Planta (Bcrl.) 74, 72--85 (1967)
HISTOLOGY AND HISTOCHEMISTI~Y OF THE COTYLEDONS O F P I S U M A R V E N S E L. D U R I N G G E R M I N A T I O N D. L. SMITH and A. M. FLI~ Botany Department, Queen's University, Belfast, l~orthern Ireland Received l~ovember 17, 1966 Summary. The mature cotyledon of Pisum arvense L. comprises several distinct tissue regions; these are the epidermis, hypodermis, storage parenchyma and procambium. The storage parenchyma includes two zones: an outer abaxial zone and an inner adaxial zone. The cells of both zones contain abundant starch grains and protein bodies. Scattered through the storage tissue but increasing in frequency towards the periphery are certain cells which differ to a slight extent from the majority of the parenchyma cells. They have a more opaque, granular cytoplasm and a higher level of cytoplasmic RI~A. The cotyledon has a complex, reticulate vascular system. Differentiation of the conducting elements from the procambium appears to begin about 12 hours and to be completed 48 hours after the commencement of imbibition. Differentiation of phloem preceeds that of xylem. The relationship between the timing of vascular differentiation and various physiological events in the cotyledon is discussed. Mobilization of the reserves in the storage parenchyma is initiated at the periphery of the cotyledon and proceeds inwards. There appears to be a correlation between the breakdown of the reserves and changes in DNA and RNA content of the cells.
Introduction Considerable i n f o r m a t i o n is n o w a v a i l a b l e on t h e physiological a n d biochemical changes which occur d u r i n g t h e g e r m i n a t i o n of pea seeds, p a r t i c u l a r l y those r e l a t i n g to t h e d i s a p p e a r a n c e of reserve proteins, carb o h y d r a t e s a n d fats from t h e cotyledons. I n f o r m a t i o n of this t y p e is of n e c e s s i t y b a s e d l a r g e l y on i n v e s t i g a t i o n s carried o u t a t t h e tissue r a t h e r t h a n a t t h e cellular level a n d m e t a b o l i c differences b e t w e e n t h e compon e n t cells are neglected. To some e x t e n t t h e s i t u a t i o n has been rectified b y r e c e n t studies concerned w i t h t h e u l t r a s t r u e t u r e of c o t y l e d o n cells d u r i n g g e r m i n a t i o n . VA~N~R a n d SCHIDLOVSKY (1963) i n v e s t i g a t e d t h e i n t r a c e l l u l a r d i s t r i b u t i o n of p r o t e i n in t h e cotyledons a n d BAI~ a n d MERCER [1966, (2)] r e l a t e d physiological changes in t h e cotyledons to u l t r a s t r u c t u r a l changes in t h e s t o r a g e cells of t h e cotyledons. The inter-relat i o n s h i p b e t w e e n t h e e m b r y o n i c axis a n d t h e cotyledons has been investig a t e d a t t h e physiological level b y V A ~ n R et al. (1963) a n d a t t h e physiological a n d u l t r a s t r u c t u r a l levels b y BAI~ a n d M ~ c E ~ [1966, (3)]. T h e y h a v e shown t h a t t h e storage reserves in d e t a c h e d cotyledons do n o t b r e a k d o w n to soluble form unless excision of t h e axis is d e l a y e d u n t i l t w o d a y s a f t e r t h e initial soaking.
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A n a c c o u n t of t h e d e v e l o p m e n t of t h e e m b r y o of P i s u m s a t i v u m L. was g i v e n b y CooPeR (1938) a n d a more detailed account, including t h e histogenesis, was g iv e n b y RE]~v~ (1948). HAYWARD (1938) described briefly t h e d e v e l o p m e n t a n d a n a t o m y of t he seed a n d cited earlier anat o m i cal work. N o n e of these previous studies has y e t p r o v i d e d a c o m p r e h e n s i v e a c c o u n t of t h e a n a t o m y of t h e m a t u r e e m b r y o an d so far little a t t e m p t has been m a d e to localize physiological e v e n t s within t h e cotyledons. The p res en t s t u d y has th r e e m a i n aims: to describe histological changes which occur during g e r m i n a t i o n , in p a r t i c u l a r t h e differentiation of t h e v a s c u l a r s y s t e m ; a n d to follow h is t o c h e m ic a l l y changes in certain cellular components. Materials and Methods Seeds of P i s u m arvense L. were soaked in water for four hours and then planted in moist vermiculite. They were then incubated in the dark at 25~ Samples were taken at 6, 8, 10, 12, 14, 15, 16, 17, 18, 20, 24, 32, 40, 42, 46 and 48 hours and 3, 5, 7, 9, and 12 days from the commencement of the initial soaking. Whole cotyledons were fixed in FAA or glutaraldehyde. Fixed cotyledons used for tracing the morphology and differentiation of the vascular system were cleared by a modification of the technique of FOSTE~ (1955) and BISALPV~A and EsAv (1964). Whole cotyledons were treated with 2 % NaOH at 600 C for 1 to 2 hours. They were then washed thoroughly and placed in an acidified solution of chloral hydrate under ~acuum at 60~ until clear (4 to 8hours). Cotyledons were examined whole in the clearing solution or selected parts were cut out, squashed and mounted in the clearing solution, and examined under polarized light. The walls of the sieve tubes and tracheary elements show conspicuous bierfringence in polarized light and this feature can be utilized in tracing the course of differentiation of the xylem and phloem (BIsALeVT~ and EsAv, 1964). For histological examination slices of cotyledons were dehydrated through an ethanol-n-butanol series and embedded in "Paraplast" wax. Sections were cut at 10 ~ and stained in a safranin-fast green combination. For the histochemical localization of cellular components i0 ~ sections were cut as above and then the following procedures were used: 1. D N A . Sections were stained by the standard Feulgen technique. 2. D N A and R N A . (a) Sections were stained using the modified methyl greenpyronin technique of KvR~IeI; (1955). This method proved unreliable as significant amounts of methyl green were retained by the cell wall. (b) Sections stained in azur B (FLAx and HIM]~s, 1952; JENSEN, 1962) proved more satisfactory. Controls were either treated for 1.5 hours with a solution of 0.1% ribonuclease in water adjusted to pH 6.8 with NaOH or were incubated for the same period in water alone at p i t 6.8. 3. Total protein. The ninhydrin-Sehiff's reagent method of YASV~A and ICnIK ~ A was used (JE~sE~, 1962). 4. Histories. Sections were stained by the fast green method of ALFERT and GEsc~n (1953). 5. Carbohydrates. Sections were stained by the standard periodic acid-Schiff's reagent technique. Iodine-potassium iodide solution was used for the specific detection of starch.
