these enzymes are relatively inactive in the peripheral region of the cotyledon but appear to be particularly active in the bundle sheath. Their function here is not entirely clear, but they also might be connected with lipid metabolism. BEvy]ms (1961) has shown that seeds undergo the glyoxylate cycle for net synthesis of carbohydrates from fatty acids. BAGLEuet al. (1963) have suggested that the pathway from lipids to soluble carbohydrate proceeds via the intermediate formation of starch. As the parenchyma of the bundle sheath is rich in lipid bodies, the activity of the starch synthesizing enzymes in this region may be an indication of their role in the pathway from lipids to soluble carbohydrate. References AVERS, C. J.: Histochemical localization of enzyme activities in root meristem cells. Amer. J. Bot. 48, 137--142 (1961). --, and R. B. Gl~n~: Intergeneric differences in the activity of glucose-6-phosphatasc in grass root epidermis. Exp. Cell Res. 16, 692--695 (1959). --, and E. E. KING: Histochemieal evidence of intracellular enzymatic heterogeneity of plant mitochondria. Amer. J. Bot. 47, 220--225 (1960). BAGLEY~ B. W., J. i. CHERRY, ~V~.L. ROLLINS, and A. M. ALTSCHUL: ~k study of protein bodies during germination in pea-nut (lrachis hypogaea) seed. Amer. J. Bot. 50, 523--532 (1963). B ~ n , J.M., and F. V. MERC~: (1) Subcellular organization of the developing cotyledons of Pisum sativum L. Aust. J. biol. Sci. 19, 49--67 (1966). -- - (2) Subcellular organization of the cotyledons in germinating seeds and seedlings of Pisum sativum L. Aust. J. biol. Sci. 19, 69--84 (1966). BEEr]ms, H.: Metabolic production of sucrose from fat. Nature (Lond.) 191, 433--436 (1961). BURSTONE,M. S. : Enzyme histochemistry and its application to the study of neoplasms. New York: Academic Press 1962. D Y e , ~ . T.: Some observations on starch synthesis in pea root tips. Amer. J. Bot. 87, 786--792 (1950). ESPADA, J. : Enzymic synthesis of adenosine diphosphate glucose from glucose-lphosphate and adenosine triphosphate. J. biol. Chem. 237, 3577--3581 (1962). GAtr~, P. B. : Histochemical evidence for the presence of lysosome-like particles in root meristem cells of Viola/aba. J. exp. Bot. 16, 350~355 (1965). --, and A. J. MAPLE: The behaviour of lysosome-like particles during cell differentiation. J. exp. Bot. 17, 151--155 (1966). HOLT, S. J. : Indigogenic staining methods for csterases. Gen. cytochem. Methods 1, 375--398 (1958). JEnSEN, W. A. : A morphological and biochemical analysis of the early phases of cellular growth in the root tip of Viola/aba. Exp. Cell Res. 8, 506--522 (1955). -Botanical histochemistry: principles and practice. San Francisco: Freeman 1962. 2b
Plan~a
(Berl.),
Bd.
75
22
A. NI. FLI~N and D. L. SMIT~: Localization of Enzymes in Cotyledons
KVXSANOV,A. L. : Metabolism and transport of organic substances in the phloem. Advane. bot. Res. 1, 209--278 (1963). NORDIlV, J. H., and S. KIRKWOOD: Biochemical aspects of plant polysaecharides. Ann. Rev. Plant Physiol. 16, 393--414 (1965). Povx, l~. : LoeMisation des phosphates et de la phosphatase acide dans les eellules des embryons de b16 (Triticum vulgate Vill.) lors de la germination. J. Microsc. 2, 557--568 (1963). Sy~IT~, D. L., and A. M. FLI~N: Histology and histoehemistry of the cotyledons of Pisum arvense L. during germination. Planta (Berl.) 74, 72--85 (1967). Dr. D. L. SmTH Department of Botany The Queen's University of Belfast Belfast 7, Northern Ireland
The Localization of Enzymes in the Cotyledons of P i s u m a r v e n s e L. during Germination A. IV[. FLrN~ a n d D. L. SMITH Botany Department, Queen's University, Belfast, Northern Ireland l~eceived December 24, 1966 S u m m a r y . Enzyme activity is not uniformly distributed through the cotyledon of P i s u m arvense. Initially the peripheral region, certain scattered cells of the
storage tissue and the procambium show a high level of activity of succinic dehydrogenase, cytochrome oxidase, acid phosphatase and esterase. Activity of acid phosphatase declines sharply after the first day of germination; activity of the other enzymes declines after about three days. In the storage tissue, where activity is lower initially, it declines after about five days and is ccrrelated with the disappearance of the reserves. The pattern of alkaline phosphat~se activity is similar except that activity is lower in the procambium but increases in the sieve-elements during differentiation of the phloem. 5-nucleotidase and glucose-6-phosphatase activity is low throughout the cotyledon but it also increases to a significant level in the sieve-elements. Activity of starch synthesizing enzymes is high in the parenchymatous bundle sheath, where they may be involved in the pathway from lipids to soluble carbohydrates.
