Planta (Berl.) 95, 314--329 (1970) 9 by Springer-Verlag 1970
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize B. E. J u ~ I r E ~ and A ~ A F~w~c~ Botany School, University of Oxford, England l~eceived August 24/September 20, 1970
Summary. Within the root cap, in maize, the cells believed to be responsible for the perception all possess large well-developed amyloplasts. They also have normal mitochondria and Golgi bodies, normal rough-surfaced Et~ with a very striking pattern of distribution, few free ribosomes, walls with an abnormal reticulate encrnsting material, irregularly distributed plasmodesmata and an as yet unidentified fine quadruple membranous system. All of these features are discussed in relation to the role of the cells in perception. Introduction The group of cells t h a t perceives gravity in maize (Zea mays) lies within the root cap. As Fig. 1 shows, a clear boundary, rarely if ever crossed by cell divisions once the structure of the root has been formed, exists between the cap and root proper. We shall call this feature, found in all grasses, the cap boundary (C.B.). The existence of the boundary makes possible the experiments of Juniper et al. (1966), since confirmed by Konings (1968) and Gibbons and Wilkins (1970) t h a t removal of the root (decapping) prevents the normally growing root from perceiving a gravitational stimulus. I t can be surmised, but not proved, that a similar situation exists in dicotyledons. The root cap of dicotyledons can only be removed by cutting (decapitating). Such experiments have been done, by Syre (1939) and Konings (1968) and confirm the general hypothesis stated above, but are open to some criticism on the grounds of wound trauma induced by the surgery. The cells of the root cap are produced by a meristem (M in Fig. 1) a group, according to Clowes (1970), of about 6600 cells of which, in some varieties of maize, 4400 divide as rapidly as once every 10 hours. These cells do not contain mature amyloplasts, possess only undifferentiated proplastids, have no special characteristics to distinguish them from any other meristematic cells and are not thought to be responsible for the perception of the stimulus. The re-establishment of the meristem, which takes place after decapping, is not sufficient to restore the ability to perceive (Schachar, 1967). Cercek (1970), working with barley
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize 315
Fig. 1. Outline drawing of the cells of the first 1 mm of the primary root of maize
(Zea) (Hordeum vulgare), found t h a t roots from which all the differentiated cells of the cap had been cut, but which still possessed a cap meristem, also failed to respond to gravity. Surrounding the whole cap is a layer two or three cells thick (P.C. in Fig. 1) of highly specialized cells. These peripheral cap cells have been described in detail b y Mollenhauer et al. (1961), Northcote and Pickett-Heaps (1966) and Juniper and Roberts (1966). These cells do not possess complete amyloplasts, nor do these incomplete amyloplasts sediment when exposed to a gravitational stimulus. The very curious structure and patterns of behaviour of these cells have already been well described. They are not thought, for reasons which will be given more fully below, to be involved in the perception of gravity, but are thought to provide protection to the root cap as a whole as the root drives through the soil. The central cap cells (C.C. in Fig. 1) on the other hand were supposed b y N6mec (1900, 1902) to be the site of the perception of gravity on the grounds that only these cells possessed movable amyloplasts, whose movements could be correlated with the imposition of a gravitational stimulus. 21"
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Following on from J u n i p e r ' s work i n 1966, Schachar (1967) was able to show t h a t deeapped roots soon recovered the ability to perceive gravity, b u t were able to do so j u s t before the whole cap was complete i.e. when the central cap cells, b u t n o t the peripheral cap cells were present. She thus confirmed Ngmec's earlier suppositions. Cereek (1970) f o u n d also t h a t in barley no more t h a n 80% of the root cap was necessary for a full 90 ~ response to a g r a v i t a t i o n a l stimulus. The region of central cap cells, those u n h a t c h e d or d o t t e d in Fig. 1, we shall call the core. W i t h i n the core each cell is replaced a b o u t every 24 hours (Clowes, 1970).
Materials and Methods Seeds of Zea mays, variety Kelvedon 33, were germinated on filter paper or in sphagnum moss and when the primary root was about 2-3 cm long were fixed and stained in the following ways. a) In 2 % glutaraldehyde in cacodylate buffer at room temperature for 2 hours followed by 2 hours in 2% OsOt also in caeodylate buffer in crushed ice. b) In 2% glutaraldehyde for 2 hours followed by 2 hours in 2% KMn04, all at room temperature, e) Directly into 2% KMnOa for 2 hours at room temperature where a rapid fixation was wanted. Some roots were taken from the sphagnum, clipped unbroken into a perspex frame and given a specific orientation for a given time before fixation. The level of the fixative was raised around the roots clamped in their determined orientation; the roots were not moved into the fixative. After fixation the roots were embedded in Epikote 812 resin, sectioned on a Cambridge Huxley or a geichert OMU2 microtome and examined in an AEI E.NL6 or Philips 200 electron microscope. For some of the measurements large numbers of individual micrographs were taken at • 1500 of 2-3 adjacent serial sections and built up into complete mosaics of the root cap, to give a final magnification of X 3 800.
Results The Amyloplasts E a c h cell of the core of the root cap contains a b o u t 35 amyloplasts ( J u n i p e r a n d Clowes, 1965). I t is interesting t h a t this is a b o u t the same n u m b e r of statoliths as observed b y Sievers (1967a a n d b) i n the rhizoids of Chara. Once they have a c c u m u l a t e d a large a m o u n t of starch (Fig. 2) t h e y do n o t seem to divide again although occasionally images are seen of amyloplasts, with a small a m o u n t of starch, a p p a r e n t l y in the process of dividing. Thus most of the divisions giving rise to their total n u m b e r s are t h o u g h t to take place in the proplastid state in the cap meristem ( J u n i p e r a n d Clowes, 1965). This mode of origin seems to differ from t h a t of chloroplasts where most divisions seem to take place in the m a t u r e state (Possingham a n d Sauer, 1969). All of the amyloplasts in the core are, therefore~ of comparable size a n d shape a n d c o n t a i n m a n y distinct starch grains (Figs. 2 a n d 3).