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D.L. SMITE and A. M. FLINN:
Anatomy and Histology of the Cotyledon The cotyledon is approximately hemispherical in shape with the flat face corresponding to the adaxial face of the leaf. I t is bounded b y an epidermis (Figs. 1,2) from which stomata are absent except in the region adjacent to the embryonic axis. Below the epidermis is a single row of cells comprising the hypodermis. The storage parenchyma makes up the bulk of the cotyledon. I t appears to consist of two distinct zones, an outer zone corresponding to the spongy parenchyma of the leaf and an inner zone corresponding to the palisade tissue. Both zones are much less distinctive than the corresponding tissues in the leaf. The cotyledon possesses a well developed vascular system which runs more or less along the boundary of the inner and outer storage tissues. In the mature embryo it consists entirely of procambium ; no mature xylem or phloem elements have been detected in it. Differentiation of vascular tissues from the procambinm occurs during the first two days of germination. The Epidermis and Hypodermis. The epidermal cells arc small, narrow and elongated. They are usually in the region of 10 ~ wide and 100 long. They are arranged parallel to one another in small groups of about eight cells (Fig. 3), each group apparently comprising the derivatives of one of the original meristematic cells of the protoderm. The orientation of these groups is not constant though they tend to be elongated along the axis of the cotyledon. The cells are not, on the whole, arranged end to end in rows as described in P. sativum by HAYWAI~D(1938), although this tendency is shown to some extent b y the cells of the adaxial epidermis. The hypodermis consists of more or less isodiametric cells which often tend to be slightly flattened in a plane parallel to the surface. Both the epidermis and the hypodermis contain cytoplasmic protein. I t does not, however, appear to be particulate but this could possibly be due to the small size of the protein bodies. Small starch grains are present in the hypodermis but have not been seen in the epidermis. In both layers the nuclei are normal in appearance ; the nucleoli stain deeply with azur B. The Storage Parenchyma. The cells of the two zones of the storage parenehyma differ from each other in size and shape. In seetions in any plane the cells of the inner storage tissue, t h a t underlying the adaxial surface, are isodiametrie and rounded in appearance. They are up to 130 ~ in diameter. In contrast, the cells of the outer storage tissue, underlying the abaxial surface, are larger and more irregular in shape. They tend to be elongated perpendicular to the surface and m a y be up to 140 ~ in diameter and 230 ~ long. The cells of both tissues are eompactly but irregularly arranged, with small triangular intercellular spaces confined to the corners of the cells (Fig. 5). During germination the size of the intercellular spaces increases
Histology and Histochemistry of the Cotyledons during Germination
75
Figs. 1 a n d 2. Sections t h r o u g h the peripheral region of a cotyledon, stained in azur B, showing the epidermis, h y p o d e r m i s a n d outer storage p a r e n c h y m a . Fig. 1. D a y 1 ; higher levels of R N A are present in the epidermis, h y p o d e r m i s a n d some of the storage cells. Fig. 2. D a y 9; the storage reserves are completely exhausted. • 200
and larger, more irregular spaces develop b y union of the small spaces initially present. This gives rise to a complex reticulum of more or less tnbu]ar spaces (Fig. 4) which penetrates the whole tissue. I t seems t h a t
76
D.L. S~ITn and A. M. FLI~N:
the walls of adjacent cells separate along the middle lamella. The cell walls are thin and nn]Jgnified. The contact areas between adjacent cells are more or less oval and show numerous primary pit fields (Fig. 4). The nuclei of both types of cell are more or less spherical in shape but have a rather diffuse appearance. The nncleoli are visible but do not stain with azur B. All the cells contain large starch grains and small protein bodies (Fig. 5). The latter are more or less spherical in shape and
Fig. 3. Surface view of p a r t of the epidermis of a cotyledon treated with N a O t I a n d chloral hydrate. The group of eight cells a p p a r e n t l y represent the derivatives of a single protodermal cell. Phase contrast. • 500 Fig. 4. Surface view of the contact area between two storage cells of a 5 day cotyledon. The u n p i t t e d wall surrounding the contact area adjoins the greatly enlarged intercellular space. Stained in periodic acid-Schiff's reagent. • 650
up to 3 ~ in diameter. They stain red-purple after treatment with ninhydrin-Schiff's reagent. The starch grains are up to 50 ~ long. Scattered through the storage tissue but increasing in frequency towards the periphery and forming an almost continuous layer under the hypodermis are cells which differ slightly from the majority of the parenehyma cells. Although the cell wall, nucleus, starch grains and protein bodies are comparable with those of other cells, these cells have a more opaque, finely granular cytoplasm. Under dark field illumination they become particularly obvious because of this opacity: they appear white or grey against a black background (Fig. 8). Their cytoplasmic R N A content, as shown by azur B staining, is relatively high (Fig. 1).
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Figs. 5--7. Sections of storage parenchyma cells showing changes in storage reserves during germination. Stained in ninhydrin-Schiff's reagent, s, intercellular space; p, protein body; g, starch grain. Fig. 5. An outer storage cell at day 1. Fig. 6. An inner storage cell of a 5 day cotyledon showing the swollen protein bodies. Fig. 7. An outer storage cell of a 5 day cotyledon showing the aggregate protein bodies containing deeply staining inclusions. Only one starch grain remains. • 650
The Vascular System. The cotyledon has a complex reticulate vascular system (Fig. 9). Two vascular strands enter the base of the cotyledon from the embryonic axis. One of these, the larger of the two, forks in the petiole and then each branch forks, thus forming four
78
D.L. SMITEand A. M. FLINN: main veins. The smaller strand entering the cotyledon forms a fifth main vein. Branching and anastomosis of these five main veins occurs as in a normal leaf. The morphological interpretation of the vascular system is complicated by the presence of two unequal strands in the base of the cotyledon. The most likely interpretation is that the larger strand represents the product of fusion of two foliar strands while the smaller represents a single stipular trace.
Fig. 8. U n s t a i n e d section of the outer storage tissue of a fixed 5 day cotyledon under dark field illumination. Only the ceils with the more opaque, granular cytoplasm are visible. • 330
The cells of the procambium are mainly long and narrow, 7 to 12 ~ in diameter and 40 to 100 ~ long. The longer, narrower cells tend to occur towards the centre of the bundle
and
are apparently
the potential
l~ig. 9. Camera lucida drawing of the vascular system of a day 1 cotyledon cleared in )~aOH-chloral
hydrate
Histology and Histochemistry of the Cotyledons during Germination
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conducting elements. The shorter cells towards the periphery give rise to the parenehyma of the bundle sheath. The walls of some of the long central cells occasionally stand out because of their stronger birefringence under polarized light. This is presumed to indicate t h a t differentiation of these cells has begun and that they will give rise to sieve-tube members of the protophloem.