Introduction The aim of the present s t u d y was to investigate changes i n the dist r i b u t i o n a n d a c t i v i t y of various enzymes i n the cotyledon of P i s u m a r v e n s e L. during g e r m i n a t i o n a n d to correlate these changes with histological a n d histochemical changes recorded previously (SMITK a n d FLIr162162 1967). The enzymes i n v e s t i g a t e d were: the respiratory enzymes succinic dehydrogenase a n d cytochrome oxidase; the h y d r o l y t i c enzymes acid phosphatase, alkaline phosphatase, 5-nucleotidase, glucose-6-phosphatase a n d esterase; a n d starch synthesizing enzymes, which m a y include starch s y n t h e t a s e a n d starch phosphorylase. Materials and Methods Seeds of P . arvense were soaked in distilled water overnight, after which they were planted in moistened vermiculite and kept in a greenhouse at about 200 C. Material was harvested 0.5, 1, 1.5, 2, 3, 5, 7, 9 and 12 days from the commencement of soaking. Fresh sections were used for all the localization procedures, the method of J~SEN (1955) being followed. A cavity was made in a block of Paraplast wax by means of a hot scalpel. A cotyledon was pressed into the cavity and the block immediately cooled in cold water. Sections were cut at 50 ~ on a rotary mierotome. Thinner sections of cotyledons proved unsatisfactory because many of the large storage parenchyma cells lost their contents during manipulation of the sections or during subsequent processing.
Localization of Enzymes in Cotyledons
11
For the histochemical localization of enzyme activity the following procedures were used: 1. Suceinie Dehydrogenase. The procedure used was the tetrazolium method of SELIGlYIA~and R~TTENBERG(JE~sE~, 1962). Controls were incubated in a medium without substrate. 2. Cytochrome Oxidase. The nadi reaction of GoMoRI (J]~Ns]~, 1962) was used. In the controls, sections were pretreated with alcohol or incubated in a reaction mixture containing 0.005 M sodium azide. 3. Esterases. The indigogenic method of HOLT (1958) was used. This procedure was also carried out with the following variations: a) Naphthol AS-D acetate was used as an alternative substrate (BURSTOI~E, 1962). b) The cotyledons were fixed in formol-calcium before sectioning (BURsTONE, 1962). c) The sections were first squashed to ensure that all the cells were damaged. d) Sections of several thicknesses, up to 100 [z, were used. 4. Acid Phosphatase. A modification of Go~oRfs lead sulphide method was used (J]~ss~, 1962). Controls were incubated in a medium without substrate or in one containing 0.001 M sodium fluoride. 5. Alkaline Phosphatase. A modification of the Go~o~I cobalt sulphide method (J]~NsE~, 1962) was used. Controls were the same as for acid phosphatase. 6. 5-Nueleotidase. The cobalt sulphide method of AvErtS (1961) was adopted. Controls were the same as for acid phosphatase. 7. Glucose-6-Phosphatase. The lead sulphide method of Av]~Rsand G n I ~ (1959) was used. Controls were the same as for acid phosphatase. 8. Starch Synthesizing Enzymes. The method used was that of DYAR (1950), which localizes enzymes catalyzing the synthesis of starch from glucose- 1-phosphate.