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Fig. 2. Electron micrograph of a group of core cells of maize fixed in KMnO 4. A amyloplasts; N nuclei; TS W transverse wall; L W longitudinal wall
Fig. 3. Electron micrograph of a group of amyloplasts from core cells fixed in glutaraldehyde, post-fixed in O s Q and stained with uranyl acetate and lead citrate. Note that the starch grains of the amyloplasts stain black with Kmn04, but react only weakly with OsO 4. V vacuoles, W wall
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Fig. 4. A single umyloplast fixed and stained as in Fig. 3. 0 osmiophilie bodies
Fig. 5. Part of a single amyloplast fixed and stained as in Fig. 3. K knot of membranes
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize 319 Apart from starch they contain only fragmentary lamellar structures usually parallel to the outer membrane. Another consistent feature is a small group of osmiophilic bodies associated with a " k n o t " of membranes of both lamellar and fibrillar form (Figs. 4 and 5). No organelle ribosomes have ever been seen in the amyloplasts although they appear to be present in the mitochondria (Fig. 6). The amyloplasts of the peripheral cap cells begin to lose their starch (Fig. 7) which m a y explain their inability, already noted, to sediment under a gravitational stimulus. The accumulation and subsequent loss of starch by the amyloplasts deserves mention. According to Clowes (1970) each root cap is totally regenerated by its own meristem within 24 hours. Therefore each of m a n y thousands of amyloplasts every day accumulates and discharges about 14/~m 3 of starch. In the normally growing vertical position the amyloplasts accumulate at the bottom of the cells. They are never seen to be wrapped by the ER.
The ER (Endoplasmic Reticulum) In the meristematic cells the EI~ is fragmentary and has no obvious preferred orientation (Fig. 10). However, as the cells cease to divide, begin to expand and to accumulate starch in their amyloplasts, the E R becomes orientated. The E R in an undisturbed root then lies preferentially parallel to the cell walls and more rarely parallel to the nuclear membrane (Fig. 2). As Juniper and Clowes (1965) have shown, the amount of Elg remains, as the cells expand, approximately constant per unit volume of cytoplasm. The core cells of the cap expand approximately fifteen fold in reaching their mature size. The E R rises from about 4 0 0 # m ~ in the meristematic cells to about 10000/~m 2 in the mature cap cells and remains constant in amount per cell after full expansion has taken place. The cap cells continue to expand almost until the margin of the core has been reached. The peripheral distribution of the E R leads to the superficial impression of an increase of E R per unit volume, since the surface area of the core cells falls, as they expand, in relation to their volume. Therefore the most mature core cells have not only the greatest absolute amount of E R but also the greatest area of EI~ parallel to the plasma membrane. If the roots suffer a gravitational disturbance the E R redistributes itself in predictable ways. This pattern of redistribution will be the subject of a later paper. However, after the stimulus, the E R returns once again to its peripherM position. As can be seen in Fig. 9 the E R lies close to and may, although this is not completely clear, make some contact with the plasmodesmata. In all positions of the root other than the vertical the amyloplasts can displace the E R from its position
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Fig. 6. P a r t of a core cell fixed and stained us in Fig. 3. QL quadruple lamellae; R ribosome-like bodies in a mi~ochondrion; F fibres in mitochondrion
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize
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Fig. 7. Part of a peripheral cap cell fixed and stained as in Fig. 3. Note the disorganised E R and the loss of starch from the amyloplast. N M nuclear membrane; V vacuoles; A amyloplast
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parallel to the wall; the E R is not displaced from the distal transverse wall of each cell in the normal growing position even though the amyloplasts are lying on top of it. Most of this E R is the so-called ' r o u g h ' form (Figs. 8 and 10). Small amounts of what m a y be smooth E R are sometimes seen close to the cell wall (Fig. 9). The E R in the peripheral cap cells becomes irregularly distributed, often forming spirals or clumps (Fig. 7) and see also Juniper and Roberts (1966). In addition to the long rough-surfaced E R membrane profiles, a few long profiles of smooth membranes appear in some of the eore cells (Fig. 6). These smooth membranes are not like ordinary smooth E g membranes, since they appear in fours, not in twos as the EI{ membranes do. None of these quadruple membranes has yet been resolved into a unit membrane structure.
The Ribosomes As already mentioned, ribosomes coat the surface of most of the E R and are found free in the cytoplasm. The varying thickness of a section in the electron mieroseope makes an exact comparison of ribosome numbers difficult, nevertheless their numbers in the cells of the cap appear to be conspicuously lower than in other root tip cells. Compare the density of cytoplasmic ribosomes in Figs. 8 and 10. This difference, if real, m a y account for the lack of basophilie response in the core cells noted by m a n y workers using the light microscope, and in particular the low absorption of PAS/toluidine blue stain, as can be seen in the light micrographs published by O'Brien and McCully (1969). I t m a y also account for the low absorption of Azure B in the cytoplasm of mature root cap cells noted by Barlow (1969). There are few, if any, ribosomes coating the outer surface of the nuclear membrane of cap cells (Fig. 7). Nuclei As the meristematic ceils pass into the core their nuclei become lobed (Fig. 10). I n their passage through the core the nuclei are always surrounded by a few profiles of E R (Fig. 2). This association of the E R and the nucleus ceases as the cells of the core pass into the peripheral cells (Fig. 7). This transition approximates to the point in time at which Et~ synthesis within the cell ceases and it m a y be that the nucleus has some role in the production of the ER. Mitochondria and Golgi The mitochondria and the Golgi bodies of the core cells are completely normal in appearance. They divide (or arise de novo) not only in the
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Fig. 8. P a r t of a core cell fixed and stained as in Fig. 3. Note the peripheral E R and the almost complete absence of vacuoles. M T microtubules; F vacuoles
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meristematie cells, but also in all the expanding cells of the core. As Juniper and Clowes (1965) have shown the rates of their divisions are approximately parallel; they fall, in relative numbers, slightly behind the rate of expansion in cytoplasmic volume, but catch up again as the rate of volume increase slows. They do not appear to multiply further once the cells have ceased to expand. The mitoehondria rise from about 170 to about 2500 per cell and the Golgi from about 30 to about 450 per cell. Some of the mitochondria appear to have structures similar to ribosomes and fine fibrillar areas within them (Fig. 6). Within the core the Golgi are relatively small and appear to be giving off only a few small vesicles. Only when they move out into the peripheral cells do they hypertrophy and become highly active in the ways which have been well described by Mollenhauer et al. (1961) and Northeote and Pickett Heaps (1966). Vacuoles The core, under normal light microscopy e.g. stained with PAS/ Schiff's reagent, toluidine blue or other cationic dyes, gives the impression of being highly vacuolate. The core cells have in fact few and very small vacuoles (Fig. 3). The statolith region in the rhizoids of Chara is similarly non-vacuolate (Sievers, 1967a and b). There are more vacuoles in the cap meristematic cells (Clowes and Juniper, 1968). These disappear as elongation gets under way. Vacuolation begins again as the cells pass into the peripheral cap region (Fig. 7), but the cells do not usually become completely vacuolated until they have been discharged from the surface of the root itself. The Microtubules
Microtubules are found occasionally in the meristematic cells of the cap, but they are very rare indeed in the expanding cells of the core. Where they are present (Fig. 8) they appear to be parallel to the long axis of the cell, but their rarity makes this observation of little value. The Plasmodesmata
Plasmodesmata are found through all the walls of the cap. Juniper and Barlow (1969) put forward a hypothesis, for maize root tips as a whole, t h a t plasmodesmata of this type are formed solely at a cell division and are subsequently diluted per unit area by the differential expansion of the cell walls. Juniper and Barlow found no evidence for the selective occlusion of any plasmodesmatu in the first 1 m m of the root tip.