Differentiation of the Vascular Strands Differentiation of the vascular tissue from the procambium was first observed 12 to 17 hours after the commencement of soaking. The first detectable change was an increase in the birefringence of the elongated cells in the centre of the strand. The first cells to differentiate were those of the protophloem of the main vein in the basal region of the cotyledon. Those of the smaller vein began shortly afterwards. Normally two or three sieve elements are recognizable before the walls of the protoxylem elements show any evident birefringence. The first mature protoxylem was seen at 16 hours. At any one point in the larger veins up to five sieve elements m a y be present by the time the first protoxylem element is fully differentiated. As far as could be determined differentiation of the phloem occurred in a strictly acropetal sequence from the basal region of the cotyledon. Differentiation of the xylem in the main veins also appeared to be acropetal but in at least some branch veins the protoxylem matured some little distance away from the main vein; subsequently differentiation occurred both towards and away from the main vein. Mature phloem was detected in the distal region of some cotyledons after 20 hours and mature xylem after 24 hours from the commencement of soaking. This, however, is probably exceptional and 30 to 32 hours is perhaps a more normal time for differentiation to have reached this region. The time taken for complete differentiation of the strands was difficult to determine precisely but strands which were apparently fully differentiated were observed at the distal end at 42 to 48 hours. In transverse section a mature strand is seen to comprise five to twenty-five xylem elements and a slightly larger group of phloem elements (Fig. 10), the wholebeing surrounded b y a parenehymatous bundle sheath. The longitudinal walls of the parenchyma cells contain abundant primary pit fields. Histoehemical Changes during Germination D N A and R N A . As has already been indicated the Feulgen positive
material of the nucleus appears to be rather diffuse in the storage parenchyma cells but compact in the cells of the epidermis, hypodermis and procambium. During germination the nucleus of the parenchyma cells increases in size and irregularity (Fig. 11), becoming conspicuously lobed
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D.L. SMIT~ and A. M. F L I ~ :
Fig. 10. Transverse section of a vascular s t r a n d of a day 1 cotyledon, stained in azur B. The I~NA content of the differentiating xylem (x) is decreasing while t h a t of the phloem (p) shows little change. • 500 Fig. 11. Section showing the ]obed nucleus of an inner storage cell of a day 5 cotyledon. Feu]gen stain. x 1200 Fig. 12. Section showing the fast green staining of the nucleus of a storage cell. x 1200 Fig. 13. Section of a storage cell of a day 5 cotyledon showing the small starch grains r o u n d the nucleus, a n d the large storage grains. Stained in periodic acid-Schiff's reagent. • 500
i n certain cases. I n i t i a l l y there is no change i n staining i n t e n s i t y associated with the increase i n size b u t as the storage reserves of the cotyledons
Histology and Histochemistry of the Cotyledons during Germination
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become depleted the staining becomes less intense. No changes were noted in the Feulgen positive material of the nuclei of the other regions of the cotyledons and no mitotic figures have been observed at any time, even in the procambium. The cells of the epidermis, hypodermis and procambium, showed heavy cytoplasmic staining with azur B. The cells of the storage parenchyma, with the exception of the scattered cells already mentioned, showed only slight staining. The parenchyma cells in the cotyledon petiole stain fairly heavily. Sections pre-treated with ribonuclease showed no cytoplasmic staining. During the first three days of germination the cells of the petiole, the epidermis and hypodermis, and the scattered parenchyma cells lose their heavy staining and have a staining density which is little different from that of the storage parenchyma cells. As the procambium undergoes differentiation into mature conducting elements the future xylem loses its staining intensity but the staining of the other cells remains unchanged or possibly even increases (Fig. 10). Nucleo]ar staining for RNA shows an interesting pattern in the cotyledon. In the early stages of germination the nucleoli of only the outer cell layers, the epidermis, hypodermis and some underlying cells, and the procambiM cells show staining with azur B. However, after the first two days the staining of the nucleolus has progressed from the periphery. The nucleoli of the outer layer of storage cells stain while the staining of those towards the periphery becomes less intense. In effect, a wave of nucleolar staining passes from the periphery towards the centre of the cotyledon. The staining is at its most intense in a given cell when breakdown of the reserves begins; it decreases as the reserves diminish. The nucleoli of the parenchyma cells of the bundle sheath are the last to lose their stainability. Protein. Initially the protein bodies (Fig. 5) in the storage parenchyma cells are up to 3 ~ in diameter and stain red-purple with ninhydrinSchiff's reagent. The inner cells appear to contain more of them per unit volume than do the outer cells. During the first two days of germination these protein bodies appear to enlarge to at least twice their original diameter (Fig. 6) and subsequently to fuse together in groups to form larger aggregate bodies (Fig. 7). These may be 25 ~ or more in diameter and they stain more intensely than did the original bodies. Also the staining changes from red-purple to a definite red. This change in intensity and colour of the staining is perhaps due to the colour reaction with small peptides rather than with large proteins. Small, very deeply staining bodies become apparent in the protein bodies after coalescence (Fig. 7). Ultimately the large aggregate bodies appear to disintegrate and to form small, more irregular bodies which finally disappear. At the tissue level, the protein first disappears from the peripheral cells and those in the 6
P l a n t a (Berl.), Bd. 74
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D.L. SMITHand A. M. F L I ~ :
basal region of the cotyledon. This is not because there is less protein in the outer cells but is attributable to a greater rate of and an earlier initiation of lysis. Thus, breakdown is initiated in the outer cells and then spreads inwards. When the aggregation stage of protein degradation has been reached in the peripheral cells the protein bodies towards the centre are still at the swollen stage. The protein disappears completely from the outer layers after about five days. After about twelve days the storage parenchyma is almost completely free of protein and only the nuclear proteins stain.
Histones. Only a brief study of histones was made using the fast green method. Only the nucleus stains (Fig. 12), there being no evident cytoplasmic staining. The nueleolus appears to stain with an intensity the same as or less than the rest of the nucleus. The appearance of the nuclei of the storage cells is similar to t h a t after Feulgen staining in t h a t the nuclear material is dispersed and t h a t after the storage reserves become depleted staining is less intense. Starch. Initially starch is present in all the cells except those of the epidermis and possibly the potential conducting cells of the procambium. I t is present in large quantities in the storage parenchyma, particularly in the inner zone. The hypodermis and the proeambinm (subsequently the parenchyma of the bundle sheath) contain only very small grains which disappear within the first three days. The pattern of degradation of starch in the storage parenchyma is similar to t h a t of protein : degradation begins at the periphery of the cotyledon and proceeds in a wave towards the centre. During the first seven days the outer storage cells become depleted of their starch and after twelve days no starch remains in the central core. During the first two days small starch grains, mostly in the size range 3 to 15 ~, appear in the storage cells. Usually, they become aggregated round the nucleus (Fig. 13). They have disappeared from the outer region of the cotyledon by day 5 and from the central core by day 7. Discussion Differentiation of the embryo during germination has been investigated in few species of plants. But in those cases which have been examined it has been shown consistently t h a t the main event is the differentiation of the vascular tissues from the procambium. The sequence of differentiation shows some variation between species, however, and there appears to be no constant pattern (BISALPUTRAand ESAU,1964). The pattern in Pisum arvense, where differentiation of phloem preceeds that of xylem, is similar to the pattern described in Helianthus, Mirabilis, Lupinus, and Ricinus (DAUPHINE et l~IvI]~]~, 1940) and Chenopodium (BISALPUTgA