Results 1. Succinic Dehydrogenase Succinic dehydrogenase is localized in small rod-like cytoplasmic particles less t h a n 1~ in length (Fig. 1). T h e y stain dark blue with the m e t h o d used. T h e y are presumed to be mitochondria. The enzyme is n o t u n i f o r m l y d i s t r i b u t e d t h r o u g h o u t the cotyledon; initially, there is a high level of a c t i v i t y in the hypodermis, the procamb i n m , the basal region a d j a c e n t to the petiole, a n d certain cells of the storage p a r e n c h y m a . The latter are of p a r t i c u l a r interest. T h e y are scattered t h r o u g h o u t the storage tissue (Fig. 2) b u t increase in frequency towards the periphery, often forming a n almost c o n t i n u o u s layer below the h y p o d e r m i s (Fig. 3). SMIT~ a n d FLIN~ (1967) showed t h a t certain cells of the cotyledon have a more opaque, g r a n u l a r cytoplasm a n d a relatively high cytoplasmic R N A c o n t e n t as shown b y azur B staining. These cells are a p p a r e n t l y identical with those h a v i n g a high level of a c t i v i t y of succinie dehydrogenase a n d of other enzymes as described below. There are two reasons for supposing this: a) their d i s t r i b u t i o n a n d frequency are comparable ; b) in both stained a n d u n s t a i n e d sections the cells are readily distinguishable u n d e r dark-field i l l u m i n a t i o n (SMIT~
12
A.M.
FLI~
and D. L. SMITR:
Figs. 1--3. Suceinic dehydrogenase, day 3. Fig. 1. Longitudinal section of a parenehyma cell of the bundle sheath. The enzyme activity is localized in cytoplasmic particles which are presumed to be mitochondria. • 1200. Fig. 2. Section of the central region of a cotyledon showing a high level of activity in only some of the storage cells. • 150. Fig. 3. Section of the peripheral region of the cotyledon showing high levels of activity in the epidermis, hypodermis and some of the storage cells. • 200
Localization of Enzymes in Cotyledons
13
and F L I ~ , 1967); in sections subjected to localization procedures only those cells so distinguishable showed heavy staining. Thus, initially, the activity of this enzyme is highest in the peripheral region of the cotyledon and falls off rapidly towards the centre, where it tends to be localized in scattered cells. The outer storage cells show some activity initially but there is a marked increase during the first two days of germination. After 3 to 5 days the peripheral cells lose much of their activity and there appears to be a degree of clumping of the mitochondria. After about 7 days these cells are no longer active. The activity in the storage cells remains high until the reserves disappear, when it falls off rapidly. I t finally disappears between the 9th and 12th days. During the differentiation of the vascular strands activity quickly disappears from the potential xylem but there is little or no change in the phloem or in the parenchyma of the bundle sheath. 2. Cytochrome Oxidase The nadi reaction for cytochrome oxidase localization stains sites of activity a bluish purple. The enzyme occurs in particles which are presumed to be mitochondria. The distribution of activity of this enzyme in the cotyledon is similar to t h a t of succiilic dehydrogenase in that initially the peripheral and basal regions, certain scattered cells of the storage tissue, and the procambium (Fig. 4) are much more active than the majority of the large storage cells. Enzyme activity in the latter increases during the first two days of germination. A striking feature of the localization of this enzyme is the reddish purple staining of globules of varying sizes. This staining is almost certainly not attributable to cytochrome oxidase activity since it occurs also in sections which have been incubated in a medium containing sodium azide. The large storage cells contain a few of these globules but the cells in the basal region are particularly rich in them. The cells near the periphery and the procambial cells contain m a n y smaller globules which~ however, are larger than mitochondria. A similar staining pattern is found in sections stained with Sudan I I I and it m a y therefore be concluded that these bodies are lipids. According to Jn~sE~ (1962) the staining of lipids in the nadi reaction is probably attributable to the non-enzymatic formation of indophenol blue from the nadi reagent, catalyzed by fat peroxides. 3. Esterases The sites of esterase activity stain a deep bluish green with the indigogenic method. The localization is very precise. The distribution
14
A.M. FLI~I~ and D. L. SMIT~:
of the enzyme appears to be mainly cytoplasmic and non-particulate. However. in some cells near the outside of the cotyledon the nuclei or the periphery of the nuclei may stain more intensely than the surrounding cytoplasm. I t is possible that this may be due to aggregation of small stained particles round the nucleus rather than to actual nuclear staining.