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Fig. 9. Part of the transverse wall of a core cell fixed and stained as in Fig. 3. Note the sparsity of free ribosomes compared with Fig. 10. SE_R smooth surfaced membranes; P osmiophilic thread running through the plasmodesmatal canal Fig. 10. Part of a cell on the edge of the meristematic zone. Note the lobed nucleus, t.he starch grains beginning to appear in the amyloplasts and the relatively abundant free ribosomes. The rough E R is not yet orientated parallel to the cell wall. S starch grain; R E R rough-surfaced E R ; F fibrils within mitochondria
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B. E. Juniper and A. French: Table 1
No. of plasmodesmata per 100 ~m2 in the meristem In the core 200 ~m distal to the cap boundary In the peripheral cells
Transverse walls
Longitudinal walls
1487~= 208a 772• 513~123
530• 108 75• 14 45~= 6
a Standard deviation. Table 2. Average thickness o/the walls in txm in di//erent regions o] the cap ~m distal to the cap boundary ~m in thickness
meristeraatie cells 0.38
50 0.39
100 0.56
200 0.71
300 0.71
400 1.39
The plane of division in the cap meristem is predominantly transverse. Therefore large numbers of plasmodesmata across the transverse walls are established within the meristem. As these cells move into the columella the direction of elongation of the walls is predominantly longitudinal, so that those plasmodesmata formed by the rare longitudinal divisions are rapidly diluted, whereas those on the transverse walls are diluted more slowly (Fig. 2). Table 1 shows their distribution. Within the cap cells there are numerous plasmodesmata eonneeting the files of cells in a longitudinal direction, but few connecting cells transversely (Fig. 2). Moreover, although the cap boundary was formed very early in the development of the root and no divisions have disturbed it since it was formed, substantial numbers of plasmodesmata cross this boundary. They are, however, restricted mainly to the region Q in Fig. 1 which is the zone bordering onto the quiescent centre of the root. This quiescent region, undergoing little division (Clowes, 1956), has imposed little wall extension on region Q, which has 576:k137 plasmodesmata per 100 #m S, whereas region F (Fig. 1) on the flanks of the quiescent centre has suffered considerable extension and the number of plasmodesmata has fallen to 152 per 100/tm 2. The walls of each plasmodesma are lined by a unit membrane similar to that of the plasmalemma and each plasmodesmatal canal is partly filled by an osmiophilic thread (Fig. 9) of variable thickness, but usually about 12 nm across. The Walls The walls of the cap meristem are soft and thin, hence the ease with which whole caps m a y be pulled away from the root tip. As the
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize 327 cells move out of the meristem they elongate and at the same time thicken their walls in all planes and in addition deposit within them an as yet unidentified substance in the form of a fine reticnlum first observed by Juniper and Roberts (1966). This substance stains with KMnO 4, but not consistently with OsO 4 or any other known stain. I t seems unlikely t h a t it can be lignin, although its appearance is similar to a staining reaction believed to be lignin seen under the electron microscope in the walls of tracheids of Coleus (Hepler et al., 1970). The deposition of this material may account for the hardness of the root caps of Gramineae and Cyperaeeae discussed by Guttenberg (1968). The surfaces of root caps always appear soft due to the partial breakdown of peripheral cap cell walls. The appearance of this entrusting material in the core walls coincides with the accumulation of starch in the amyloplasts, the orientation of the E R and the cessation of division. I t persists until the core cells reach the periphery of the cap and its disappearance coincides with the loss of starch from the amyloplasts, the disorganization of the ER, the increase in activity of the Golgi bodies and the partial breakdown of the cell walls. Discussion I n Guttenberg's review of root caps, their functions are listed as protecting the meristem from damage and serving as a drilling tissue through the earth. Both these functions are served by the two or three layers of cells forming the cap periphery ; a third function, the perception of gravity, is served by the cells of the core. Only certain of the characteristics of core cells appear to have any obvious relevance to such a function. They m a y be stiffened, so that they do not distort under earth pressure, by some entrusting substance in the walls. They appear to have a low density of cytoplasm and a relative absence of vacuoles. These features might serve to facilitate the rapid movement of the amyloplasts which are believed b y most authors but doubted by others (Anker, 1968) to act in some way as statoliths (Iversen et al., 1968) and see also the review by Wilkins (1966). The amyloplasts themselves are relatively few in number, but large and able to accumulate starch rapidly as soon as the cells eease meristematie activity. Thus cells immediately distal to the meristem could serve as statocytes for the small number and large size of the amyloplasts renders them most suitable objects as statoliths, compared to the large number and small size of the other cytoplasmic organelles. In spite of the rapid turnover of carbohydrate within each amyloplast (Northcote and Piekett-tIeaps, 1966) the starch, while in the core region, is almost completely resistant to remobilisation (Audus,