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and EsAu, 1964). I n Chenopodium, as in Pisum, the protophloem is partly differentiated in the mature, dormant embryo but in Lupinus fully differentiated sieve elements are present. The timing of the vascular differentiation in relation to physiological events in the cotyledon is of particular interest. Differentiation of the phloem was first observed 12 hours after the commencement of soaking and differentiation of the xylem apparently began about 4 hours later. A continuous conducting system through the cotyledon was present after 24 to 32 hours and differentiation was complete after 40 to 48 hours. The period from 16 to 32 hours is probably particularly important since during this period the differentiation of xylem is initiated and a continuous conducting system is laid down. VARNE~ et M. (1963) have shown t h a t m a n y of the metabolic events which occur in the cotyledons of P. sativum are triggered off and controlled by stimuli from the embryonic axis. If cotyledons are excised during the first 24 to 48 hours they undergo a rapid decline in respiration rate and fail to develop their characteristic enzymatic activities. At the ultrastructural level B ~ N and MERCEa [1966, (3)] have shown t h a t the completion of the mitochondria and the development of the endoplasmic reticulum of the storage cells is accomplished in the period between 24 and 48 hours and t h a t completion can occur only in the presence of axis tissue. According to V ~ N E g et al. these various events are initiated by some factor which passes from the axis into the cotyledons sometime after the first 18 hours of germination. The timing of this movement thus coincides approximately with the differentiation of the first xylem in the cotyledon. I t seems likely t h a t until a continuous strand of vascular tissue is present the movement of substances into the cotyledon would not occur or would be slow, and t h a t the passage of the stimulus from the axis is at least partly dependent on the differentiation of the vascular system. The increase in size of the nuclei of the storage cells during the first few days of germination and their subsequent decrease in staining intensity are in agreement with quantitative changes in DNA recorded by LEHMn_N~ and GaRz (1962) in the cotyledon of P. sativum and by C ~ E ~ Y (1963) in the cotyledon of Arachis. They found that the DNA content doubles, during the first few days, after which there is a decline. The decrease in azur B staining is also consistent with the results of LEKMA~N and GA~Z which indicate t h a t the RNA content of the cotyledon decreases from the beginning of germination. The scattered cells with a high initial I~NA content are a puzzling feature of the cotyledons. B ~ r and ME~CE~ [1966, (1)] described certain cells in the cotyledon of P. sativum in which the contents became disorganized to some extent during maturation of the seed. I t seems likely that these are identical with the scattered cells described here. BaI~ and 6*
84
D.L. SMIT]~and A. M. FLINN:
M]~Rc~,~ suggested t h a t they might be concerned with the transport of materials through the bulky cotyledon tissue. Their occurrence predominantly in the peripheral region, which is in close proximity to the vascular system, is rather against this suggestion. So also is the fact that the cells are scattered among the normal storage cells and, except near the periphery, are not usually in contact with one another. An alternative possibility is t h a t they are specialized as sites of enzyme synthesis. This view is based on the fact t h a t they show a relatively high activity of certain respiratory and hydrolytic enzymes (FLINN and SMITIt, in preparation). The zonation of reserve degradation in P. arvense differs from that found in some other legume cotyledons. 0PIK (1966) has shown that in Phaseolus degradation begins in the cells furthest removed from the vascular bundles and from the epidermis ; after 4 days the central regions have been exhausted while the cells around the vascular bundles and under the epidermis are still packed with reserves. Similarly, in Arachis cotyledons, BAGLEY et al. (1963) reported that breakdown of the reserves was delayed in cells round the vascular bundles. In contrast, in P. arvense degradation begins at the periphery and does not appear to show any correlation with the distance from the vascular bundles. The pattern of breakdown of the protein bodies by swelling, coalescence and subsequent fragmentation is similar to t h a t described in Arachis cotyledons b y BAGLEY et al. (1963) and in the embryo and perisperm of Yucca seeds b y HOR~ER and A ~ O T T (1965). In Phaseolus the protein bodies are reported to swell and coalesce and then to be replaced b y vacuoles ((JPIK, 1966). However, BMN and MERCER [1966, (2)] indicate t h a t the protein material in the protein bodies of the cotyledons of P i s u m sativum simply decreases during early germination. They do not mention any aggregation of the bodies. That such differences should exist between two closely related species is perhaps surprising but on the other hand HorNIeR and AI~NOTT (1965) have shown t h a t in seeds of Yucca schidigera the protein bodies in the embryo coalesce before breaking down whereas the protein from those in the perisperm disappears directly. The possibility cannot yet be excluded, however, t h a t the differences observed in the breakdown of the bodies in P. sativum and P. arvense could be attributable to differences in technique or even to differences in the growing conditions. Literature
ALF:EI~T,M., and I. I. GESC~WIN]): A selective staining method for the basic proteins of cell nuclei. Proc. nat. Acad. Sci. (Wash.) 39, 991--999 (1953). BAOL]~Y,B. W., J. H. C~E~RY, M. L. ROLLINS, and A. M. ALTSC~UL: A study of protein bodies during germination in pea-nut (Arachis hypogaea) seed. Amer. J. Bot. 50, 523--532 (1963).
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BAIN, J. M., and F. V. MERCER: (1) Subcellular organization of the developing cotyledons of P i s u m sativum L. Aust. J. biol. Sci. 19, 49--67 (1966). - - - - (2) Subce]lular organization of the cotyledons in germinating seeds and seedlings of P i s u m sativum L. Aust. J. biol. Sci. 19, 69--84 (1966). - - (3) The relationship of the axis and the cotyledons in germinating seeds and seedlings of P i s u m sativum L. Aust. J. biol. Sci. 19, 85--96 (1966). BISALPUTRA,It., and K. EsAv: Polarized light study of phloem differentiation in embryo of Chenopodium album. Bot. Gaz. 125, 1--7 (1964). C~ERRu J. H. : Nucleic acid, mitochondria and enzyme changes in cotyledons of peanut seeds during germination. Plant Physiol. 38, 440~446 (1963). COOPER, D. C.: Embryology of P i s u m sativum. Bot. Gaz. 100, 123--132 (1938). DAUPt~NE, A., et S. ]~IVIERE: Sur la pr6sence des tubes cribl6es darts des embryons de graines non germ6es. C. R. Acad. Sci. (Paris) 211, 359--361 (1940). FLAx, M. H., and M. H. HI~ES: Microspectrophotometric analysis of metachromatic staining of nucleic acids. Physiol. Zool. 25, 297--311 (1952). FLINN, A. M., and D. L. S~IT~: The localization of enzymes in the cotyledons of P i s u m arvense L. during germination. Planta (In Press). FOSTER, A. S. : Comparative morphology of the foliar sclereids in Borouella Baill. J. Arnold Arboretum (Harvard Univ.) 36, 189--198 (1955). HAYWAR]), H, E. : The structure of economic plants. New York: Macmillan 1938. HOR~ER, H. T., and H. J. AR~OTT: A histochemical and ultrastructural study of Yucca seed proteins. Amer. J. Bot. 52, 1027--1038 (1965). JEI~SElV, W. A.: Botanical histochemistry, principles and practice. San Francisco: Freeman 1962. I~URNICK, N. B. : Pyronin Y in the methyl-green-pyronin histological stain. Stain Technol. 30, 213--230 (1955). LEH~A~N, K., and J. G ~ z : Untersuchnngen fiber den Umsatz der phosphathaltigen Substanzen und des Eiwei2es in den Kotyledonen keimender Erbsensamen. Flora (Jena) 152, 516--522 (1962). 0P~K, H. : Changes in cell fine structure in the cotyledons of Phaseolus vulgaris L. during germination. J. exp. Bot. 17, 4 2 7 ~ 3 9 (1966). REEVE, R. M. : Late embryogeny and histogenesis in Pisum. Amer. J. Bot. 3~, 591--602 (1948). V_4_RNER,J. E., L. V. BALCE, and R. C. HUANO: Senescence of cotyledons of germinating peas. Influence of axis tissue. Plant Physiol. 38, 88--92 (1963). - - , and G. SCHIDI,OVSKY: Intracellular distribution of proteins in pea cotyledons. Plant Physiol. 38, 139--143 (1963). -
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Dr. D. L. SMITH Department of Botany, The Queen's University Belfast 7, Northern Ireland
HISTOLOGY AND HISTOCHEMISTI~Y OF THE COTYLEDONS O F P I S U M A R V E N S E L. D U R I N G G E R M I N A T I O N D. L. SMITH and A. M. FLI~ Botany Department, Queen's University, Belfast, l~orthern Ireland Received l~ovember 17, 1966 Summary. The mature cotyledon of Pisum arvense L. comprises several distinct tissue regions; these are the epidermis, hypodermis, storage parenchyma and procambium. The storage parenchyma includes two zones: an outer abaxial zone and an inner adaxial zone. The cells of both zones contain abundant starch grains and protein bodies. Scattered through the storage tissue but increasing in frequency towards the periphery are certain cells which differ to a slight extent from the majority of the parenchyma cells. They have a more opaque, granular cytoplasm and a higher level of cytoplasmic RI~A. The cotyledon has a complex, reticulate vascular system. Differentiation of the conducting elements from the procambium appears to begin about 12 hours and to be completed 48 hours after the commencement of imbibition. Differentiation of phloem preceeds that of xylem. The relationship between the timing of vascular differentiation and various physiological events in the cotyledon is discussed. Mobilization of the reserves in the storage parenchyma is initiated at the periphery of the cotyledon and proceeds inwards. There appears to be a correlation between the breakdown of the reserves and changes in DNA and RNA content of the cells.