Fig. 4. Cytochrome oxidase, day 1. Longitudinal section of the procambium, where activity is much higher than in the surrounding storage cells. X 75 Fig. 5. Esterase, day 5. Section of the central region of the cotyledon, showing a high level of activity in some of the storage cells. • 150 The distribution of the enzyme through the cotyledon is similar to that of the respiratory enzymes (Fig. 5), though there is a more striking difference between cells of high activity and those of low activity. Only very slight staining is detectable in the bulk of the storage cells. I n view of the peculiar distribution of activity of this and other enzymes in the storage tissue it is essential to consider the possibility that the oeeurrenee of high levels of activity in only some of the cells is an artifact. The possibility exists that the pattern of enzyme distribution in this tissue could be interpreted as the result of variation in uptake of the substrates by the cells or as a wound response. The following
Localization of Enzymes in Cotyledons
15
evidence, which is based on a more detailed investigation of esterase activity, is opposed to this view: a) The pattern of enzyme activity is similar with both the substrates used. b) The pattern is similar in fixed and unfixed sections. c) The pattern is similar in sections which had been squashed before treatment. This tends to eliminate the possibility t h a t only undamaged cells are showing high activity. d) The proportion of cells showing high activity is more or less constant in sections of different thickness. H a d the effect been a wound response the proportion should have been lower in thicker sections, since the proportion of cut cells would have been reduced. This tends to eliminate the possibility that only damaged cells are showing high activity. e) Although it is difficult to determine conclusively whether a particular cell is intact or has been cut slightly during sectioning, both stained and unstained cells which appear to be intact can be found in sections. The intensity of staining in the peripheral cells decreases rapidly after 5 to 7 days. As the reserves of the storage cells become exhausted their staining intensity also decreases. Activity is still present in the centre of the cotyledon on the 9th day but is undetcctablc by the 12th day. In the vascular strands much of the activity is lost during the first 2 days, when the mature conducting elements are undergoing differentiation. The parenchyma of the bundle sheath remains highly active until a late stage and some activity is still present on the 12th day.
4. Acid Phosphatase Acid phosphatase was found to be localized in cytoplasmic particles (Fig. 6). These appeared to be considerably larger than the particles staining with the localization methods for the respiratory enzymes and presumed to be mitochondria. I t is possible, though unlikely, that these larger particles m a y be aggregates of smaller bodies formed during the processing of the sections. The peripheral cells and scattered cells of the storage tissue appear to be more active than the majority of the storage cells, but not markedly so. The activity is only apparent during the first 2 days of germination, and after the first day the particles are difficult to see. Initially, activity is fairly high in the vascular strands. I t disappears quickly from the differentiating xylem and possibly from the phloem but remains
16
A.M. F L I ~ and D. L. SMIT~:
Fig. 6. Acid phosphatase, day 0.5. Section of the outer storage tissue, showing the particulate distribution of the enzyme. • 300 Fig. 7. Alkaline phosphatase, day 2. Longitudinal section of ]?art of a vascular strand, showing the localization of enzyme activity in the sieve-elements of the phloem. • 580 Fig. 8. Glucose-6-phosphatase, day 3. Longitudinal section of a vascular strand, showing a high level of activity in the sieve-elements. • 580
Localization of E n z y m e s in C o t y l e d o n s
17
detectable for a few days in the parenchyma of the strands as weaklystaining particles.