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1962). However total elimination of the starch from the amyloplasts elimhlates the geotropic response (Iversen, 1969). The s y m m e t r i c a l d i s t r i b u t i o n of the E R within the columella offers possibilities to the cell to create w i t h i n it gradients of a c t i v i t y as a result of a g r a v i t a t i o n a l stimulus, which could in some way be t r a n s l a t e d into a perceived a n d t r a n s m i t t e d stimulus. Moreover, although the role of p l a s m o d e s m a t a in the root cap is u n k n o w n , their d i s t r i b u t i o n could serve as a n excellent l o n g i t u d i n a l channel of c o m m u n i c a t i o n w i t h i n the core cells of the cap where the stimulus is perceived, a n d from the core cells to the elongating zone of the root proper where the response occurs. Relerenees
Ankcr, L. : On gravi-sensitivity in plants. Acta bot. n6erl. 17, 385-389 (1968). Audus, L. J.: The mechanism of the perception of gravity by plants. "Biological receptor mechanisms." Syrup. Soc. exp. Biol. 16, 197-226 (1962). Barlow, 1). W. : Organisation in root meristems. 1)h. D. Thesis of the University of Oxford (1969). Cercek, L.: Effect of X-ray irradiation on regeneration and geotropic function of barley root caps. Int. J. Radiat. Biol. 17, 187-194 (1970). Clowes, F. A. L. : Nucleic acids in root apical meristems of Zea. New Phytol. 55, 29-34 (1956). - - Duration of the mitotic cycle in a meristem. J. exp. Bot. 12, 283-293 (1961). The proportion of cells that divide in meristems. Ann. Bot. (in press) (1970}. - - Juniper, B. E. : Plant cells. Oxford: Blackwells Sci. Publ. 1968. Gibbons, G. S. B., Wilkins, M. B. : Growth inhibitor production by root caps in relation to geotropic response. Nature (Lond.) 226, 558-559 (1970). Guttenberg, H.: Der primare Ban der Angiospermenwurzel. Handbuch der Pflanzenanatomie, Bd. 8, Tell 5, S. 138-141. Berlin: Gebrfider Borntraeger 1968. Hepler, P. K., Fosket, D. E., Newcomb, E. H. : Lignification during secondary wall formation in Coleus--an electron microscope study. Amer. J. Bot. 57, 85-92 (1970). Iversen, T. H. : Elimination of geotropic responsiveness in roots of cress (Lepidium satiwem) by removal of statolith starch. Physiol. 1)lantarum (Cph.) 22, ]2511262 (1969). - - 1)edersen, K., Larsen, 1). : Movement of amyloplasts in the root cap cells of geotropically sensitive roots. Physiol. 1)lantarum (Cph.) 21, 811-819 (1968). Juniper, B. E., Barlow, 1). W. : The distribution of plasmodesmata in the root tip of maize. Planta (Berl.) 89, 352-360 (1969). --Clowes, F. A.L.: Cytoplasmic organelles and cell growth. Nature (Lond.) 208, 864-865 (1965). - - Groves, S., Landau-Schachar, B., Audus, L. J. : The root cap and the perception of gravity. Nature (Lond.) 209, 93-94 (1966). -Roberts, R. M. : 1)olysaccharide synthesis and the fine structure of root cap cells. J. roy. micr. Soc. 85, 63-72 (1966). Konings, tt. : The significance of the root cap for geotropism. Acta hot. n4erl. 17, 203-221 (1968). Mollenhauer, It. H., Whaley, W. G., Leech, J. H. : A function of the Golgi apparatus in outer root cap cells. J. Ultrastruct. Res. 5, 193-200 (1961).
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize 329 N~mec, B.: ~ber die Art dcr Wahrnehmung des Schwerkraftreizes bei den Pflanzen. Ber. dtseh, bot. Ges. 18, 241-245 (1900). - - Die Perception des Schwerkraftreizes bei den Pflanzen. Ber. dtsch, bot. Ges. 20, 339-354 (1902). Northcote, D.H., Pickett-Heaps, J . D . : A function of the Golgi apparatus in polysaccharide synthesis and transport in the root-cap cells of wheat. Biochem. J. 98, 159-167 (1966). O'Brien, T.P., McCully, M.E.: Plant structure and development. London: Macmillan Company/Collier-Macmillan, Ltd. 1969. Possingham, J. V., Sauer, W. : Changes in chloroplast number per cell during leaf development in spinach. Planta (Berl.) 86, 186-194 (1969). Schachar, B. Landau: The root cap and its significance in graviperception. Ph. D. Thesis of the University of London (1967). Sievers, A.: Elektronenmikroskopische Untersuchungcn zur geotropischcn Reaktion. II. Die polare Organisation des normal wachsenden Rhizoids von Chara /oetida. Protoplasma (Wien) 64, 225-253 (1967a). - - Elektronenmikroskopische Untersuchungen zur geotropischen Reaktion. I. Die transversMe Polarisierung der Rhizoid-Spitze yon Chara/oetida nach 5--10 Minuten ttorizontallage. Z. Pflanzenphysiol. 57, 462473 (1967b). Syre, H. : Untersuchungen fiber Statolithenst~rke und Wuehsstoff an vorbehandelten Wurzeln. Z. Bot. 88, 129-182 (1939). Wilkins, M. B. : Geotropism. Ann. Roy. Plant. Physiol. 17, 379-408 (1966). Dr. B. E. Juniper The Botany School Oxford University South Parks Road Oxford, OX 1 3 RA, England
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The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize B. E. J u ~ I r E ~ and A ~ A F~w~c~ Botany School, University of Oxford, England l~eceived August 24/September 20, 1970