Introduction Considerable i n f o r m a t i o n is n o w a v a i l a b l e on t h e physiological a n d biochemical changes which occur d u r i n g t h e g e r m i n a t i o n of pea seeds, p a r t i c u l a r l y those r e l a t i n g to t h e d i s a p p e a r a n c e of reserve proteins, carb o h y d r a t e s a n d fats from t h e cotyledons. I n f o r m a t i o n of this t y p e is of n e c e s s i t y b a s e d l a r g e l y on i n v e s t i g a t i o n s carried o u t a t t h e tissue r a t h e r t h a n a t t h e cellular level a n d m e t a b o l i c differences b e t w e e n t h e compon e n t cells are neglected. To some e x t e n t t h e s i t u a t i o n has been rectified b y r e c e n t studies concerned w i t h t h e u l t r a s t r u e t u r e of c o t y l e d o n cells d u r i n g g e r m i n a t i o n . VA~N~R a n d SCHIDLOVSKY (1963) i n v e s t i g a t e d t h e i n t r a c e l l u l a r d i s t r i b u t i o n of p r o t e i n in t h e cotyledons a n d BAI~ a n d MERCER [1966, (2)] r e l a t e d physiological changes in t h e cotyledons to u l t r a s t r u c t u r a l changes in t h e s t o r a g e cells of t h e cotyledons. The inter-relat i o n s h i p b e t w e e n t h e e m b r y o n i c axis a n d t h e cotyledons has been investig a t e d a t t h e physiological level b y V A ~ n R et al. (1963) a n d a t t h e physiological a n d u l t r a s t r u c t u r a l levels b y BAI~ a n d M ~ c E ~ [1966, (3)]. T h e y h a v e shown t h a t t h e storage reserves in d e t a c h e d cotyledons do n o t b r e a k d o w n to soluble form unless excision of t h e axis is d e l a y e d u n t i l t w o d a y s a f t e r t h e initial soaking.
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A n a c c o u n t of t h e d e v e l o p m e n t of t h e e m b r y o of P i s u m s a t i v u m L. was g i v e n b y CooPeR (1938) a n d a more detailed account, including t h e histogenesis, was g iv e n b y RE]~v~ (1948). HAYWARD (1938) described briefly t h e d e v e l o p m e n t a n d a n a t o m y of t he seed a n d cited earlier anat o m i cal work. N o n e of these previous studies has y e t p r o v i d e d a c o m p r e h e n s i v e a c c o u n t of t h e a n a t o m y of t h e m a t u r e e m b r y o an d so far little a t t e m p t has been m a d e to localize physiological e v e n t s within t h e cotyledons. The p res en t s t u d y has th r e e m a i n aims: to describe histological changes which occur during g e r m i n a t i o n , in p a r t i c u l a r t h e differentiation of t h e v a s c u l a r s y s t e m ; a n d to follow h is t o c h e m ic a l l y changes in certain cellular components. Materials and Methods Seeds of P i s u m arvense L. were soaked in water for four hours and then planted in moist vermiculite. They were then incubated in the dark at 25~ Samples were taken at 6, 8, 10, 12, 14, 15, 16, 17, 18, 20, 24, 32, 40, 42, 46 and 48 hours and 3, 5, 7, 9, and 12 days from the commencement of the initial soaking. Whole cotyledons were fixed in FAA or glutaraldehyde. Fixed cotyledons used for tracing the morphology and differentiation of the vascular system were cleared by a modification of the technique of FOSTE~ (1955) and BISALPV~A and EsAv (1964). Whole cotyledons were treated with 2 % NaOH at 600 C for 1 to 2 hours. They were then washed thoroughly and placed in an acidified solution of chloral hydrate under ~acuum at 60~ until clear (4 to 8hours). Cotyledons were examined whole in the clearing solution or selected parts were cut out, squashed and mounted in the clearing solution, and examined under polarized light. The walls of the sieve tubes and tracheary elements show conspicuous bierfringence in polarized light and this feature can be utilized in tracing the course of differentiation of the xylem and phloem (BIsALeVT~ and EsAv, 1964). For histological examination slices of cotyledons were dehydrated through an ethanol-n-butanol series and embedded in "Paraplast" wax. Sections were cut at 10 ~ and stained in a safranin-fast green combination. For the histochemical localization of cellular components i0 ~ sections were cut as above and then the following procedures were used: 1. D N A . Sections were stained by the standard Feulgen technique. 2. D N A and R N A . (a) Sections were stained using the modified methyl greenpyronin technique of KvR~IeI; (1955). This method proved unreliable as significant amounts of methyl green were retained by the cell wall. (b) Sections stained in azur B (FLAx and HIM]~s, 1952; JENSEN, 1962) proved more satisfactory. Controls were either treated for 1.5 hours with a solution of 0.1% ribonuclease in water adjusted to pH 6.8 with NaOH or were incubated for the same period in water alone at p i t 6.8. 3. Total protein. The ninhydrin-Sehiff's reagent method of YASV~A and ICnIK ~ A was used (JE~sE~, 1962). 4. Histories. Sections were stained by the fast green method of ALFERT and GEsc~n (1953). 5. Carbohydrates. Sections were stained by the standard periodic acid-Schiff's reagent technique. Iodine-potassium iodide solution was used for the specific detection of starch.