5. Alkaline Phosphatase The localization of alkaline phosphatase and 5-nucleotidase is made more difficult by the non-enzymatic formation of cobalt sulphide within the cell walls. The nucleoli of some cells also stain deeply but this too appears to be non-enzymatic since it also occurs in the controls. However, the non-particulate, cytoplasmic localization of these enzymes appears to be genuine. Initially, the activity of alkaline phosphatase is at its highest in the peripheral region of the cotyledon. Activity in this region increases during the first 2 or 3 days of germination and subsequently a wave of activity progresses from the periphery towards the centre of the cotyledon. Activity in the outer storage parenchyma reaches its peak between days 3 and 5 and in the inner storage parenchyma between days 5 and 7. Thereafter, the cytoplasmic staining decreases as the reserves become depleted. Activity is also initially present, but only at a low level, in the procambium. As differentiation proceeds the phloem elements become conspicuously active (Fig. 7) but all activity is lost from the xylem. Some activity persists in the bundle sheath. 6. 5-Nucleotidase The activity of 5-nueleotidase is very low in all the cells of the cotyledon. Little change can be detected in the peripheral or storage regions during germination. A low level of activity is present initially in the procambium but during and after differentiation a rather higher level is detectable in the sieve elements of the phloem. 7. Glucose-6-Phosphatase This enzyme, like alkaline phosphatase and 5-nucleotidase, has a non-particulate, cytoplasmic distribution. No activity is detectable in the peripheral or storage regions of the cotyledon. The localization procedure used results in slight staining of the cell walls but as this occurs also in the controls it cannot be attributed to enzyme activity. Such wall staining would probably make difficult the detection of low levels of activity in the cytoplasm. Some activity is detectable in the procambium during the first day of germination and by the second day the sieve tubes of the phloem show a high level of activity (Fig. 8). This activity persists until about the seventh day, after which it declines and is nndetectable by the ninth. 2a Planta (Berl.), Bd. 75
18
A.M. FLINN and D. L. SM~Tg:
8. Starch Synthesizing Enzymes The method used here was t h a t devized by DYAR (1950) for the localization of phosphorylase. The tissue is supplied with glucose-lphosphate and subsequently stained with iodine-potassium iodide solution. New starch, formed during the localization procedure, has few branched chains and stains a reddish brown eolour. The specificity of this method for the localization of phosphory]ase is questionable since it has now been established t h a t in vivo starch is synthesised from uridine diphosphate glucose and adenosine diphosphate glucose by the action of starch synthetase (NOI~DIN and K m ~ w o o D , 1965). I t is likely t h a t this enzyme would also be localized b y the procedure used here, since adenosine diphosphate glucose is probably synthesized in vivo from glucose- 1-phosphate and ATP (ESPADA, 1962). The starch synthesizing enzymes are not demonstrably active in the majority of the cotyledon cells. However, the cells at the periphery of the procambium show a high aetiFig. 9. Starch synthesizing enzymes, v i t y and this persists in the parday 3. Section of a vascular strand, enchyma of the bundle sheath after showing enzyme activity in the paren- differentiation of the vascular strands chyma of the bundle sheath. The new, (Fig. 9). The hypodermis and some of small starch grains, formed during the localization procedure, appear paler the underlying storage cells appear than the much larger old grains. • 500 to have a very slight activity. Discussion Although the cells of the storage parenchyma of the cotyledon of Pisum appear morphologically similar under the light microscope recent work has shown t h a t they are not homogenous at the ultrastructural and physiological levels. I n P. sativum, BAI~ and MEI~CS,Ir (1966, 1) have shown t h a t in certain cells the protoplast breaks down during maturation and consequently the storage products, fragmented plastids and mitochondria are dispersed through a vesiculated cytoplasm. I n P. arvense, as described above, certain cells of the storage tissue have a higher cytoplasmic R N A content and a higher level of enzyme activity than the other cells. Although there is no direct evidence for it, it seems
Localization of Enzymes in Cotyledons
19