Summary. Within the root cap, in maize, the cells believed to be responsible for the perception all possess large well-developed amyloplasts. They also have normal mitochondria and Golgi bodies, normal rough-surfaced Et~ with a very striking pattern of distribution, few free ribosomes, walls with an abnormal reticulate encrnsting material, irregularly distributed plasmodesmata and an as yet unidentified fine quadruple membranous system. All of these features are discussed in relation to the role of the cells in perception. Introduction The group of cells t h a t perceives gravity in maize (Zea mays) lies within the root cap. As Fig. 1 shows, a clear boundary, rarely if ever crossed by cell divisions once the structure of the root has been formed, exists between the cap and root proper. We shall call this feature, found in all grasses, the cap boundary (C.B.). The existence of the boundary makes possible the experiments of Juniper et al. (1966), since confirmed by Konings (1968) and Gibbons and Wilkins (1970) t h a t removal of the root (decapping) prevents the normally growing root from perceiving a gravitational stimulus. I t can be surmised, but not proved, that a similar situation exists in dicotyledons. The root cap of dicotyledons can only be removed by cutting (decapitating). Such experiments have been done, by Syre (1939) and Konings (1968) and confirm the general hypothesis stated above, but are open to some criticism on the grounds of wound trauma induced by the surgery. The cells of the root cap are produced by a meristem (M in Fig. 1) a group, according to Clowes (1970), of about 6600 cells of which, in some varieties of maize, 4400 divide as rapidly as once every 10 hours. These cells do not contain mature amyloplasts, possess only undifferentiated proplastids, have no special characteristics to distinguish them from any other meristematic cells and are not thought to be responsible for the perception of the stimulus. The re-establishment of the meristem, which takes place after decapping, is not sufficient to restore the ability to perceive (Schachar, 1967). Cercek (1970), working with barley
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize 315
Fig. 1. Outline drawing of the cells of the first 1 mm of the primary root of maize
(Zea) (Hordeum vulgare), found t h a t roots from which all the differentiated cells of the cap had been cut, but which still possessed a cap meristem, also failed to respond to gravity. Surrounding the whole cap is a layer two or three cells thick (P.C. in Fig. 1) of highly specialized cells. These peripheral cap cells have been described in detail b y Mollenhauer et al. (1961), Northcote and Pickett-Heaps (1966) and Juniper and Roberts (1966). These cells do not possess complete amyloplasts, nor do these incomplete amyloplasts sediment when exposed to a gravitational stimulus. The very curious structure and patterns of behaviour of these cells have already been well described. They are not thought, for reasons which will be given more fully below, to be involved in the perception of gravity, but are thought to provide protection to the root cap as a whole as the root drives through the soil. The central cap cells (C.C. in Fig. 1) on the other hand were supposed b y N6mec (1900, 1902) to be the site of the perception of gravity on the grounds that only these cells possessed movable amyloplasts, whose movements could be correlated with the imposition of a gravitational stimulus. 21"
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Following on from J u n i p e r ' s work i n 1966, Schachar (1967) was able to show t h a t deeapped roots soon recovered the ability to perceive gravity, b u t were able to do so j u s t before the whole cap was complete i.e. when the central cap cells, b u t n o t the peripheral cap cells were present. She thus confirmed Ngmec's earlier suppositions. Cereek (1970) f o u n d also t h a t in barley no more t h a n 80% of the root cap was necessary for a full 90 ~ response to a g r a v i t a t i o n a l stimulus. The region of central cap cells, those u n h a t c h e d or d o t t e d in Fig. 1, we shall call the core. W i t h i n the core each cell is replaced a b o u t every 24 hours (Clowes, 1970).
Materials and Methods Seeds of Zea mays, variety Kelvedon 33, were germinated on filter paper or in sphagnum moss and when the primary root was about 2-3 cm long were fixed and stained in the following ways. a) In 2 % glutaraldehyde in cacodylate buffer at room temperature for 2 hours followed by 2 hours in 2% OsOt also in caeodylate buffer in crushed ice. b) In 2% glutaraldehyde for 2 hours followed by 2 hours in 2% KMn04, all at room temperature, e) Directly into 2% KMnOa for 2 hours at room temperature where a rapid fixation was wanted. Some roots were taken from the sphagnum, clipped unbroken into a perspex frame and given a specific orientation for a given time before fixation. The level of the fixative was raised around the roots clamped in their determined orientation; the roots were not moved into the fixative. After fixation the roots were embedded in Epikote 812 resin, sectioned on a Cambridge Huxley or a geichert OMU2 microtome and examined in an AEI E.NL6 or Philips 200 electron microscope. For some of the measurements large numbers of individual micrographs were taken at • 1500 of 2-3 adjacent serial sections and built up into complete mosaics of the root cap, to give a final magnification of X 3 800.
Results The Amyloplasts E a c h cell of the core of the root cap contains a b o u t 35 amyloplasts ( J u n i p e r a n d Clowes, 1965). I t is interesting t h a t this is a b o u t the same n u m b e r of statoliths as observed b y Sievers (1967a a n d b) i n the rhizoids of Chara. Once they have a c c u m u l a t e d a large a m o u n t of starch (Fig. 2) t h e y do n o t seem to divide again although occasionally images are seen of amyloplasts, with a small a m o u n t of starch, a p p a r e n t l y in the process of dividing. Thus most of the divisions giving rise to their total n u m b e r s are t h o u g h t to take place in the proplastid state in the cap meristem ( J u n i p e r a n d Clowes, 1965). This mode of origin seems to differ from t h a t of chloroplasts where most divisions seem to take place in the m a t u r e state (Possingham a n d Sauer, 1969). All of the amyloplasts in the core are, therefore~ of comparable size a n d shape a n d c o n t a i n m a n y distinct starch grains (Figs. 2 a n d 3).