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D.L. SMITE and A. M. FLINN:
Anatomy and Histology of the Cotyledon The cotyledon is approximately hemispherical in shape with the flat face corresponding to the adaxial face of the leaf. I t is bounded b y an epidermis (Figs. 1,2) from which stomata are absent except in the region adjacent to the embryonic axis. Below the epidermis is a single row of cells comprising the hypodermis. The storage parenchyma makes up the bulk of the cotyledon. I t appears to consist of two distinct zones, an outer zone corresponding to the spongy parenchyma of the leaf and an inner zone corresponding to the palisade tissue. Both zones are much less distinctive than the corresponding tissues in the leaf. The cotyledon possesses a well developed vascular system which runs more or less along the boundary of the inner and outer storage tissues. In the mature embryo it consists entirely of procambium ; no mature xylem or phloem elements have been detected in it. Differentiation of vascular tissues from the procambinm occurs during the first two days of germination. The Epidermis and Hypodermis. The epidermal cells arc small, narrow and elongated. They are usually in the region of 10 ~ wide and 100 long. They are arranged parallel to one another in small groups of about eight cells (Fig. 3), each group apparently comprising the derivatives of one of the original meristematic cells of the protoderm. The orientation of these groups is not constant though they tend to be elongated along the axis of the cotyledon. The cells are not, on the whole, arranged end to end in rows as described in P. sativum by HAYWAI~D(1938), although this tendency is shown to some extent b y the cells of the adaxial epidermis. The hypodermis consists of more or less isodiametric cells which often tend to be slightly flattened in a plane parallel to the surface. Both the epidermis and the hypodermis contain cytoplasmic protein. I t does not, however, appear to be particulate but this could possibly be due to the small size of the protein bodies. Small starch grains are present in the hypodermis but have not been seen in the epidermis. In both layers the nuclei are normal in appearance ; the nucleoli stain deeply with azur B. The Storage Parenchyma. The cells of the two zones of the storage parenehyma differ from each other in size and shape. In seetions in any plane the cells of the inner storage tissue, t h a t underlying the adaxial surface, are isodiametrie and rounded in appearance. They are up to 130 ~ in diameter. In contrast, the cells of the outer storage tissue, underlying the abaxial surface, are larger and more irregular in shape. They tend to be elongated perpendicular to the surface and m a y be up to 140 ~ in diameter and 230 ~ long. The cells of both tissues are eompactly but irregularly arranged, with small triangular intercellular spaces confined to the corners of the cells (Fig. 5). During germination the size of the intercellular spaces increases
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75
Figs. 1 a n d 2. Sections t h r o u g h the peripheral region of a cotyledon, stained in azur B, showing the epidermis, h y p o d e r m i s a n d outer storage p a r e n c h y m a . Fig. 1. D a y 1 ; higher levels of R N A are present in the epidermis, h y p o d e r m i s a n d some of the storage cells. Fig. 2. D a y 9; the storage reserves are completely exhausted. • 200
and larger, more irregular spaces develop b y union of the small spaces initially present. This gives rise to a complex reticulum of more or less tnbu]ar spaces (Fig. 4) which penetrates the whole tissue. I t seems t h a t
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D.L. S~ITn and A. M. FLI~N:
the walls of adjacent cells separate along the middle lamella. The cell walls are thin and nn]Jgnified. The contact areas between adjacent cells are more or less oval and show numerous primary pit fields (Fig. 4). The nuclei of both types of cell are more or less spherical in shape but have a rather diffuse appearance. The nncleoli are visible but do not stain with azur B. All the cells contain large starch grains and small protein bodies (Fig. 5). The latter are more or less spherical in shape and
Fig. 3. Surface view of p a r t of the epidermis of a cotyledon treated with N a O t I a n d chloral hydrate. The group of eight cells a p p a r e n t l y represent the derivatives of a single protodermal cell. Phase contrast. • 500 Fig. 4. Surface view of the contact area between two storage cells of a 5 day cotyledon. The u n p i t t e d wall surrounding the contact area adjoins the greatly enlarged intercellular space. Stained in periodic acid-Schiff's reagent. • 650
up to 3 ~ in diameter. They stain red-purple after treatment with ninhydrin-Schiff's reagent. The starch grains are up to 50 ~ long. Scattered through the storage tissue but increasing in frequency towards the periphery and forming an almost continuous layer under the hypodermis are cells which differ slightly from the majority of the parenehyma cells. Although the cell wall, nucleus, starch grains and protein bodies are comparable with those of other cells, these cells have a more opaque, finely granular cytoplasm. Under dark field illumination they become particularly obvious because of this opacity: they appear white or grey against a black background (Fig. 8). Their cytoplasmic R N A content, as shown by azur B staining, is relatively high (Fig. 1).
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Figs. 5--7. Sections of storage parenchyma cells showing changes in storage reserves during germination. Stained in ninhydrin-Schiff's reagent, s, intercellular space; p, protein body; g, starch grain. Fig. 5. An outer storage cell at day 1. Fig. 6. An inner storage cell of a 5 day cotyledon showing the swollen protein bodies. Fig. 7. An outer storage cell of a 5 day cotyledon showing the aggregate protein bodies containing deeply staining inclusions. Only one starch grain remains. • 650
The Vascular System. The cotyledon has a complex reticulate vascular system (Fig. 9). Two vascular strands enter the base of the cotyledon from the embryonic axis. One of these, the larger of the two, forks in the petiole and then each branch forks, thus forming four
78
D.L. SMITEand A. M. FLINN: main veins. The smaller strand entering the cotyledon forms a fifth main vein. Branching and anastomosis of these five main veins occurs as in a normal leaf. The morphological interpretation of the vascular system is complicated by the presence of two unequal strands in the base of the cotyledon. The most likely interpretation is that the larger strand represents the product of fusion of two foliar strands while the smaller represents a single stipular trace.
Fig. 8. U n s t a i n e d section of the outer storage tissue of a fixed 5 day cotyledon under dark field illumination. Only the ceils with the more opaque, granular cytoplasm are visible. • 330
The cells of the procambium are mainly long and narrow, 7 to 12 ~ in diameter and 40 to 100 ~ long. The longer, narrower cells tend to occur towards the centre of the bundle
and
are apparently
the potential
l~ig. 9. Camera lucida drawing of the vascular system of a day 1 cotyledon cleared in )~aOH-chloral
hydrate
Histology and Histochemistry of the Cotyledons during Germination
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conducting elements. The shorter cells towards the periphery give rise to the parenehyma of the bundle sheath. The walls of some of the long central cells occasionally stand out because of their stronger birefringence under polarized light. This is presumed to indicate t h a t differentiation of these cells has begun and that they will give rise to sieve-tube members of the protophloem.