likely t h a t these cells are similar to the ones described by BAI~ and MERCE}~ (1966, I). In view of the high IINA content and high levels of enzyme activity it appears t h a t the cells must be specialized as sites of enzyme synthesis. I t is possible that they are concerned with the initiation of breakdown of the storage reserves in the cotyledon. Some of the enzymes, such as acid phosphatase and the esterases, must be synthesised during the maturation of the embryo since their activity is high from the beginning of germination. As in the case of the scattered cells of the storage tissue, other cells which have been shown to have a high level of cytoplasmic RNA (SMITIt and FLI~N, 1967) have now been shown to have a high level of enzyme activity. Thus the general pattern of enzyme activity in the cotyledon coincides with the pattern of high levels of RNA. The region showing the highest activity initially is the peripheral region, comprising the epidermis, hypodermis and underlying storage cells. I t has previously been shown (SMITH and FLINN,1967) t h a t this is also the region in which breakdown of the reserve starch and protein is initiated. The decrease in lgNA content of the cells coincides with the disappearance of the reserves during the second or third day of germination; enzyme activity in this region persists up to the fifth day and then declines rapidly. Although the majority of enzymes conform to the general tissue activity pattern already indicated, the different enzymes show interesting variations. Cytochrome oxidase and succinic dehydrogenase are enzymes generally associated with mitochondrial respiration. Both enzymes increase in activity in the storage cells during the first 3 days. Thus the m a x i m u m activity is reached as the reserves visibly begin to disappear. A fall in the activity of these enzymes is apparent in those cells which have lost all their reserves. Significantly, the cells of the phloem and the bundle sheath are the last to show a fall off in activity. BAIN and gm~CEl~ (1966, 2) have shown t h a t in P . sativum the mitochondria assume their normal functional morphology during the first few days of germination. Thus, the increase in activity of these enzymes is not only a function of synthesis but of organelle completion at the ultrastructura] level. The phosphatases - - acid phosphatase, alkaline phosphatase, 5-nueleotidase and glucose-6-phosphatase - - are hydrolytic enzymes under physiological conditions. Acid phosphatase was found to be localized in cytoplasmic particles apparently bigger than mitochondria. These disappeared rapidly in most cells, the exception being some of the cells of the vascular strands. The association of the enzyme with cytoplasmic particles is in agreement with previous observations (AVERS and KI~O, 2*
20
A.M. F L ~ and D. L. SMIT~:
1960; AvErs, 1961). GA~A~ (1965) has demonstrated t h a t a considerable period of incubation is necessary to demonstrate the presence of such particles in frozen sections of root tips of V i c i a . This implies t h a t the enzyme is present in an inactive form. I t therefore seems likely that the acid phosphatase in this form is present within a membrane. GAI~A~ and MArL~ (1966) produced evidence for the concept that self digestion of the cell contents occurs b y the release of hydrolytic enzymes, in particular acid phosphatase, from lysosomes. They showed t h a t this happens in the developing protoxylem elements in root tips of V i c i a . I t seems reasonable to conclude, on this evidence, t h a t in cotyledons of P . arvense the acid phosphatase is localized initially in lysosome-like particles and is then released during the early stages of germination to aid in some way in the mobilization of the storage reserves. I t must be borne in mind, however, that no organelles comparable to the lysosomes of animal cells have yet been identified in plant cells. The scheme suggested here is different from the one proposed b y P o v x (1963), where the protein bodies of T r i t i c u m embryos contain acid phosphatase in a form which becomes activated only at germination. I n P . arvense no evidence of acid phosphatase activity within the protein bodies was obtained. Alkaline phosphatase, 5-nueleotidase and glueose-6-phosphatase do not have a particulate distribution. There is a significant increase in activity of alkaline phosphatase throughout the cotyledon during the first few days of germination. The low activity of 5-nueleotidase in the cotyledons is perhaps surprising since there is probably a considerable turnover of I~NA in the storage tissue, related to enzyme synthesis. Of special interest is the increase in activity of all three enzymes in the sieve-elements during the differentiation of the phloem. As a high level of activity is retained until the storage reserves are exhausted it indicates a probable involvement of the enzymes in the transport of materials through the phloem. On evidence from other sources, it has been suggested t h a t phosphatase activity in phloem is associated with passage of sugars into the sieve-elements and it is possible also t h a t the processes of phosphorylation and dephosphorylation m a y be involved in the subsequent transport of sugars through the sieve-plates (Kt;~SA~OV, 1963). The localization procedure used for esterases probably reveals a range of these enzymes. The initial distribution is predominantly in the peripheral region and in the procambium, subsequently in the bundle sheath. These regions have a high lipid content and it is probable t h a t one of the functions of the csterases is to break down the lipids to their constituents. They might also be involved in the destruction of organelle membranes, including those of the protein bodies.