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize
317
Fig. 2. Electron micrograph of a group of core cells of maize fixed in KMnO 4. A amyloplasts; N nuclei; TS W transverse wall; L W longitudinal wall
Fig. 3. Electron micrograph of a group of amyloplasts from core cells fixed in glutaraldehyde, post-fixed in O s Q and stained with uranyl acetate and lead citrate. Note that the starch grains of the amyloplasts stain black with Kmn04, but react only weakly with OsO 4. V vacuoles, W wall
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Fig. 4. A single umyloplast fixed and stained as in Fig. 3. 0 osmiophilie bodies
Fig. 5. Part of a single amyloplast fixed and stained as in Fig. 3. K knot of membranes
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize 319 Apart from starch they contain only fragmentary lamellar structures usually parallel to the outer membrane. Another consistent feature is a small group of osmiophilic bodies associated with a " k n o t " of membranes of both lamellar and fibrillar form (Figs. 4 and 5). No organelle ribosomes have ever been seen in the amyloplasts although they appear to be present in the mitochondria (Fig. 6). The amyloplasts of the peripheral cap cells begin to lose their starch (Fig. 7) which m a y explain their inability, already noted, to sediment under a gravitational stimulus. The accumulation and subsequent loss of starch by the amyloplasts deserves mention. According to Clowes (1970) each root cap is totally regenerated by its own meristem within 24 hours. Therefore each of m a n y thousands of amyloplasts every day accumulates and discharges about 14/~m 3 of starch. In the normally growing vertical position the amyloplasts accumulate at the bottom of the cells. They are never seen to be wrapped by the ER.
The ER (Endoplasmic Reticulum) In the meristematic cells the EI~ is fragmentary and has no obvious preferred orientation (Fig. 10). However, as the cells cease to divide, begin to expand and to accumulate starch in their amyloplasts, the E R becomes orientated. The E R in an undisturbed root then lies preferentially parallel to the cell walls and more rarely parallel to the nuclear membrane (Fig. 2). As Juniper and Clowes (1965) have shown, the amount of Elg remains, as the cells expand, approximately constant per unit volume of cytoplasm. The core cells of the cap expand approximately fifteen fold in reaching their mature size. The E R rises from about 4 0 0 # m ~ in the meristematic cells to about 10000/~m 2 in the mature cap cells and remains constant in amount per cell after full expansion has taken place. The cap cells continue to expand almost until the margin of the core has been reached. The peripheral distribution of the E R leads to the superficial impression of an increase of E R per unit volume, since the surface area of the core cells falls, as they expand, in relation to their volume. Therefore the most mature core cells have not only the greatest absolute amount of E R but also the greatest area of EI~ parallel to the plasma membrane. If the roots suffer a gravitational disturbance the E R redistributes itself in predictable ways. This pattern of redistribution will be the subject of a later paper. However, after the stimulus, the E R returns once again to its peripherM position. As can be seen in Fig. 9 the E R lies close to and may, although this is not completely clear, make some contact with the plasmodesmata. In all positions of the root other than the vertical the amyloplasts can displace the E R from its position
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Fig. 6. P a r t of a core cell fixed and stained us in Fig. 3. QL quadruple lamellae; R ribosome-like bodies in a mi~ochondrion; F fibres in mitochondrion
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize
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Fig. 7. Part of a peripheral cap cell fixed and stained as in Fig. 3. Note the disorganised E R and the loss of starch from the amyloplast. N M nuclear membrane; V vacuoles; A amyloplast
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parallel to the wall; the E R is not displaced from the distal transverse wall of each cell in the normal growing position even though the amyloplasts are lying on top of it. Most of this E R is the so-called ' r o u g h ' form (Figs. 8 and 10). Small amounts of what m a y be smooth E R are sometimes seen close to the cell wall (Fig. 9). The E R in the peripheral cap cells becomes irregularly distributed, often forming spirals or clumps (Fig. 7) and see also Juniper and Roberts (1966). In addition to the long rough-surfaced E R membrane profiles, a few long profiles of smooth membranes appear in some of the eore cells (Fig. 6). These smooth membranes are not like ordinary smooth E g membranes, since they appear in fours, not in twos as the EI{ membranes do. None of these quadruple membranes has yet been resolved into a unit membrane structure.
The Ribosomes As already mentioned, ribosomes coat the surface of most of the E R and are found free in the cytoplasm. The varying thickness of a section in the electron mieroseope makes an exact comparison of ribosome numbers difficult, nevertheless their numbers in the cells of the cap appear to be conspicuously lower than in other root tip cells. Compare the density of cytoplasmic ribosomes in Figs. 8 and 10. This difference, if real, m a y account for the lack of basophilie response in the core cells noted by m a n y workers using the light microscope, and in particular the low absorption of PAS/toluidine blue stain, as can be seen in the light micrographs published by O'Brien and McCully (1969). I t m a y also account for the low absorption of Azure B in the cytoplasm of mature root cap cells noted by Barlow (1969). There are few, if any, ribosomes coating the outer surface of the nuclear membrane of cap cells (Fig. 7). Nuclei As the meristematic ceils pass into the core their nuclei become lobed (Fig. 10). I n their passage through the core the nuclei are always surrounded by a few profiles of E R (Fig. 2). This association of the E R and the nucleus ceases as the cells of the core pass into the peripheral cells (Fig. 7). This transition approximates to the point in time at which Et~ synthesis within the cell ceases and it m a y be that the nucleus has some role in the production of the ER. Mitochondria and Golgi The mitochondria and the Golgi bodies of the core cells are completely normal in appearance. They divide (or arise de novo) not only in the
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Fig. 8. P a r t of a core cell fixed and stained as in Fig. 3. Note the peripheral E R and the almost complete absence of vacuoles. M T microtubules; F vacuoles
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meristematie cells, but also in all the expanding cells of the core. As Juniper and Clowes (1965) have shown the rates of their divisions are approximately parallel; they fall, in relative numbers, slightly behind the rate of expansion in cytoplasmic volume, but catch up again as the rate of volume increase slows. They do not appear to multiply further once the cells have ceased to expand. The mitoehondria rise from about 170 to about 2500 per cell and the Golgi from about 30 to about 450 per cell. Some of the mitochondria appear to have structures similar to ribosomes and fine fibrillar areas within them (Fig. 6). Within the core the Golgi are relatively small and appear to be giving off only a few small vesicles. Only when they move out into the peripheral cells do they hypertrophy and become highly active in the ways which have been well described by Mollenhauer et al. (1961) and Northeote and Pickett Heaps (1966). Vacuoles The core, under normal light microscopy e.g. stained with PAS/ Schiff's reagent, toluidine blue or other cationic dyes, gives the impression of being highly vacuolate. The core cells have in fact few and very small vacuoles (Fig. 3). The statolith region in the rhizoids of Chara is similarly non-vacuolate (Sievers, 1967a and b). There are more vacuoles in the cap meristematic cells (Clowes and Juniper, 1968). These disappear as elongation gets under way. Vacuolation begins again as the cells pass into the peripheral cap region (Fig. 7), but the cells do not usually become completely vacuolated until they have been discharged from the surface of the root itself. The Microtubules
Microtubules are found occasionally in the meristematic cells of the cap, but they are very rare indeed in the expanding cells of the core. Where they are present (Fig. 8) they appear to be parallel to the long axis of the cell, but their rarity makes this observation of little value. The Plasmodesmata
Plasmodesmata are found through all the walls of the cap. Juniper and Barlow (1969) put forward a hypothesis, for maize root tips as a whole, t h a t plasmodesmata of this type are formed solely at a cell division and are subsequently diluted per unit area by the differential expansion of the cell walls. Juniper and Barlow found no evidence for the selective occlusion of any plasmodesmatu in the first 1 m m of the root tip.