Differentiation of the Vascular Strands Differentiation of the vascular tissue from the procambium was first observed 12 to 17 hours after the commencement of soaking. The first detectable change was an increase in the birefringence of the elongated cells in the centre of the strand. The first cells to differentiate were those of the protophloem of the main vein in the basal region of the cotyledon. Those of the smaller vein began shortly afterwards. Normally two or three sieve elements are recognizable before the walls of the protoxylem elements show any evident birefringence. The first mature protoxylem was seen at 16 hours. At any one point in the larger veins up to five sieve elements m a y be present by the time the first protoxylem element is fully differentiated. As far as could be determined differentiation of the phloem occurred in a strictly acropetal sequence from the basal region of the cotyledon. Differentiation of the xylem in the main veins also appeared to be acropetal but in at least some branch veins the protoxylem matured some little distance away from the main vein; subsequently differentiation occurred both towards and away from the main vein. Mature phloem was detected in the distal region of some cotyledons after 20 hours and mature xylem after 24 hours from the commencement of soaking. This, however, is probably exceptional and 30 to 32 hours is perhaps a more normal time for differentiation to have reached this region. The time taken for complete differentiation of the strands was difficult to determine precisely but strands which were apparently fully differentiated were observed at the distal end at 42 to 48 hours. In transverse section a mature strand is seen to comprise five to twenty-five xylem elements and a slightly larger group of phloem elements (Fig. 10), the wholebeing surrounded b y a parenehymatous bundle sheath. The longitudinal walls of the parenchyma cells contain abundant primary pit fields. Histoehemical Changes during Germination D N A and R N A . As has already been indicated the Feulgen positive
material of the nucleus appears to be rather diffuse in the storage parenchyma cells but compact in the cells of the epidermis, hypodermis and procambium. During germination the nucleus of the parenchyma cells increases in size and irregularity (Fig. 11), becoming conspicuously lobed
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D.L. SMIT~ and A. M. F L I ~ :
Fig. 10. Transverse section of a vascular s t r a n d of a day 1 cotyledon, stained in azur B. The I~NA content of the differentiating xylem (x) is decreasing while t h a t of the phloem (p) shows little change. • 500 Fig. 11. Section showing the ]obed nucleus of an inner storage cell of a day 5 cotyledon. Feu]gen stain. x 1200 Fig. 12. Section showing the fast green staining of the nucleus of a storage cell. x 1200 Fig. 13. Section of a storage cell of a day 5 cotyledon showing the small starch grains r o u n d the nucleus, a n d the large storage grains. Stained in periodic acid-Schiff's reagent. • 500
i n certain cases. I n i t i a l l y there is no change i n staining i n t e n s i t y associated with the increase i n size b u t as the storage reserves of the cotyledons
Histology and Histochemistry of the Cotyledons during Germination
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become depleted the staining becomes less intense. No changes were noted in the Feulgen positive material of the nuclei of the other regions of the cotyledons and no mitotic figures have been observed at any time, even in the procambium. The cells of the epidermis, hypodermis and procambium, showed heavy cytoplasmic staining with azur B. The cells of the storage parenchyma, with the exception of the scattered cells already mentioned, showed only slight staining. The parenchyma cells in the cotyledon petiole stain fairly heavily. Sections pre-treated with ribonuclease showed no cytoplasmic staining. During the first three days of germination the cells of the petiole, the epidermis and hypodermis, and the scattered parenchyma cells lose their heavy staining and have a staining density which is little different from that of the storage parenchyma cells. As the procambium undergoes differentiation into mature conducting elements the future xylem loses its staining intensity but the staining of the other cells remains unchanged or possibly even increases (Fig. 10). Nucleo]ar staining for RNA shows an interesting pattern in the cotyledon. In the early stages of germination the nucleoli of only the outer cell layers, the epidermis, hypodermis and some underlying cells, and the procambiM cells show staining with azur B. However, after the first two days the staining of the nucleolus has progressed from the periphery. The nucleoli of the outer layer of storage cells stain while the staining of those towards the periphery becomes less intense. In effect, a wave of nucleolar staining passes from the periphery towards the centre of the cotyledon. The staining is at its most intense in a given cell when breakdown of the reserves begins; it decreases as the reserves diminish. The nucleoli of the parenchyma cells of the bundle sheath are the last to lose their stainability. Protein. Initially the protein bodies (Fig. 5) in the storage parenchyma cells are up to 3 ~ in diameter and stain red-purple with ninhydrinSchiff's reagent. The inner cells appear to contain more of them per unit volume than do the outer cells. During the first two days of germination these protein bodies appear to enlarge to at least twice their original diameter (Fig. 6) and subsequently to fuse together in groups to form larger aggregate bodies (Fig. 7). These may be 25 ~ or more in diameter and they stain more intensely than did the original bodies. Also the staining changes from red-purple to a definite red. This change in intensity and colour of the staining is perhaps due to the colour reaction with small peptides rather than with large proteins. Small, very deeply staining bodies become apparent in the protein bodies after coalescence (Fig. 7). Ultimately the large aggregate bodies appear to disintegrate and to form small, more irregular bodies which finally disappear. At the tissue level, the protein first disappears from the peripheral cells and those in the 6
P l a n t a (Berl.), Bd. 74
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D.L. SMITHand A. M. F L I ~ :
basal region of the cotyledon. This is not because there is less protein in the outer cells but is attributable to a greater rate of and an earlier initiation of lysis. Thus, breakdown is initiated in the outer cells and then spreads inwards. When the aggregation stage of protein degradation has been reached in the peripheral cells the protein bodies towards the centre are still at the swollen stage. The protein disappears completely from the outer layers after about five days. After about twelve days the storage parenchyma is almost completely free of protein and only the nuclear proteins stain.
Histones. Only a brief study of histones was made using the fast green method. Only the nucleus stains (Fig. 12), there being no evident cytoplasmic staining. The nueleolus appears to stain with an intensity the same as or less than the rest of the nucleus. The appearance of the nuclei of the storage cells is similar to t h a t after Feulgen staining in t h a t the nuclear material is dispersed and t h a t after the storage reserves become depleted staining is less intense. Starch. Initially starch is present in all the cells except those of the epidermis and possibly the potential conducting cells of the procambium. I t is present in large quantities in the storage parenchyma, particularly in the inner zone. The hypodermis and the proeambinm (subsequently the parenchyma of the bundle sheath) contain only very small grains which disappear within the first three days. The pattern of degradation of starch in the storage parenchyma is similar to t h a t of protein : degradation begins at the periphery of the cotyledon and proceeds in a wave towards the centre. During the first seven days the outer storage cells become depleted of their starch and after twelve days no starch remains in the central core. During the first two days small starch grains, mostly in the size range 3 to 15 ~, appear in the storage cells. Usually, they become aggregated round the nucleus (Fig. 13). They have disappeared from the outer region of the cotyledon by day 5 and from the central core by day 7. Discussion Differentiation of the embryo during germination has been investigated in few species of plants. But in those cases which have been examined it has been shown consistently t h a t the main event is the differentiation of the vascular tissues from the procambium. The sequence of differentiation shows some variation between species, however, and there appears to be no constant pattern (BISALPUTRAand ESAU,1964). The pattern in Pisum arvense, where differentiation of phloem preceeds that of xylem, is similar to the pattern described in Helianthus, Mirabilis, Lupinus, and Ricinus (DAUPHINE et l~IvI]~]~, 1940) and Chenopodium (BISALPUTgA