Localization of Enzymes in Cotyledons
21
Of the enzymes which are probably localized by the starch method of DYA~ (1950) starch synthetase is probably the one responsible for starch synthesis in vivo. I n vitro, phosphorylase can catalyze the synthesis of starch from glucose-l-phosphate but in vivo it is now considered to be an agent only of degradation (NoRDIN and KncKwoo]), 1965). Unlike most of the other enzymes investigated, these enzymes are relatively inactive in the peripheral region of the cotyledon but appear to be particularly active in the bundle sheath. Their function here is not entirely clear, but they also might be connected with lipid metabolism. BEvy]ms (1961) has shown that seeds undergo the glyoxylate cycle for net synthesis of carbohydrates from fatty acids. BAGLEuet al. (1963) have suggested that the pathway from lipids to soluble carbohydrate proceeds via the intermediate formation of starch. As the parenchyma of the bundle sheath is rich in lipid bodies, the activity of the starch synthesizing enzymes in this region may be an indication of their role in the pathway from lipids to soluble carbohydrate. References AVERS, C. J.: Histochemical localization of enzyme activities in root meristem cells. Amer. J. Bot. 48, 137--142 (1961). --, and R. B. Gl~n~: Intergeneric differences in the activity of glucose-6-phosphatasc in grass root epidermis. Exp. Cell Res. 16, 692--695 (1959). --, and E. E. KING: Histochemieal evidence of intracellular enzymatic heterogeneity of plant mitochondria. Amer. J. Bot. 47, 220--225 (1960). BAGLEY~ B. W., J. i. CHERRY, ~V~.L. ROLLINS, and A. M. ALTSCHUL: ~k study of protein bodies during germination in pea-nut (lrachis hypogaea) seed. Amer. J. Bot. 50, 523--532 (1963). B ~ n , J.M., and F. V. MERC~: (1) Subcellular organization of the developing cotyledons of Pisum sativum L. Aust. J. biol. Sci. 19, 49--67 (1966). -- - (2) Subcellular organization of the cotyledons in germinating seeds and seedlings of Pisum sativum L. Aust. J. biol. Sci. 19, 69--84 (1966). BEEr]ms, H.: Metabolic production of sucrose from fat. Nature (Lond.) 191, 433--436 (1961). BURSTONE,M. S. : Enzyme histochemistry and its application to the study of neoplasms. New York: Academic Press 1962. D Y e , ~ . T.: Some observations on starch synthesis in pea root tips. Amer. J. Bot. 87, 786--792 (1950). ESPADA, J. : Enzymic synthesis of adenosine diphosphate glucose from glucose-lphosphate and adenosine triphosphate. J. biol. Chem. 237, 3577--3581 (1962). GAtr~, P. B. : Histochemical evidence for the presence of lysosome-like particles in root meristem cells of Viola/aba. J. exp. Bot. 16, 350~355 (1965). --, and A. J. MAPLE: The behaviour of lysosome-like particles during cell differentiation. J. exp. Bot. 17, 151--155 (1966). HOLT, S. J. : Indigogenic staining methods for csterases. Gen. cytochem. Methods 1, 375--398 (1958). JEnSEN, W. A. : A morphological and biochemical analysis of the early phases of cellular growth in the root tip of Viola/aba. Exp. Cell Res. 8, 506--522 (1955). -Botanical histochemistry: principles and practice. San Francisco: Freeman 1962. 2b
Plan~a
(Berl.),
Bd.
75
22
A. NI. FLI~N and D. L. SMIT~: Localization of Enzymes in Cotyledons
KVXSANOV,A. L. : Metabolism and transport of organic substances in the phloem. Advane. bot. Res. 1, 209--278 (1963). NORDIlV, J. H., and S. KIRKWOOD: Biochemical aspects of plant polysaecharides. Ann. Rev. Plant Physiol. 16, 393--414 (1965). Povx, l~. : LoeMisation des phosphates et de la phosphatase acide dans les eellules des embryons de b16 (Triticum vulgate Vill.) lors de la germination. J. Microsc. 2, 557--568 (1963). Sy~IT~, D. L., and A. M. FLI~N: Histology and histoehemistry of the cotyledons of Pisum arvense L. during germination. Planta (Berl.) 74, 72--85 (1967). Dr. D. L. SmTH Department of Botany The Queen's University of Belfast Belfast 7, Northern Ireland