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize
325
Fig. 9. Part of the transverse wall of a core cell fixed and stained as in Fig. 3. Note the sparsity of free ribosomes compared with Fig. 10. SE_R smooth surfaced membranes; P osmiophilic thread running through the plasmodesmatal canal Fig. 10. Part of a cell on the edge of the meristematic zone. Note the lobed nucleus, t.he starch grains beginning to appear in the amyloplasts and the relatively abundant free ribosomes. The rough E R is not yet orientated parallel to the cell wall. S starch grain; R E R rough-surfaced E R ; F fibrils within mitochondria
326
B. E. Juniper and A. French: Table 1
No. of plasmodesmata per 100 ~m2 in the meristem In the core 200 ~m distal to the cap boundary In the peripheral cells
Transverse walls
Longitudinal walls
1487~= 208a 772• 513~123
530• 108 75• 14 45~= 6
a Standard deviation. Table 2. Average thickness o/the walls in txm in di//erent regions o] the cap ~m distal to the cap boundary ~m in thickness
meristeraatie cells 0.38
50 0.39
100 0.56
200 0.71
300 0.71
400 1.39
The plane of division in the cap meristem is predominantly transverse. Therefore large numbers of plasmodesmata across the transverse walls are established within the meristem. As these cells move into the columella the direction of elongation of the walls is predominantly longitudinal, so that those plasmodesmata formed by the rare longitudinal divisions are rapidly diluted, whereas those on the transverse walls are diluted more slowly (Fig. 2). Table 1 shows their distribution. Within the cap cells there are numerous plasmodesmata eonneeting the files of cells in a longitudinal direction, but few connecting cells transversely (Fig. 2). Moreover, although the cap boundary was formed very early in the development of the root and no divisions have disturbed it since it was formed, substantial numbers of plasmodesmata cross this boundary. They are, however, restricted mainly to the region Q in Fig. 1 which is the zone bordering onto the quiescent centre of the root. This quiescent region, undergoing little division (Clowes, 1956), has imposed little wall extension on region Q, which has 576:k137 plasmodesmata per 100 #m S, whereas region F (Fig. 1) on the flanks of the quiescent centre has suffered considerable extension and the number of plasmodesmata has fallen to 152 per 100/tm 2. The walls of each plasmodesma are lined by a unit membrane similar to that of the plasmalemma and each plasmodesmatal canal is partly filled by an osmiophilic thread (Fig. 9) of variable thickness, but usually about 12 nm across. The Walls The walls of the cap meristem are soft and thin, hence the ease with which whole caps m a y be pulled away from the root tip. As the
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize 327 cells move out of the meristem they elongate and at the same time thicken their walls in all planes and in addition deposit within them an as yet unidentified substance in the form of a fine reticnlum first observed by Juniper and Roberts (1966). This substance stains with KMnO 4, but not consistently with OsO 4 or any other known stain. I t seems unlikely t h a t it can be lignin, although its appearance is similar to a staining reaction believed to be lignin seen under the electron microscope in the walls of tracheids of Coleus (Hepler et al., 1970). The deposition of this material may account for the hardness of the root caps of Gramineae and Cyperaeeae discussed by Guttenberg (1968). The surfaces of root caps always appear soft due to the partial breakdown of peripheral cap cell walls. The appearance of this entrusting material in the core walls coincides with the accumulation of starch in the amyloplasts, the orientation of the E R and the cessation of division. I t persists until the core cells reach the periphery of the cap and its disappearance coincides with the loss of starch from the amyloplasts, the disorganization of the ER, the increase in activity of the Golgi bodies and the partial breakdown of the cell walls. Discussion I n Guttenberg's review of root caps, their functions are listed as protecting the meristem from damage and serving as a drilling tissue through the earth. Both these functions are served by the two or three layers of cells forming the cap periphery ; a third function, the perception of gravity, is served by the cells of the core. Only certain of the characteristics of core cells appear to have any obvious relevance to such a function. They m a y be stiffened, so that they do not distort under earth pressure, by some entrusting substance in the walls. They appear to have a low density of cytoplasm and a relative absence of vacuoles. These features might serve to facilitate the rapid movement of the amyloplasts which are believed b y most authors but doubted by others (Anker, 1968) to act in some way as statoliths (Iversen et al., 1968) and see also the review by Wilkins (1966). The amyloplasts themselves are relatively few in number, but large and able to accumulate starch rapidly as soon as the cells eease meristematie activity. Thus cells immediately distal to the meristem could serve as statocytes for the small number and large size of the amyloplasts renders them most suitable objects as statoliths, compared to the large number and small size of the other cytoplasmic organelles. In spite of the rapid turnover of carbohydrate within each amyloplast (Northcote and Piekett-tIeaps, 1966) the starch, while in the core region, is almost completely resistant to remobilisation (Audus,