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and EsAu, 1964). I n Chenopodium, as in Pisum, the protophloem is partly differentiated in the mature, dormant embryo but in Lupinus fully differentiated sieve elements are present. The timing of the vascular differentiation in relation to physiological events in the cotyledon is of particular interest. Differentiation of the phloem was first observed 12 hours after the commencement of soaking and differentiation of the xylem apparently began about 4 hours later. A continuous conducting system through the cotyledon was present after 24 to 32 hours and differentiation was complete after 40 to 48 hours. The period from 16 to 32 hours is probably particularly important since during this period the differentiation of xylem is initiated and a continuous conducting system is laid down. VARNE~ et M. (1963) have shown t h a t m a n y of the metabolic events which occur in the cotyledons of P. sativum are triggered off and controlled by stimuli from the embryonic axis. If cotyledons are excised during the first 24 to 48 hours they undergo a rapid decline in respiration rate and fail to develop their characteristic enzymatic activities. At the ultrastructural level B ~ N and MERCEa [1966, (3)] have shown t h a t the completion of the mitochondria and the development of the endoplasmic reticulum of the storage cells is accomplished in the period between 24 and 48 hours and t h a t completion can occur only in the presence of axis tissue. According to V ~ N E g et al. these various events are initiated by some factor which passes from the axis into the cotyledons sometime after the first 18 hours of germination. The timing of this movement thus coincides approximately with the differentiation of the first xylem in the cotyledon. I t seems likely t h a t until a continuous strand of vascular tissue is present the movement of substances into the cotyledon would not occur or would be slow, and t h a t the passage of the stimulus from the axis is at least partly dependent on the differentiation of the vascular system. The increase in size of the nuclei of the storage cells during the first few days of germination and their subsequent decrease in staining intensity are in agreement with quantitative changes in DNA recorded by LEHMn_N~ and GaRz (1962) in the cotyledon of P. sativum and by C ~ E ~ Y (1963) in the cotyledon of Arachis. They found that the DNA content doubles, during the first few days, after which there is a decline. The decrease in azur B staining is also consistent with the results of LEKMA~N and GA~Z which indicate t h a t the RNA content of the cotyledon decreases from the beginning of germination. The scattered cells with a high initial I~NA content are a puzzling feature of the cotyledons. B ~ r and ME~CE~ [1966, (1)] described certain cells in the cotyledon of P. sativum in which the contents became disorganized to some extent during maturation of the seed. I t seems likely that these are identical with the scattered cells described here. BaI~ and 6*
84
D.L. SMIT]~and A. M. FLINN:
M]~Rc~,~ suggested t h a t they might be concerned with the transport of materials through the bulky cotyledon tissue. Their occurrence predominantly in the peripheral region, which is in close proximity to the vascular system, is rather against this suggestion. So also is the fact that the cells are scattered among the normal storage cells and, except near the periphery, are not usually in contact with one another. An alternative possibility is t h a t they are specialized as sites of enzyme synthesis. This view is based on the fact t h a t they show a relatively high activity of certain respiratory and hydrolytic enzymes (FLINN and SMITIt, in preparation). The zonation of reserve degradation in P. arvense differs from that found in some other legume cotyledons. 0PIK (1966) has shown that in Phaseolus degradation begins in the cells furthest removed from the vascular bundles and from the epidermis ; after 4 days the central regions have been exhausted while the cells around the vascular bundles and under the epidermis are still packed with reserves. Similarly, in Arachis cotyledons, BAGLEY et al. (1963) reported that breakdown of the reserves was delayed in cells round the vascular bundles. In contrast, in P. arvense degradation begins at the periphery and does not appear to show any correlation with the distance from the vascular bundles. The pattern of breakdown of the protein bodies by swelling, coalescence and subsequent fragmentation is similar to t h a t described in Arachis cotyledons b y BAGLEY et al. (1963) and in the embryo and perisperm of Yucca seeds b y HOR~ER and A ~ O T T (1965). In Phaseolus the protein bodies are reported to swell and coalesce and then to be replaced b y vacuoles ((JPIK, 1966). However, BMN and MERCER [1966, (2)] indicate t h a t the protein material in the protein bodies of the cotyledons of P i s u m sativum simply decreases during early germination. They do not mention any aggregation of the bodies. That such differences should exist between two closely related species is perhaps surprising but on the other hand HorNIeR and AI~NOTT (1965) have shown t h a t in seeds of Yucca schidigera the protein bodies in the embryo coalesce before breaking down whereas the protein from those in the perisperm disappears directly. The possibility cannot yet be excluded, however, t h a t the differences observed in the breakdown of the bodies in P. sativum and P. arvense could be attributable to differences in technique or even to differences in the growing conditions. Literature
ALF:EI~T,M., and I. I. GESC~WIN]): A selective staining method for the basic proteins of cell nuclei. Proc. nat. Acad. Sci. (Wash.) 39, 991--999 (1953). BAOL]~Y,B. W., J. H. C~E~RY, M. L. ROLLINS, and A. M. ALTSC~UL: A study of protein bodies during germination in pea-nut (Arachis hypogaea) seed. Amer. J. Bot. 50, 523--532 (1963).
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BAIN, J. M., and F. V. MERCER: (1) Subcellular organization of the developing cotyledons of P i s u m sativum L. Aust. J. biol. Sci. 19, 49--67 (1966). - - - - (2) Subce]lular organization of the cotyledons in germinating seeds and seedlings of P i s u m sativum L. Aust. J. biol. Sci. 19, 69--84 (1966). - - (3) The relationship of the axis and the cotyledons in germinating seeds and seedlings of P i s u m sativum L. Aust. J. biol. Sci. 19, 85--96 (1966). BISALPUTRA,It., and K. EsAv: Polarized light study of phloem differentiation in embryo of Chenopodium album. Bot. Gaz. 125, 1--7 (1964). C~ERRu J. H. : Nucleic acid, mitochondria and enzyme changes in cotyledons of peanut seeds during germination. Plant Physiol. 38, 440~446 (1963). COOPER, D. C.: Embryology of P i s u m sativum. Bot. Gaz. 100, 123--132 (1938). DAUPt~NE, A., et S. ]~IVIERE: Sur la pr6sence des tubes cribl6es darts des embryons de graines non germ6es. C. R. Acad. Sci. (Paris) 211, 359--361 (1940). FLAx, M. H., and M. H. HI~ES: Microspectrophotometric analysis of metachromatic staining of nucleic acids. Physiol. Zool. 25, 297--311 (1952). FLINN, A. M., and D. L. S~IT~: The localization of enzymes in the cotyledons of P i s u m arvense L. during germination. Planta (In Press). FOSTER, A. S. : Comparative morphology of the foliar sclereids in Borouella Baill. J. Arnold Arboretum (Harvard Univ.) 36, 189--198 (1955). HAYWAR]), H, E. : The structure of economic plants. New York: Macmillan 1938. HOR~ER, H. T., and H. J. AR~OTT: A histochemical and ultrastructural study of Yucca seed proteins. Amer. J. Bot. 52, 1027--1038 (1965). JEI~SElV, W. A.: Botanical histochemistry, principles and practice. San Francisco: Freeman 1962. I~URNICK, N. B. : Pyronin Y in the methyl-green-pyronin histological stain. Stain Technol. 30, 213--230 (1955). LEH~A~N, K., and J. G ~ z : Untersuchnngen fiber den Umsatz der phosphathaltigen Substanzen und des Eiwei2es in den Kotyledonen keimender Erbsensamen. Flora (Jena) 152, 516--522 (1962). 0P~K, H. : Changes in cell fine structure in the cotyledons of Phaseolus vulgaris L. during germination. J. exp. Bot. 17, 4 2 7 ~ 3 9 (1966). REEVE, R. M. : Late embryogeny and histogenesis in Pisum. Amer. J. Bot. 3~, 591--602 (1948). V_4_RNER,J. E., L. V. BALCE, and R. C. HUANO: Senescence of cotyledons of germinating peas. Influence of axis tissue. Plant Physiol. 38, 88--92 (1963). - - , and G. SCHIDI,OVSKY: Intracellular distribution of proteins in pea cotyledons. Plant Physiol. 38, 139--143 (1963). -
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Dr. D. L. SMITH Department of Botany, The Queen's University Belfast 7, Northern Ireland