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1962). However total elimination of the starch from the amyloplasts elimhlates the geotropic response (Iversen, 1969). The s y m m e t r i c a l d i s t r i b u t i o n of the E R within the columella offers possibilities to the cell to create w i t h i n it gradients of a c t i v i t y as a result of a g r a v i t a t i o n a l stimulus, which could in some way be t r a n s l a t e d into a perceived a n d t r a n s m i t t e d stimulus. Moreover, although the role of p l a s m o d e s m a t a in the root cap is u n k n o w n , their d i s t r i b u t i o n could serve as a n excellent l o n g i t u d i n a l channel of c o m m u n i c a t i o n w i t h i n the core cells of the cap where the stimulus is perceived, a n d from the core cells to the elongating zone of the root proper where the response occurs. Relerenees
Ankcr, L. : On gravi-sensitivity in plants. Acta bot. n6erl. 17, 385-389 (1968). Audus, L. J.: The mechanism of the perception of gravity by plants. "Biological receptor mechanisms." Syrup. Soc. exp. Biol. 16, 197-226 (1962). Barlow, 1). W. : Organisation in root meristems. 1)h. D. Thesis of the University of Oxford (1969). Cercek, L.: Effect of X-ray irradiation on regeneration and geotropic function of barley root caps. Int. J. Radiat. Biol. 17, 187-194 (1970). Clowes, F. A. L. : Nucleic acids in root apical meristems of Zea. New Phytol. 55, 29-34 (1956). - - Duration of the mitotic cycle in a meristem. J. exp. Bot. 12, 283-293 (1961). The proportion of cells that divide in meristems. Ann. Bot. (in press) (1970}. - - Juniper, B. E. : Plant cells. Oxford: Blackwells Sci. Publ. 1968. Gibbons, G. S. B., Wilkins, M. B. : Growth inhibitor production by root caps in relation to geotropic response. Nature (Lond.) 226, 558-559 (1970). Guttenberg, H.: Der primare Ban der Angiospermenwurzel. Handbuch der Pflanzenanatomie, Bd. 8, Tell 5, S. 138-141. Berlin: Gebrfider Borntraeger 1968. Hepler, P. K., Fosket, D. E., Newcomb, E. H. : Lignification during secondary wall formation in Coleus--an electron microscope study. Amer. J. Bot. 57, 85-92 (1970). Iversen, T. H. : Elimination of geotropic responsiveness in roots of cress (Lepidium satiwem) by removal of statolith starch. Physiol. 1)lantarum (Cph.) 22, ]2511262 (1969). - - 1)edersen, K., Larsen, 1). : Movement of amyloplasts in the root cap cells of geotropically sensitive roots. Physiol. 1)lantarum (Cph.) 21, 811-819 (1968). Juniper, B. E., Barlow, 1). W. : The distribution of plasmodesmata in the root tip of maize. Planta (Berl.) 89, 352-360 (1969). --Clowes, F. A.L.: Cytoplasmic organelles and cell growth. Nature (Lond.) 208, 864-865 (1965). - - Groves, S., Landau-Schachar, B., Audus, L. J. : The root cap and the perception of gravity. Nature (Lond.) 209, 93-94 (1966). -Roberts, R. M. : 1)olysaccharide synthesis and the fine structure of root cap cells. J. roy. micr. Soc. 85, 63-72 (1966). Konings, tt. : The significance of the root cap for geotropism. Acta hot. n4erl. 17, 203-221 (1968). Mollenhauer, It. H., Whaley, W. G., Leech, J. H. : A function of the Golgi apparatus in outer root cap cells. J. Ultrastruct. Res. 5, 193-200 (1961).
The Fine Structure of the Cells that Perceive Gravity in the Root Tip of Maize 329 N~mec, B.: ~ber die Art dcr Wahrnehmung des Schwerkraftreizes bei den Pflanzen. Ber. dtseh, bot. Ges. 18, 241-245 (1900). - - Die Perception des Schwerkraftreizes bei den Pflanzen. Ber. dtsch, bot. Ges. 20, 339-354 (1902). Northcote, D.H., Pickett-Heaps, J . D . : A function of the Golgi apparatus in polysaccharide synthesis and transport in the root-cap cells of wheat. Biochem. J. 98, 159-167 (1966). O'Brien, T.P., McCully, M.E.: Plant structure and development. London: Macmillan Company/Collier-Macmillan, Ltd. 1969. Possingham, J. V., Sauer, W. : Changes in chloroplast number per cell during leaf development in spinach. Planta (Berl.) 86, 186-194 (1969). Schachar, B. Landau: The root cap and its significance in graviperception. Ph. D. Thesis of the University of London (1967). Sievers, A.: Elektronenmikroskopische Untersuchungcn zur geotropischcn Reaktion. II. Die polare Organisation des normal wachsenden Rhizoids von Chara /oetida. Protoplasma (Wien) 64, 225-253 (1967a). - - Elektronenmikroskopische Untersuchungen zur geotropischen Reaktion. I. Die transversMe Polarisierung der Rhizoid-Spitze yon Chara/oetida nach 5--10 Minuten ttorizontallage. Z. Pflanzenphysiol. 57, 462473 (1967b). Syre, H. : Untersuchungen fiber Statolithenst~rke und Wuehsstoff an vorbehandelten Wurzeln. Z. Bot. 88, 129-182 (1939). Wilkins, M. B. : Geotropism. Ann. Roy. Plant. Physiol. 17, 379-408 (1966). Dr. B. E. Juniper The Botany School Oxford University South Parks Road Oxford, OX 1 3 RA, England
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